The rice plants of anti-insectForeword
The present invention relates to control the method for rice insect insect, the rice insect insect comprises lepidopterans paddy rice moth stem insect, and paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Especially be selected from insect: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee with next group, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; Wherein main is striped rice borer, and white standing grain snout moth's larva belongs to incertulas, and brush must belong to patnalis, cnaphalocrocis medinalls guenee by wild snout moth's larva; The Cry9C albumen that this method relates to the present invention's definition is applied to protected area and plant.In the concrete case study on implementation that the present invention optimizes, Cry9C albumen be by stable integration in rice genome the rice plant of one section proteic DNA of coding Cry9C express.
The invention still further relates to the rice plant that possesses anti-rice grub by the proteic expression of Cry9C, relate to and have the active insecticide component of anti-rice grub, specifically comprise lepidopterans paddy rice moth stem insect, paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Especially comprise the insect that is selected from next group: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; Wherein main is striped rice borer, and white standing grain snout moth's larva belongs to incertulas, and brush must belong to patnalis by wild snout moth's larva, and cnaphalocrocis medinalls guenee, insecticidal constituent comprise the Cry9C crystal, crystallin, or wherein as the active part of active ingredient.
The invention still further relates to and use the coding proteic section of DNA sequence of Cry9C of the pesticidal of definition from here, make rice plants obtain anti-insect property.
Background of invention (i) invention field
Bacillus thuringiensis (" Bt ") is a kind of gram positive bacterium, and it can produce crystal in gemma.Form crystalline protein and be proved to be toxicity with anti-insect larvae.According to amino acid whose sequence to these crystallins and corresponding gene thereof classify (Crifkmore etc., 1998).Cry9C albumen is a kind of insecticidal proteins, finds (Lambert etc., 1996) from bacillus thuringiensis at first.Have the activity of interesting anti-corn insect pest owing to express the proteic transgenic corns of Cry9C, it is by the insect invasion and attack (Jansens etc., 1997) that are used for preventing corn field of success.PCT patent publications WO 94/05771 is seen in description to Cry9C albumen and mutation and their expression products in several plant, and WO 94/24264, and WO 99/00407, and United States Patent (USP) 5,885,571 and 5,861,543 (its content is incorporated this paper into as a reference).
Insect pest is the critical limitation factor that rice is produced, and all can take place at the environment of all paddy growths.Insect seriously reduces yield of brown rice, it is reported that (except that China) is because the underproduction that insect is caused is about 31.5% (Heinrichs, 1994) in the Asia.(ii) relevant speciality technical specification
By in plant, expressing Cry1Ab or Cry1Ac albumen (e.g., Fujimoto etc., 1993 of the pesticidal in the coding bacillus thuringiensis; Wunn etc., 1996; Wu etc., 1997; Ghareyazie etc., 1997; Nayak etc., 1997; With Cheng etc., 1998), phytohaemagglutinin (Rao etc., 1998) or proteinase inhibitor (Xu etc., 1996; Duan etc., 1996) gene has the paddy rice of several types to obtain insect resistibility to some deleterious lepidopteran class rice insect insects.Isolated crystallin from bacillus thuringiensis, to lepidopteran class rice insect insect toxic component be assessed as Cry1Aa, Cry1Ab, Cry1Ac, Cry1B, Cry1C, Cry1D, Cry1E, Cry1F, Cry1G, and Cry2A albumen (Karim etc., 1997; Lee etc., 1997b).
Form in order to prevent or to postpone to continue to be exposed to the resistance of having expressed the insect pest in the proteic plant of Bt, preferably have different sources and at the higher Bt albumen of rice plants content, preferably in a strain plant, can make up the Bt albumen of expression simultaneously to having mode of action resistant gene not of the same race.The invention provides and express the proteic novel paddy rice of Cry9C, this albumen has produced the high dosage influence to relevant rice insect insect, and formed the commercial rice plants of new variety, this kind can be defendd the infringement of insect better, prevents and postpone the formation of resistance in the insect simultaneously.
In the rice biological technology International Programme conference that Phuket, THA is held, Cohen etc. have delivered the operating strategy (referring to 25 pages of summaries) of optimizing the additional defence of Bt paddy rice high dosage resistance in 20-24 in September (1999).
Brief summary of the invention
The invention provides the rice plants that following insect is had resistance: 1) lepidopterans paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus and or paddy rice hinged joint worm; 2) insect that is selected from next group comprises: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; 3) insect that is selected from next group comprises: striped rice borer, and white standing grain snout moth's larva belongs to incertulas, and brush must belong to patnalis, cnaphalocrocis medinalls guenee by wild snout moth's larva.This class rice plants comprises the dna sequence dna that can in plant express of one section stable integration in Plant Genome, its encoded protein comprises shown in the SEQ ID NO:2 in the sequence 658 the aminoacid sequence from amino acid sites 44 to amino acid sites, perhaps comprises the aminoacid sequence shown in the SEQ ID NO:4.The present invention also provides the seed of the rice plants that comprises one section proteic dna sequence dna of Cry9C of can encoding, cereal and the cereal through processing.
In another concrete case study on implementation of the present invention, in the genome of this class rice plants, also comprise the section of DNA sequence, its encoded protein is to be selected from as next histone: Cry1B, Cry1C, Cry1D, Cry1E, Cry1Aa, Cry1Ab, Cry1Ac, Cry1I, Cry1J, Cry2A, Cry6B, Cry3A, proteinase inhibitor, the cowpea insulin inhibitor, proteinase inhibitor II, cystatin, GNA phytohaemagglutinin, Xhenorhabdus or Photorhabdus spp. insecticidal proteins, the protein that careless ammonium phosphine is had resistance.
The present invention also provides a kind of method to be used to obtain following several groups of insects are had the rice plant and the offspring thereof of resistance: 1) lepidopterans paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus and or paddy rice hinged joint worm; 2) insect that is selected from next group comprises: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; 3) insect that is selected from next group comprises: striped rice borer, and white standing grain snout moth's larva belongs to incertulas, and brush must belong to patnalis, cnaphalocrocis medinalls guenee by wild snout moth's larva; Method comprises utilizes section of DNA sequence rice transformation, and wherein dna sequence dna can be expressed in plant, and its is encoded by the proteic dna sequence dna that 658 aminoacid sequence is formed from amino acid sites 44 to amino acid sites in the sequence shown in the SEQ ID NO:2; Or the protein DNA sequence that can encode and form by the aminoacid sequence shown in the SEQ ID NO:4.
The present invention also provides a kind of method to be used for producing to following several groups of insects: 1) lepidopterans paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus and or paddy rice hinged joint worm; 2) insect that is selected from next group comprises: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; 3) insect that is selected from next group comprises: striped rice borer, and white standing grain snout moth's larva belongs to incertulas, and brush must belong to patnalis by wild snout moth's larva, cnaphalocrocis medinalls guenee, rice plant and reproductive material with resistance are as seed.Method comprises utilizes section of DNA sequence rice transformation, and wherein dna sequence dna can be expressed in plant, and its coded albumen comprise by in the sequence shown in the SEQ ID NO:2 from amino acid sites 44 to amino acid sites 658 aminoacid sequence; Or can encode and comprise the protein DNA sequence of the aminoacid sequence shown in the SEQ ID NO:4.
In another concrete case study on implementation, provide the method for the following several groups of insects of control: 1) lepidopterans paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus and or paddy rice hinged joint worm; 2) insect that is selected from next group comprises: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, the America mythimna separata, straw borer spp zacconius, and rice hesperiidae; 3) insect that is selected from next group comprises: striped rice borer, white standing grain snout moth's larva belongs to incertulas, brush must belong to patnalis by wild snout moth's larva, cnaphalocrocis medinalls guenee, method comprises that the protein that utilizes desinsection dosage contacts with above-mentioned insect, wherein protein comprises shown in the SEQ ID NO:2 in the sequence from amino acid sites 44 to amino acid sites 658 aminoacid sequence or comprises the aminoacid sequence shown in the SEQ ID NO:4, also comprise following step simultaneously in this method: utilize the above-mentioned protein DNA sequence rice transformation of coding, in the rice field, cultivate above-mentioned plant and the offspring thereof of containing above-mentioned dna sequence dna.And the present invention also provides plant, seed and the cereal that obtains by above any method.Optimize the detailed description of concrete case study on implementation
Novel transgenic paddy rice plant provided by the present invention, especially various in the Oryza (Oryza), preferred Oryza sativa, show that they are fit to prevent the infringement of following several class pests: lepidopterans specifically comprises lepidopterans paddy rice moth stem insect, paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Better suited striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer, the white standing grain snout moth's larva of rice by wild snout moth's larva, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, America mythimna separata, straw borer spp zacconius, and rice hesperiidae, especially striped rice borer; The optimal straw borer spp that comprises is various, and brush must wild snout moth's larva belongs to variously, and wild snout moth's larva belongs to various, and white standing grain snout moth's larva belongs to the insect in various.By in paddy rice, expressing coding Cry9C protein DNA, can obtain such transgenic paddy rice.Utilize plant of the present invention can control lepidopterans rice insect insect well, especially the lepidopterans paddy rice eats into the stem insect, and paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Especially striped rice borer, yellow rice borer, twill is coveted noctuid, brush must belong to patnalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis by wild snout moth's larva, paddy case-bearer, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, and cnaphalocrocis medinalls guenee.Can also utilize Cry9C albumen of the present invention to control other relevant insect pest, for example rice borer rice snout moth's larva and other lepidopteran class storage insect.
In the concrete case study on implementation that the present invention optimizes, Cry9C albumen is used to control the rice insect insect, as striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp Polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, rice hesperiidae; Striped rice borer preferably, yellow rice borer, brush must belong to patnalis by wild snout moth's larva, and cnaphalocrocis medinalls guenee.The selected objective target of Cry9 control is a lepidopteran class rice insect, and the insect that wherein belongs to this type of has paddy rice moth stem insect, and paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm.
Cry9C can separate from several Bt bacterial strains according to the conventional method and obtains, and perhaps obtains by recombinant expressed, and as PCT patent publications WO 94/05771, WO 94/24264, and WO 99/00407, and United States Patent (USP) 5,885,571 and 5,861,543 description.
Term cited herein " Cry9C albumen " is meant and comprises shown in the SEQ ID NO:2 in the sequence insect-killing protein of 658 aminoacid sequence from amino acid sites 44 to amino acid sites, or comprise shown in the SEQID NO:4 in the sequence insect-killing protein of 625 aminoacid sequence from amino acid sites 1 to amino acid sites, and these two kinds of proteic variants that keep insecticidal activity, as the L-Ala variant of the protease resistant that describes in detail at PCT patent publications WO 94/24264.This term comprises that crystallin, preceding toxin or they have the form of insecticidal effect, the toxin that for example utilizes the preceding toxin of protease digestion to obtain, perhaps have in the aminoacid sequence shown in the SEQ ID NO:2 from a kind of protein of the aminoacid sequence that forms to an amino acid sites amino acid sites 658 and 666 at an amino acid sites between amino acid sites 1 and 44, also comprise fusion rotein or its varient simultaneously with protease resistant.Proteic some amino acid of known Cry9C can be replaced by other amino acid of equal value, and has still kept whole or some insecticidal activity, referring to for example PCT patent publications WO 99/00407 in the reference.These albumen all are included in the Cry9C albumen scope.Most preferred Cry9C albumen form is to comprise shown in the SEQ ID NO:2 in the sequence albumen of the aminoacid sequence 658 from amino acid sites 44 to amino acid sites among the present invention, or comprises the protein of aminoacid sequence shown in the SEQ ID NO:4.
" cry9C DNA " cited herein is meant the proteic any dna sequence dna of coding Cry9C; comprise natural; reorganization; semisynthetic or synthetic dna sequence dna; and comprise regulation domain or other zone of encoding amino acid sequence not, as intron, leader sequence or tailer sequence, promotor and 3 ' transcribe end and poly-adenylylation sequence." Cry9C coding region " is meant from initiator codon and begins to finish and the proteic any nucleotide sequence of the Cry9C that can encode to terminator codon.Because the degeneracy of genetic code, some amino acid code can not change proteinic aminoacid sequence with other codon replacement.The embodiment of relevant cry9C DNA is the DNA shown in the sequence SEQ IDNO:3 among the present invention." chimeric cry9C gene " cited herein is meant by both sides and connects the cry9C DNA that the cry9C coding region of regulatory element constitutes; regulatory element such as promotor and transcribe end sequence and poly-adenylylation sequence; they can make Cry9C albumen plant, especially express in the cell of rice plants.For other crystallin or the toxin fragment of bacillus thuringiensis that is applied to this, represent with " Cry ", as the description (1998) of Crickmore etc.When after Cry, only using a numeral and one alphabetical, as Cry1B, represent all with the listed numeral and the correlated series of beginning of letter, toxin also comprised any segment that insecticidal effect is arranged before wherein both having comprised, as the toxin (for example, " Cry1B " comprises that the form of all Cry1B is as " Cry1Ba1 " and " Cry1Bb1 ") that obtains through protease treatment.
The plant that insect is had " resistibility " or " resistance " cited herein, and infecting or attack plant to insect with " resistibility " or " resistance ", be meant by artificial action, concrete is to transform through DNA of the present invention, perhaps spray the plant that sterilant of the present invention forms, they demonstrate the obvious controllability that has strengthened insect, otherwise these insects will encroach on this kind of plant.Comprise the obvious inhibition of ingesting that strengthened, the lethality rate of the insect of growth-inhibiting or feeding plant.In the most preferred concrete case study on implementation of the present invention, the insect-resistance of transgenic plant among the present invention is meant the complete defence capability to insect, it can be by (for example being measured the less infringement that plant causes by this class insect, concerning moth stem insect, do not have passage in the cane of paddy rice), insect-resistance or be meant and do not have the similarity condition of insect infringement to be issued to much at one output.In most preferred case study on implementation, the insect-resistance of paddy rice of the present invention was meant within ten days, was preferably within 5 days, killed at least 90%, and preferred at least 95% with the Cry9C paddy rice of the present invention insect that is food.
Among the present invention, plant-effable dna sequence dna is the proteinic section of DNA sequence of gene product that can express in the plant.In the most preferred concrete case study on implementation of the present invention, this dna sequence dna comprises regulatory element, and regulatory element can make the encoding sequence that is introduced into Plant Genome correctly transcribe and translate and obtain needed protein.Representative, the promoter region that plant of the present invention-effable dna sequence dna can be transcribed in the cell of vegetable cell especially paddy rice.Plant-effable dna sequence dna can also comprise that other element is as 3 ' transcription termination sequence and poly-adenylylation sequence or intron.
The plant that this paper uses or " genome " of vegetable cell are meant the whole genetic material that are present in plant or the vegetable cell, include but are not limited to: plastosome, chloroplast(id) and nuclear DNA.The DNA of " stable integration " in genome that this paper uses is meant the DNA that integrates in the mode that can entail the plant offspring, and preferred mode is that the genome that DNA is incorporated into plant is especially examined among the DNA." offspring " of the plant that this paper uses is meant the next generation of plant, comprise the seed that obtains from plant, offspring plant and by comprise plant propagation can available from or take from cell, tissue or the whole plant of plant, can obtain to have the more plant or the following generation of identical or different genetic material by them.
" treating grain " that this paper uses is meant the cereal that utilizes one or more working methods to handle, and especially is used to the treating grain of feed and food.The course of processing of cereal includes but not limited to grinding, pulverizing, half-matureization, decortication etc.From proteic processing of paddy rice Cry9C of the present invention and undressed cereal, and the mosaic gene of the present invention that is comprised in the cereal, all comprise within the scope of the invention.
In a concrete case study on implementation of the present invention, Cry9C albumen combines with other insecticidal proteins, and the ability of the relevant rice grub of better defence is provided, and prevents and postpone the resistance formation of insect simultaneously.Preferred albumen is selected from next histone: Cry1B, Cry1C, Cry1D, Cry1E, Cry1Aa, Cry1Ab, Cry1Ac, Cry1I, Cry1J, Cry2A, Cry6B, Cry3A, proteinase inhibitor such as Cowpea Trypsin Inhibitor (CpTI, Xu etc., 1996), proteinase inhibitor II (pinII, Duan etc., 1996) or cystatin (Irie etc., 1996), GNA phytohaemagglutinin (Galanthus nivalis agglutinin, Rao etc., 1998), (1998) ffrench-Constant and Bowen such as Xhenorhabdus or Photorhabdus spp insecticidal proteins such as Bowen.(1999), PCT patent publications WO 98/08388, WO 99/03328, WO 95/00647, and WO 97/17432, and WO 98/05212, or the description among the WO 98/08932, or bacillus thuringiensis or bacillus cereus derived protein that rice grub is had insecticidal activity, the rice grub that is primarily aimed at comprises paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Specifically be various at straw borer spp, wild snout moth's larva belongs to various, and the wild snout moth's larva of brush palpus belongs to various, and white standing grain snout moth's larva belongs to various, more specifically is at striped rice borer, and brush must belong to patnalis by wild snout moth's larva, cnaphalocrocis medinalls guenee, and yellow rice borer.In the concrete case study on implementation of optimizing, Cry9C albumen is mainly by the expression of uniting in transgenic paddy rice, with at least a be selected from that following albumen combines or these protein in activeconstituents combine: Cry1Ab, Cry1B, Cry2A, Cry1C (preferred Cry1Ca4).
And, in order to screen genetically modified vegetable cell and/or to make the field plant have resistance to weedicide, optimized encoding has the anti-proteic dna sequence dna of careless ammonium phosphine, therefore comprise this proteic vegetable cell or plant can carry out normal growth in the poisonous concentration of careless ammonium phosphine molecule, specifically comprise phosphinothricin Transacetylase from streptomyces hygroscopicus or green color-producing streptomycete, as at PCT patent publications WO 87/0569, U.S. Pat 5,561,236 and US5, description in 276,268.
For intestinal bacteria, express Cry9C albumen in other bacterium or the plant, can introduce suitable restriction enzyme site in the both sides of DNA, this operation can utilize well-known method (Stanssens etc., 1989; White etc., 1989) finish by rite-directed mutagenesis.
In addition, because Cry9C albumen can be combined on the acceptor different with the Cry1A type Bt albumen on the insect goldbeater's skin, this point can be used for carrying out mating with express a kind of protein that has with the different binding sites of Cry9C albumen of proteic transgenic paddy rice of Cry9C and expression in the target insect as Cry1A or the proteic transgenic plant of Cry2A.Equally, has protein DNA in can utilizing cry9C DNA and being coded in the target insect with the different binding sites of Cry9C albumen, the Cry1Ab5 albumen that comprises brachymemma as cry1A DNA with insecticidal effect, carry out the conversion of paddy rice, control proteic transgenic plant or its offspring so that obtain to express these two kinds of insects.Coding cry1A protein DNA, can be from being included in successful expression paddy rice or the corn arbitrarily plant, and screen in any protein DNA sequence among the Cry1A that lists by people such as Crickmore that can encode and obtain that (1998 (about its full length sequence, can be referring to the numbering of reference of quoting or database)) include but not limited to these proteinic desinsection segments, the Cry1Ab of preferred this dna sequence encoding or people's such as Cry1Ac albumen, especially Hofte Cry1Ab5 albumen (1986) or it have the segment of pesticidal.In U.S. Pat 5,866, reported that the combination that several method is used to obtain the Bt toxin of at least two kind of plant expresses in 784, these methods are included in the research range of the present invention.
Can be with the cry9C gene, cry9C mosaic gene preferably, stably be inserted in the conventional mode in the nuclear gene group of rice cell, the rice plant and the offspring (seed and the next generation) thereof of the conversion of anti-insect can be regenerated and be produced to cell transformed in the conventional mode like this.In this, unloading in the Agrobacterium tumefaciens (disarmed) Ti-plasmids, owing to comprise and have the corresponding cry9C gene element of desinsection, it can be used to the rice transformation cell, utilize the method for describing among the WO 92/09696 that the PCT patent delivers then, from the rice cell that transforms regenerate the rice plant of conversion.Utilize edaphic bacillus to transform institute and preferably organize including, but not limited to sophisticated seed-deutero-callus immature plumule-deutero-callus, and immature plumule.Before cultivating altogether with edaphic bacillus, it is injured that these are organized, and utilize Syringylethanone or other the plant oxybenzene compound as describing among the PCT patent WO 98/37212, carries out pre-induction.Each all comprises the cry9C mosaic gene of Ti-plasmids DNA preferred Ti-plasmids carrier, and it or is positioned at the left side of right-hand member edge sequence at least between the edge sequence.Certainly, the carrier of other type also can be used to transformed plant cells or plant, the method that adopts comprises that direct gene shifts (as at EP 0,233, the description in 247), the conversion of pollen-mediated is (as at EP 0,270,356, PCT WO 85/01856, and U.S. Pat 4, description in 684,611), the virus-mediated conversion of plant RNA is (as at EP 0,067,553 and U.S. Pat 4,407,956 in description), liposome-mediated conversion is (as in U.S. Pat 4, description in 536,475), and other method comprise the rice conversion method of (1990) such as (1989) such as Shimanoto and Datta, with the different methods that is used for transforming monocots, as the description in PCTWO 93/21335 and WO 92/09696.Any known method that is used for rice transformation all is suitable for obtaining transgenic paddy rice of the present invention.Resulting transgenic paddy rice can be supported method for planting according to traditional plant and is used for producing the transgenic paddy rice that more has same characteristic features, perhaps the cry9C gene is introduced other paddy rice varient or relevant plant variety.Preferred paddy rice comprises the plant of each kind of Oryza, especially Oryza sativa, preferably Japanese (japonica), no matter the paddy rice of India (indica) or Java (javanica) kind is at soil, water, the highland, rainfed lowland, deep water, the paddy rice of floating growth, still irrigable paddy rice.Seed from the rice plants that transforms obtains comprises the cry9C gene, and it is to be present in the genome of nuclear, plastosome or chloroplast(id) as stable genomic parenthesis.The cell of rice transformation can be cultivated in the conventional mode, produces the Cry9C albumen with insecticidal effect.
With the genome of Cry9C DNA insertion rice cell, make the DNA of insertion be positioned at the downstream (i.e. 3 ' end) of promotor, and be subjected to the control of promotor, promotor can instruct the DNA in the vegetable cell to express.This operation is preferably inserted the cry9C mosaic gene and is finished in the vegetable cell genome.Preferred promotor comprises: cauliflower mosaic virus isolate CM 1841 (Gardner etc., 1981), the strong composing type 35S promoter (" 35S promoters ") among CabbB-S (Franck etc., 1980) and the CabbB-JI (Hull and Howell, 1987); The promotor of ubiquitin family (as, the corn ubiquitin promotor of Christensen etc., 1992, referring to Cornejo etc., 1993); Gos2 promotor (Pater etc., 1992); Emu promotor (Last etc., 1990); The promotor (1991) that rice actin promotor such as Zhang etc. describe; TR1 ' promotor and TR2 ' promotor (" TR1 ' promotor " and " TR2 ' promotor " is separately independently), 1 among their each self-driven T-DNA ' and 2 ' expression of gene (Velten etc., 1984).Perhaps, can also utilize the promotor of non-composing type, but they to one or more tissues of plant or organ (as, leaf, stem, pith and/or root tissue) have specificity, make the 9C Gene Partial of inserting in specific tissue or organ, to express.For example, the cry9C Gene Partial with insecticidal effect can optionally be expressed in the blade of paddy rice and stem, or by in pollen, there not being active promotor to express.Other promotor comprises the ribulose-1,5-bisphosphate of light-inductive promotor such as paddy rice, and the promotor of 5-bisphosphate carboxylase small ylidene gene, or the promotor of this gene of another kind of plant such as pea are as the description in patent US 5,254,799.Other promotor that can select to use be express can the inductive promotor (as because the insect that ingests, what the injury that temperature or chemical factor cause caused induces).Certainly, anyly knownly can may be used to the present invention in paddy rice especially leaf and stem camber expression promoter.
Cry9C DNA is inserted the genome of rice cell, make the Gene Partial of insertion be positioned at the upstream (promptly 5 ' hold) of genomic suitable 3 ' terminal transcriptional regulatory signal (promptly transcribe and form and poly-adenylylation signal).This operation is preferably inserted the cry9C mosaic gene and is finished in the vegetable cell genome.Preferably gather adenylylation and transcribe the formation signal and comprise octopine synthase gene (Gielen etc.; 1984) and T-DNA gene 7 (Velten and Schell; 1985) two kinds of signals of this in, these two kinds of genes in the rice transformation plant as 3 '-the non-transcribed dna sequence dna.
Cry9C DNA can be used as heterozygous genes and is optionally inserted (Vaeck etc., 1987) in the Plant Genome, and is subjected to the control of the promotor identical with the selected marker, so plant can be expressed the fusion rotein that keeps two kinds of protein actives simultaneously.Like this, can go out the Cry9C albumen of high expression level by the concentration screening that improves selected marker reagent in the substratum.
The present invention also provides a kind of method to be used to prepare the paddy rice of anti-following insect: lepidopteran class insect, especially striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, brush must belong to patnalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, brush must belong to ruralis by wild snout moth's larva, paddy case-bearer, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogramma licarisalis, rice green caterpillar, the rice satyrid, rice hesperiidae; Preferably straw borer spp is various, and wild snout moth's larva belongs to various, and the wild snout moth's larva of brush palpus belongs to various, and white standing grain snout moth's larva belongs to; Most preferably striped rice borer is brushed and must belong to patnalis, cnaphalocrocis medinalls guenee, and yellow rice borer by wild snout moth's larva.The characteristics of this method are to utilize the proteic dna sequence dna of coding Cry9C, with known step rice transformation.In a preferred method of the invention, the plant that utilizes the proteic dna sequence dna rice transformation of coding Cry9C to be produced can be resisted lepidopterans paddy rice moth stem insect, and paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, the perhaps infringement of paddy rice hinged joint worm.
Another concrete case study on implementation of the present invention provides a kind of method that is used to produce paddy rice and reproductive material such as seed, and they can resist the infringement that is selected from next group insect: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, brush must belong to patnalis by wild snout moth's larva, brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, rice hesperiidae; Preferably straw borer spp is various, and wild snout moth's larva belongs to various, and the wild snout moth's larva of brush palpus belongs to various, and white standing grain snout moth's larva belongs to; Most preferably striped rice borer is brushed and must belong to patnalis by wild snout moth's larva, cnaphalocrocis medinalls guenee, and yellow rice borer, and method comprises utilizes the proteic dna sequence dna rice transformation of coding Cry9C cell, produces the plant and the reproductive material that include above-mentioned dna sequence dna again.In preferred concrete case study on implementation, the method that provides is used to produce paddy rice and reproductive material such as seed, they can resist lepidopterans paddy rice moth stem insect, paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, the perhaps infringement of paddy rice hinged joint worm, method comprise the proteic dna sequence dna rice transformation of utilization coding Cry9C cell, produce the plant and the reproductive material that include above-mentioned dna sequence dna again.
The present invention also provides the method for the following insect of control: lepidopterans paddy rice moth stem insect, and paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm; Striped rice borer particularly, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, brush must belong to patnalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, rice hesperiidae; Preferably straw borer spp is various, and wild snout moth's larva belongs to various, and the wild snout moth's larva of brush palpus belongs to various, and white standing grain snout moth's larva belongs to; Most preferably striped rice borer is brushed and must belong to patnalis, cnaphalocrocis medinalls guenee, and yellow rice borer by wild snout moth's larva; Method comprises that the Cry9C albumen that utilizes desinsection dosage contacts with above-mentioned insect.In most preferred concrete case study on implementation, by carrying out this contact to express Cry9C albumen at field sowing, cultivation or farming rice transformation.In this case, only attempting with the paddy rice is that the rice grub that eats just is subjected to the proteic influence of Cry9C.
In another concrete case study on implementation of the present invention, the proteic composition of the Cry9C that contains insecticidal effect dosage can be sowed on paddy rice, so that these insects contact with Cry9C albumen.The conventional formulation of this composition is to utilize Cry9C albumen as active ingredient, adds appropriate carriers, diluent, emulsifying agent and/or dispersion agent (as the description of Bernhard and Utz, 1993).Parasiticidal composition can be mixed with moistening powder, pill, and particle or grit, or utilize water or anhydrous solvent to make liquid formulations such as foam, gel, suspension, concentrated solution etc.The proteic concentration of Cry9C depends on the attribute of prescription and final application mode in these compositions.Usually, can utilize insect-killing composition of the present invention to protect rice terrace, use the infringement that once can continue 1 to 2 week opposing lepidopteran class insect.For the more protection of the degree of depth (as, whole growth season), under the situation of using spray composition, the composition of the additional consumption of use that should be regular.Bt cell or crystal, crystallin, preceding toxin, toxin, and have the component of preceding toxin of insecticidal effect and other sterilant, and also have mycocide, sterilant, weedicide and chemical fertilizer can use with Cry9C, can obtain extra benefit.Usually, the proteic concentration of Cry9C accounts at least 0.1% to 100% of prescription gross weight in the composition, and concentration more commonly used accounts for prescription gross weight 0.15% to 0.8%.
The present invention also provides the method for a kind of process for processing and raw water paddy kernel, is included in field planting Cry9C paddy rice of the present invention, and results and processing seed, obtains processing and raw water paddy kernel.Output through conversion or treated water rice plants among the present invention obtains to improve.
Can utilize the albumen toxicity of the anti-following insect of program determination of having set up: lepidopterans paddy rice moth stem insect, paddy rice is hesperided, the paddy rice cutworm, the paddy rice mythimna separata, rice echinococcus, perhaps paddy rice hinged joint worm, striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, brush must belong to patnalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, brush must belong to ruralis, paddy case-bearer, the white standing grain snout moth's larva of rice by wild snout moth's larva, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, rice hesperiidae.Be applicable to that measuring method of the present invention is to soak one deck rice paddy seed or blade with the solution that contains albumen (determining concentration).Then larva is placed on seed or the blade, places after the certain hour, calculate the number of dead larva and the larva of survival is weighed.With in once testing, suitable control sample is set so that the background mortality ratio under the mensuration identical conditions and the larval growth situation of standard.Utilization has added or has added proteinic artificial food or added proteinic rice paddy seed or rice leaf can carry out similar test, as the description of (1998) such as (1997a) such as Lee or Theunis.According to routine operation, select for use suitable contrast as with the stem of one section cutting of in vitro tests or complete plant checking system system in contrast, can measure and express the toxicity of the proteinic transgenic paddy rice of the present invention, as the description of (1997) such as (1997) such as Nayak or Ghareyazie insect.
Can in rice terrace, catch rice grub and can culture in accordance with known methods (referring to as Kamano and Sato, 1985, the breed of striped rice borer) the incertulas kind that belongs to for white standing grain snout moth's larva for example, can be used the cultural method of (1998) descriptions such as Yannian.The taxonomy of lepidopteran class rice insect insect can be measured by the known method of the those of ordinary skill in this field, referring to, as Barrion and Litsinger (1994).
Be used to control the method for following insect among the present invention: lepidopterans paddy rice moth stem insect, paddy rice is hesperided, paddy rice cutworm, paddy rice mythimna separata, rice echinococcus, perhaps paddy rice leaffolders; Particularly be selected from insect: striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee with next group, brush must belong to patnalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis, paddy case-bearer by wild snout moth's larva, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, Hereitogrammalicarisalis, rice green caterpillar, rice satyrid, rice hesperiidae; Preferably straw borer spp is various, Cnaphalocrocis spp, and the wild snout moth's larva of brush palpus belongs to various, and white standing grain snout moth's larva belongs to; Most preferably striped rice borer is brushed and must belong to patnalis, cnaphalocrocis medinalls guenee, and yellow rice borer by wild snout moth's larva; Preferably include the Cry9C albumen of desinsection dosage used (for example, spray or in transgenic plant, express) to shielded field or area.Shielded field or area can comprise, for example, and the habitat of insect pest, the field of paddy growth.Or area that will rice cultivation.
The following example is used to illustrate the present invention, and is not intended to limit the scope of the invention.
The sequence table that relates in specification sheets and embodiment is as follows:
Sequence table:
Abiogenous Cry9C albumen of SEQ ID NO:1-and cry9C dna sequence dna
The proteic aminoacid sequence of Cry9C shown in the SEQ ID NO:2-SEQ ID NO:1
Cry9C DNA that SEQ ID NO:3-expresses in paddy rice and Cry9C aminoacid sequence
The proteic aminoacid sequence of Cry9C shown in the SEQ ID NO:4-SEQ ID NO:3
In an embodiment except as otherwise noted, the method of used preparation and operation recombinant DNA is carried out according to the standard method of following document description: Sambrook etc., molecular cloning-laboratory manual (MolecularCloning-A Laboratory Manual), second edition (Second Ed), Cold Spring HarborLaboratory Press, NY (1989), volume such as Ausubel 1 and volume 2 molecular biology current methods (Current Protocols in Molecular Biology), current methods (CurrentProtocols) USA.The description that is used for the material of molecular biology of plants and method is referring to the molecular biology of plants (Plant Molecular Biology Labfax) by BIOS scientific publication company limited (UK) and Blackwell Science Press (UK) combined publication, the R.R.D.Croy work.
EXAMPLE Example 1:Cry9C albumen is to the pesticidal of rice insect insect
By the insect ingestion experiment, assessment Cry9Ca albumen is to the toxicity of the rice grub that selects.
Utilize the dipping test of paddy rice leaf, check paddy rice hinged joint worm, cnaphalocrocis medinalls guenee, brush must belong to the patnalis kind by wild snout moth's larva, the various paddy rice leaves that are subject to the hinged joint influence are used for measuring Cry9Ca (R164K) albumen (LC50) (a kind of Cry9Ca albumen of purifying, therefore the arginine in 164 sites is replaced by Methionin in its aminoacid sequence, has increased the stability to proteolytic enzyme) toxic limit medium dose.Born of the same parents are immersed in 0,0.025 with blade, 0.1,0.39,1.56,6.25,25 and the Cry9C through trypsinase-processing (R164K) the active toxin body lotion of 100 micrograms/a series of concentration of ml in.All diluting solns are all used and are contained 0.01%Triton X-100 sterile distilled water and prepare, behind air-dry 30 minutes of the blade, be positioned in the culture dish and 10 newborn larvaes of adding in the sample of every kind of concentration, every kind of concentration is used five leaves, a blade representative once repeats, and adds up mortality ratio after infecting 96 hours.Utilize field test/farming research management software (ARM version 6.0.2; Gylling DataManagement (GDM), 1999) calculate toxic limit medium dose (LC50) by minimum probability variance method (Probit-Least SequareMethod).
For cnaphalocrocis medinalls guenee, the value of LC50 is that (confidence limit is in 95% degree of confidence: 0.64-1.52 microgram/ml) for 0.956 microgram/ml, must wild snout moth's larva belong to the patnalis kind for brush, the LC50 value is that (confidence limit is in 95% degree of confidence: 0.82-1.92 microgram/ml) for 1.230 micrograms/ml.Therefore Cry9C albumen is to paddy rice hinged joint worm, and cnaphalocrocis medinalls guenee and the wild snout moth's larva of brush palpus belong to the patnalis kind and have higher toxicity.Utilization is to the leaf sheath of the various paddy rice of moth stem insect sensitivity, dipping test by paddy rice detects speckle moth stem insect and striped rice borer, leaf sheath is immersed in the diluting soln of Cry9C (R163k) active toxin of a series of trypsinase-processing, five concentration have been measured, negative control is: 0 microgram/ml Cry9Ca albumen, all diluting solns are all used and are contained 0.01%Triton X-100 sterile distilled water and prepare, after leaf sheath is air-dry, it is positioned in the culture dish, and in the sample of each concentration, add five newborn larvaes, 96 as a child back statistics mortality ratio, biological detection repeats 10 times.Utilize field test/farming research management software (ARM version 6.0.2; Gylling Data Management (GDM), 1999) calculate toxic limit medium dose (LC50) by minimum probability variance method.The value of LC50 is 6.44 micrograms/ml (95% degree of confidence, confidence limit: 5.74-7.23), can find that therefore Cry9C albumen has than high toxicity speckle moth stem insect.
Natural Cry9Ca albumen (SEQ ID ND:2) is applied to above insect obtains similar result.
In the similar insect blade dipping ingestion experiment that utilizes newborn larvae to carry out, find that also Cry1Ca4 albumen is to the paddy rice cutworm, twill is coveted the noctuid kind, and ((confidence limit is in 95% degree of confidence: 0.02-0.62) have higher toxicity for LC50:019 μ g/ml, select a series of concentration (0.1 of Cry1Ca4 solution, 0.39,1.56,6.25,25 and 100 μ g/ml sterilized waters add 0.01%Triton X-100) be used to detect the activity of the greedy noctuid kind newborn larvae of twill.(diameter 6cm) Folium Ricini of will boring a hole is soaked in Cry1C solution and air-dry, and the blade that will handle is positioned in the culture dish of being furnished with moist filter paper pads again, and attacks with 10 newborn larvaes in each repeat samples.After cultivating 96 hours, measure mortality ratio.The S.litura newborn larvae is to hatch under 25 ℃ (+/-1 ℃) by the same a collection of ovum that produces in a day to obtain, with the identity of guaranteeing to hatch.Utilize other Cry1Ca albumen to carry out this test and can obtain similar result.
Utilize Cry9Ca (R164K) toxic protein segment that the greedy noctuid kind of twill is carried out same test, the result who obtains in the revision test several times of leaf dipping test is: the value of LC50 is 19.63 μ g/ml (95% degree of confidence, confidence limits: 12.61-33.55)
Therefore, for the paddy rice cutworm, twill is coveted the noctuid kind, and Cry1C and Cry9C have more highly toxic albumen.Embodiment 2: utilize cry9C mosaic gene rice transformation
The carrier system that can utilize Deblaere etc. (1985,1987) to describe carries out the conversion of rice plant.Carrier system comprises bacterial strain and two plasmid components of an edaphic bacillus: non-carcinogenic Ti-plasmid 1) middle cloning vector and 2).
Middle cloning vector comes down to (the Cornelissen andVandewiele from pGSC1700,1989) derive and obtain, it comprises a synthetical T district, is made of left side and the right side boundary sequence of the TL-DNA among the pTiB6S3 and polylinker cloning site that mosaic gene is inserted between the tumor-necrosis factor glycoproteins on T-DNA border.Be inserted into T-DNA coding two genes, i.e. a chimeric excellent gene and a chimeric cry9C gene between the tumor-necrosis factor glycoproteins on T-DNA border.Chimeric excellent gene; resistance to careless ammonium phosphine or phpsphinothricin is provided; it is by CaMV35S promotor (Odell etc.; 1985) coding region of the phpsphinothricin acetyl transferase gene of Kong Zhi streptomyces hygroscopicus (the PCT patent is published WO 87/05629); CaMV35S3 ' transcribes terminal and poly-adenylylation district (Mogen etc., 1990) are formed.Chimeric excellent gene can be used as selected marker, and provide for be applied to the field with the resistance of careless ammonium phosphine as the commercialization weedicide of active ingredient.Chimeric cry9C gene is the coding region by the SEQ IDNO:3 of CaMV35S promotor (Odell etc., 1985) control, and both sides connect 3 of CaMV35S ' and transcribe terminal and poly-adenylylation district (Mogen etc., 1990) composition.
Acceptor edaphic bacillus bacterial strain is loaded with a non-carcinogenic (unloading) Ti-plasmids, and it is that the deletion of the T-DNA in the Ti-plasmids has been kept normal viral metastatic gene.This plasmid also has a homologous region, can form cointegrates with middle cloning vector.Intermediate carrier makes up in intestinal bacteria, is loaded with the colibacillary three parental plant mating of shifting the assistance plasmid by comprising one, and intermediate carrier is transferred to acceptor Agrobacterium tumefaciens bacterial strain (Van Haute etc., 1983, Deblaere etc., 1987).The structure of T-DNA in the edaphic bacillus bacterial strain that is obtained can be measured (Deblaere etc., 1985) by southern blotting technique hybridization.
Utilize agrobacterium-mediated transgenosis, middle cloning vector is transferred to the dna segment between the tumor-necrosis factor glycoproteins of T-DNA border of rice genome.With being derived from the prematurity embryo or the callus of the rice cropping mutation of Japan and India, be cut into small pieces, as the target tissue that transforms, the technology of employing is referring to the description of PCT patent WO 92/09696.This tissue and edaphic bacillus are cultivated altogether, then careless ammonium phosphine is added in the rice tissue substratum, filter out the rice cell that is transformed.
The Calli that is grown on the careless ammonium phosphine is transferred to regeneration culture medium, after the paddy rice seedling bears root and bud, it is transferred to place the greenhouse to cultivate in the soil.Transformation can be applied to the careless ammonium phosphine of blade by the test of phosphinothricin Transacetylase, and quantitative ELISA, and methods such as PCR and southern blotting technique analysis are identified.Carry out behind the plant blossom from being pollinated the ripe seed of results later on.
Also can carry out electroporation by embryogenetic callus and obtain the Cry9C transformant injured densification, as U.S. Pat 5,679,558 description.Remove above-mentioned mosaic gene, the chimeric cry9C gene that can also make up other is expressed in paddy rice, comprise different can be in plant expression promoter such as ubiquitin and gos2 promotor (Christensen etc., 1992, dePater etc., 1992).Utilize above-mentioned soil root fungus Transformation Program, these mosaic genes also can be used to rice transformation.
Can utilize the southern blotting technique method that the rice plant with single cry9c mosaic gene that is incorporated into rice genome that copies is screened, utilize ELISA test and western blotting that the proteic expression level of the Cry9C in blade and the stem is analyzed.Proteic expression of Cry9C and insect-resistance in the plant that detection is grown up to by the seed of successful plants transformed.According to above method, utilize chimeric cry9C gene also can transform to the indica-type rice plant, after plants transformed is transferred to the greenhouse, changing effect and the insect-resistant of assessment plant.
(aminoacid sequence with Hofte etc. is from amino acid sites 2 to 616 with above-mentioned Cry9C indica-type rice kind system and the Cry1Ab5 albumen by the brachymemma of can encoding, begin with methionine(Met) and L-Ala) the indica-type rice kind system that transforms of mosaic gene carry out mating, with the resistance of raising plant, and prevent and postpone the formation of insect-resistant to insect.As mentioned above, comprise ubiquitin and gos2 promotor in the proteic mosaic gene of Cry1Ab5 of coding brachymemma, can utilize this gene to obtain the rice plant that Cry1Ab5-expresses.According to above explanation, can utilize the mosaic gene rice transformation that to encode Cry1Ca4 protein-active component and in plant, express equally.Embodiment 3: the insect-resistant of expressing the Japanese type rice transformation of Cry9C
Japanese type paddy rice from utilize the embodiment 2 that the 35S-cry9C mosaic gene transforms obtains has the anti-lepidopteran class of ELISA male rice grub insecticidal activity and the active T1 offspring of an antiweed plant, is to be undertaken by ingest Chilo spp larvae growth velocity and the mortality ratio of this class plant of record in the greenhouse at first to their assessment.The result of gained is compared with growth velocity and the mortality ratio of the larva of the rice varieties Chiyonishiki blade of the unconverted of ingesting.
In twice greenhouse test, finish four conversions (ROS-012-2805, the-2401 ,-2702, the-3703rd, be proved and expressed the proteic transformed plant of Cry9C) and a negative control, the Chiyonishiki paddy rice is assessed their resistances to striped rice borer.The rice strain that transforms is sowed in the greenhouse, when waiting to grow the 4th leaf, on plant, sprayed careless ammonium phosphine to distinguish non-transgenic plant and transfer-gen plant.
When plant begins to tiller, plant to be infected in continuous two days, each plant is used pieces of an egg.After infecting 20-35 days and 70 days, the insect pest degree of plant is not estimated to the such grade of 3 (being serious insect pest) with 0 (promptly having insect pest) respectively.Only before the plant heading, infect just and can repeat.After infecting 44-52 days, estimate outside suffered insect pest to stem.After infecting 70 days, dissect the quantity that plant and calculating contain the stem in insect tunnel.The insect pest degree that is subjected to of Chiyonishiki plant reaches the 2.5-3.0 level, and it is the insect pest that successful and unconverted plant is subjected to having higher degree that the insect that proves growth phase is infected.This conclusion infecting of last stage that be equally applicable to ear is because most of Chiyonishiki adjoining tree demonstrates the tunnel of striped rice borer.Through after growth phase infects, in the rice transformation strain three kinds, i.e. ROS12-2805 ,-2401 ,-2702 show the very low insect pest degree that is subjected to, and wherein Zui Da plant insect pest grade is 0.25.
The quantity of the cunicular stem that all transgenosis Cry9C rice plants demonstrate is lower than the Chiyonishiki adjoining tree.The ROS12-3703 of one of conversion strain of being assessed is not subjected to insect pest and cane not to have the tunnel to exist fully at all.
Compare with the straw borer spp larva of the blade of the unconverted plant that ingests, the larva that can express the proteic rice transformation plant of Cry9C that ingests has higher significantly mortality ratio.And, contain the proteic plant of higher concentration Cry9C and can better defend insect than the plant that contains low concentration.In independently insect toxicity is tested, the proteic transgenosis Cry9C plant of expression high-content Cry9C can be good at suppressing other the insect that is used to test such as lepidopteran class paddy rice moth stem insect and paddy rice hinged joint worm, especially yellow rice borer is brushed the infringement that the wild snout moth's larva of palpus belongs to patnalis and cnaphalocrocis medinalls guenee.
Transformed plant also can be used to obtain the resistance to other insect: other lepidopteran class such as paddy rice are hesperided, pink rice borer, and twill is coveted noctuid kind, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, or rice hesperiidae.Embodiment 4: the insect-resistant of expressing the indica-type rice transformation of Cry9C
Indica-type rice from utilize the embodiment 2 that Pgos2-cry9C or Pubi-cry9C mosaic gene transform obtains has the anti-lepidopteran class of ELISA male rice grub insecticidal activity and the active T1 offspring of an antiweed plant, is to be undertaken by the ingest yellow rice borer of this class plant and the larval growth speed and the mortality ratio of cnaphalocrocis medinalls guenee of record in the greenhouse at first to their assessment.The result of gained is compared with growth velocity and the mortality ratio of the larva of the blade of the same indica rice kind of the unconverted of ingesting.
In greenhouse test, the rice strain of seven conversions (be proved to be and expressed the proteic transformed plant of Cry9C) and a unconverted negative control referring to subordinate list 1, indica-type rice is assessed their resistances to yellow rice borer and cnaphalocrocis medinalls guenee.The rice strain that transforms is sowed in the greenhouse, when transplanting and waiting to grow the 4th leaf, on plant, sprayed careless ammonium phosphine to distinguish non-transgenic plant and transfer-gen plant.
Gather the larva of yellow rice borer in the field, make them under lab grow up to adult, and gather their pieces of an egg.
Transplant 4-5 after week, tillering of each plant infected with five pieces of an egg or the larva of yellow rice borer.Infect after 20-35 days by the tiller number that occurs giving up the idea of statistics and write down the degree that plant is subjected to the yellow rice borer infringement with respect to the overall number of plant tillering, tillering the characteristic feature that plant eaten into the stem insect pest infestation occurs giving up the idea.After having write down the insect pest rate, dissection plant and mensuration are subjected to the quantity (result is referring to following subordinate list 1) of the recovery larva on the insect pest plant.Unconverted indica-type rice plant has the higher insect pest grade (giving up the idea appears in the 50-100% of the sum of tillering) that is subjected to, it is the insect pest that successful and unconverted plant is subjected to higher degree that the insect of proof growth phase is infected, for the plant that is subjected to insect pest, each plant can be reclaimed 2-4 larva.
After the process growth phase infects, in the rice transformation strain three kinds, i.e. ROS045-01001, ROS046-00401, ROS048-00207 shows the very low insect pest degree that is subjected to, and each strain only has 1-2 to tiller and occur giving up the idea in the sum of tillering of all plant.Other strain defends to be subjected to the infringement of yellow rice borer fully.
Transplant 6-8 after week, tillering of each plant infected with five pieces of an egg or the larva of cnaphalocrocis medinalls guenee.Utilization is not added up the infringement degree that plant is subjected to cnaphalocrocis medinalls guenee from 0 (having insect pest) to the such grade of 9 (whole plant is withered).After having write down the insect pest rate, dissection plant and mensuration are subjected to the quantity (result is referring to following subordinate list 1) of the recovery larva on the insect pest plant.
Unconverted indica-type rice plant reaches 8 grades the higher insect pest degree that is subjected to, and it is the insect pest that successful and unconverted plant is subjected to higher degree that the insect that proves growth phase is infected, and for the plant that is subjected to insect pest, each plant can be reclaimed 9-11 larva.
Through after growth phase infects, all transform the very low insect pest ratio that is subjected to that strains demonstrate the 1-2 level, show that they can prevent to be subjected to the infringement of cnaphalocrocis medinalls guenee fully.Some slight infringements are normal, because insect need could be experienced the proteic toxicity of Cry9C by the material of digestion rice plant.
Therefore, infect test by these greenhouse plant and confirm that compare with the lepidopteran class larva of the unconverted plant that ingests, the lepidopteran class larva that can express the proteic rice transformation plant of Cry9C that ingests has higher significantly mortality ratio.
Much less, the present invention's Cry9C albumen of being not limited to above-mentioned rice plant and specifically being employed.In addition, the present invention also comprises proteic any mutant of Cry9C or varient, their keep or have improved insecticidal activity to lepidopteran class insect pest, as a kind of protein that has the proteic primary amino acid sequence of Cry9C and keep the proteic basic insecticidal activity of Cry9C (for example, a kind of pesticidal albumen and the pulsating protein that contains sequence shown in SEQ ID NO:2 or 4 or insecticidal effect wherein have 90% amino acid sequence identity at least, at least have 95% identity preferably, pesticidal wherein is at striped rice borer and yellow rice borer kind).And the present invention also is not limited to the above-mentioned rice plant of enumerating, and also comprises rice varieties arbitrarily, hybridization, selfing or original seed strain.And any plant that is vulnerable to following insect pest infestation all is included within the present invention, and as the preferred target that utilizes the proteic DNA of coding Cry9C to transform, insect comprises striped rice borer, yellow rice borer, pink rice borer, cnaphalocrocis medinalls guenee, Hereitogramma licarisalis, rice green caterpillar, rice satyrid, brush must belong to patanalis by wild snout moth's larva, and brush must belong to exigua by wild snout moth's larva, and brush must belong to ruralis by wild snout moth's larva, paddy case-bearer, the white standing grain snout moth's larva of rice, twill is coveted noctuid, straw borer spp polychrysus, the white snout moth's larva of South America rice, little sugarcane bar crambid, fall army worm, secret Noctua unipuncta, straw borer spp zacconius, and rice hesperiidae, especially striped rice borer, can use plant that method of the present invention controls these insects also including, but not limited to other grass, as water oat, sugarcane, Chinese sorghum, wheat, millet, autoecious paddy rice and India's corn.The present invention also provides a kind of method to be used to protect plant except that paddy rice to avoid the infringement of above-mentioned insect, and this method comprises utilizes the proteic DNA of coding Cry9C to transform plant, perhaps Cry9C albumen is applied to these plants.
The proteic DNA of Cry9C that utilizes the coded insect-killing effect among the present invention transforms rice cell and is not limited to finish by using the Agrobacterium tumefaciens Ti-plasmids.Other the known technology that is used for rice conversion, for example by liposome, the direct gene that electroporation or particle gun bombardment are carried out shifts, or utilizes the carrier system based on plant virus or pollen, can be used for the cry9C mosaic gene paddy rice being transformed.Expression can be carried out in the rice plant cell of stable conversion, and wherein cry9C is incorporated into genome, especially in the nuclear gene group, perhaps also can carry out in the rice plant cell of of short duration conversion.
Can be sure of to be used for rice transformation and make paddy rice express enough proteic any methods of Cry9C, can both be used to develop the rice plant of anti-insect property.In this, should first-selectedly in individual plant, express at least two kinds of non-composing types in conjunction with Bt albumen, as Cry1Aa, Cry1Ab, Cry1C or Cry1B add Cry9C albumen, to prevent and to postpone the formation of insect-resistant.Table 1
| Project | Cry 9C in the leaf tissue (percentage of extracting Tot Prot) | The insect pest that causes by yellow rice borer | The insect pest that causes by cnaphalocrocis medinalls guenee |
| (tiller number that occurs giving up the idea)/(sum of tillering) | The larva number that every strain is reclaimed | The insect pest rate | The larva number that every strain is reclaimed |
| ??ROS045- ??01001 | ?1.91+/-0.4 ????4 | ????1/34 | ????ND | ????1-2 | ????ND |
| ??ROS046- ??00301 | ?2.37+/-0.5 ????3 | ????0/91 | ????ND | ????1 | ????ND |
| ??ROS046- ??00401 | ?1.32+/-0.4 ????4 | ????2/57 | ????ND | ????1 | ????ND |
| ??ROS045- ??02401 | ?0.92+/-0.3 ????4 | ????0/39 | ????ND | ????1 | ????ND |
| ??ROS048- ??00207 | ?1.16+/-0.4 ????9 | ????1/55 | ????ND | ????1-2 | ????ND |
| ??ROS053- ??00202 | ?1.15+/-0.3 ????8 | ????0/32 | ????ND | ????1 | ????ND |
| ??ROS053- ??00306 | ?1.42+/-0.1 ????9 | ????0/36 | ????ND | ????1 | ????ND |
| Unconverted contrast | ????0 | ????46/58 | ????2-3 | ????7-8 | ????9-10 |
ND: undetermined
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Sequence table＜110〉Aventis Cropscience GmbH＜120〉paddy rice＜130〉9CRICE＜140 of anti-insect〉＜141＜160〉4＜170〉patent version 2 .0＜210〉1＜211〉3474＜212〉DNA＜213〉bacillus thuringiensis＜220〉＜221〉CDS＜222〉(1) .. (3474)＜400〉1atg aat cga aat aat caa aat gaa tat gaa att att gat gcc ccc cat 48Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His 15 10 15tgt ggg tgt cca tca gat gac gat gtg agg tat cct ttg gca agt gac 96Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp
20??????????????????25??????????????????30cca?aat?gca?gcg?tta?caa?aat?atg?aac?tat?aaa?gat?tac?tta?caa?atg????144Pro?Asn?Ala?Ala?Leu?Gln?Asn?Met?Asn?Tyr?Lys?Asp?Tyr?Leu?Gln?Met
35??????????????????40??????????????????45aca?gat?gag?gac?tac?act?gat?tct?tat?ata?aat?cct?agt?tta?tct?att????192Thr?Asp?Glu?Asp?Tyr?Thr?Asp?Ser?Tyr?Ile?Asn?Pro?Ser?Leu?Ser?Ile
50??????????????????55??????????????????60agt?ggt?aga?gat?gca?gtt?cag?act?gcg?ctt?act?gtt?gtt?ggg?aga?ata????240Ser?Gly?Arg?Asp?Ala?Val?Gln?Thr?Ala?Leu?Thr?Val?Val?Gly?Arg?Ile?65??????????????????70??????????????????75??????????????????80ctc?ggg?gct?tta?ggt?gtt?ccg?ttt?tct?gga?caa?ata?gtg?agt?ttt?tat????288Leu?Gly?Ala?Leu?Gly?Val?Pro?Phe?Ser?Gly?Gln?Ile?Val?Ser?Phe?Tyr
85??????????????????90??????????????????95caa?ttc?ctt?tta?aat?aca?ctg?tgg?cca?gtt?aat?gat?aca?gct?ata?tgg????336Gln?Phe?Leu?Leu?Asn?Thr?Leu?Trp?Pro?Val?Asn?Asp?Thr?Ala?Ile?Trp
100?????????????????105?????????????????110gaa?gct?ttc?atg?cga?cag?gtg?gag?gaa?ctt?gtc?aat?caa?caa?ata?aca????384Glu?Ala?Phe?Met?Arg?Gln?Val?Glu?Glu?Leu?Val?Asn?Gln?Gln?Ile?Thr
115?????????????????120?????????????????125gaa?ttt?gca?aga?aat?cag?gca?ctt?gca?aga?ttg?caa?gga?tta?gga?gac????432Glu?Phe?Ala?Arg?Asn?Gln?Ala?Leu?Ala?Arg?Leu?Gln?Gly?Leu?Gly?Asp
130?????????????????135?????????????????140tct?ttt?aat?gta?tat?caa?cgt?tcc?ctt?caa?aat?tgg?ttg?gct?gat?cga????480Ser?Phe?Asn?Val?Tyr?Gln?Arg?Ser?Leu?Gln?Asn?Trp?Leu?Ala?Asp?Arg145?????????????????150?????????????????155?????????????????160aat?gat?aca?cga?aat?tta?agt?gtt?gtt?cgt?gct?caa?ttt?ata?gct?tta????528Asn?Asp?Thr?Arg?Asn?Leu?Ser?Val?Val?Arg?Ala?Gln?Phe?Ile?Ala?Leu
165?????????????????170?????????????????175gac?ctt?gat?ttt?gtt?aat?gct?att?cca?ttg?ttt?gca?gta?aat?gga?cag????576Asp?Leu?Asp?Phe?Val?Asn?Ala?Ile?Pro?Leu?Phe?Ala?Val?Asn?Gly?Gln
180?????????????????185?????????????????190cag?gtt?cca?tta?ctg?tca?gta?tat?gca?caa?gct?gtg?aat?tta?cat?ttg????624Gln?Val?Pro?Leu?Leu?Ser?Val?Tyr?Ala?Gln?Ala?Val?Asn?Leu?His?Leu
195?????????????????200?????????????????205tta?tta?tta?aaa?gat?gca?tct?ctt?ttt?gga?gaa?gga?tgg?gga?ttc?aca????672Leu?Leu?Leu?Lys?Asp?Ala?Ser?Leu?Phe?Gly?Glu?Gly?Trp?Gly?Phe?Thr
210?????????????????215?????????????????220cag?ggg?gaa?att?tcc?aca?tat?tat?gac?cgt?caa?ttg?gaa?cta?acc?gct????720Gln?Gly?Glu?Ile?Ser?Thr?Tyr?Tyr?Asp?Arg?Gln?Leu?Glu?Leu?Thr?Ala225?????????????????230?????????????????235?????????????????240aag?tac?act?aat?tac?tgt?gaa?act?tgg?tat?aat?aca?ggt?tta?gat?cgt????768Lys?Tyr?Thr?Asn?Tyr?Cys?Glu?Thr?Trp?Tyr?Asn?Thr?Gly?Leu?Asp?Arg
245?????????????????250?????????????????255tta?aga?gga?aca?aat?act?gaa?agt?tgg?tta?aga?tat?cat?caa?ttc?cgt????816Leu?Arg?Gly?Thr?Asn?Thr?Glu?Ser?Trp?Leu?Arg?Tyr?His?Gln?Phe?Arg
260?????????????????265?????????????????270aga?gaa?atg?act?tta?gtg?gta?tta?gat?gtt?gtg?gcg?cta?ttt?cca?tat????864Arg?Glu?Met?Thr?Leu?Val?Val?Leu?Asp?Val?Val?Ala?Leu?Phe?Pro?Tyr
275?????????????????280?????????????????285tat?gat?gta?cga?ctt?tat?cca?acg?gga?tca?aac?cca?cag?ctt?aca?cgt????912Tyr?Asp?Val?Arg?Leu?Tyr?Pro?Thr?Gly?Ser?Asn?Pro?Gln?Leu?Thr?Arg
290?????????????????295?????????????????300gag?gta?tat?aca?gat?ccg?att?gta?ttt?aat?cca?cca?gct?aat?gtt?gga????960Glu?Val?Tyr?Thr?Asp?Pro?Ile?Val?Phe?Asn?Pro?Pro?Ala?Asn?Val?Gly305?????????????????310?????????????????315?????????????????320ctt?tgc?cga?cgt?tgg?ggt?act?aat?ccc?tat?aat?act?ttt?tct?gag?ctc????1008Leu?Cys?Arg?Arg?Trp?Gly?Thr?Asn?Pro?Tyr?Asn?Thr?Phe?Ser?Glu?Leu
325?????????????????330?????????????????335gaa?aat?gcc?ttc?att?cgc?cca?cca?cat?ctt?ttt?gat?agg?ctg?aat?agc????1056Glu?Asn?Ala?Phe?Ile?Arg?Pro?Pro?His?Leu?Phe?Asp?Arg?Leu?Asn?Ser
340?????????????????345?????????????????350tta?aca?atc?agc?agt?aat?cga?ttt?cca?gtt?tca?tct?aat?ttt?atg?gat????1104Leu?Thr?Ile?Ser?Ser?Asn?Arg?Phe?Pro?Val?Ser?Ser?Asn?Phe?Met?Asp
355?????????????????360?????????????????365tat?tgg?tca?gga?cat?acg?tta?cgc?cgt?agt?tat?ctg?aac?gat?tca?gca????1152Tyr?Trp?Ser?Gly?His?Thr?Leu?Arg?Arg?Ser?Tyr?Leu?Asn?Asp?Ser?Ala
370?????????????????375?????????????????380gta?caa?gaa?gat?agt?tat?ggc?cta?att?aca?acc?aca?aga?gca?aca?att????1200Val?Gln?Glu?Asp?Ser?Tyr?Gly?Leu?Ile?Thr?Thr?Thr?Arg?Ala?Thr?Ile385?????????????????390?????????????????395?????????????????400aat?ccc?gga?gtt?gat?gga?aca?aac?cgc?ata?gag?tca?acg?gca?gta?gat????1248Asn?Pro?Gly?Val?Asp?Gly?Thr?Asn?Arg?Ile?Glu?Ser?Thr?Ala?Val?Asp
405?????????????????410?????????????????415ttt?cgt?tct?gca?ttg?ata?ggt?ata?tat?ggc?gtg?aat?aga?gct?tct?ttt????1296Phe?Arg?Ser?Ala?Leu?Ile?Gly?Ile?Tyr?Gly?Val?Asn?Arg?Ala?Ser?Phe
420?????????????????425?????????????????430gtc?cca?gga?ggc?ttg?ttt?aat?ggt?acg?act?tct?cct?gct?aat?gga?gga????1344Val?Pro?Gly?Gly?Leu?Phe?Asn?Gly?Thr?Thr?Ser?Pro?Ala?Asn?Gly?Gly
435?????????????????440?????????????????445tgt?aga?gat?ctc?tat?gat?aca?aat?gat?gaa?tta?cca?cca?gat?gaa?agt????1392Cys?Arg?Asp?Leu?Tyr?Asp?Thr?Asn?Asp?Glu?Leu?Pro?Pro?Asp?Glu?Ser
450?????????????????455?????????????????460acc?gga?agt?tca?acc?cat?aga?cta?tct?cat?gtt?acc?ttt?ttt?agc?ttt????1440Thr?Gly?Ser?Ser?Thr?His?Arg?Leu?Ser?His?Val?Thr?Phe?Phe?Ser?Phe465?????????????????470?????????????????475?????????????????480caa?act?aat?cag?gct?gga?tct?ata?gct?aat?gca?gga?agt?gta?cct?act????1488Gln?Thr?Asn?Gln?Ala?Gly?Ser?Ile?Ala?Asn?Ala?Gly?Ser?Val?Pro?Thr
485?????????????????490?????????????????495tat?gtt?tgg?acc?cgt?cgt?gat?gtg?gac?ctt?aat?aat?acg?att?acc?cca????1536Tyr?Val?Trp?Thr?Arg?Arg?Asp?Val?Asp?Leu?Asn?Asn?Thr?Ile?Thr?Pro
500?????????????????505?????????????????510aat?aga?att?aca?caa?tta?cca?ttg?gta?aag?gca?tct?gca?cct?gtt?tcg????1584Asn?Arg?Ile?Thr?Gln?Leu?Pro?Leu?Val?Lys?Ala?Ser?Ala?Pro?Val?Ser
515?????????????????520?????????????????525ggt?act?acg?gtc?tta?aaa?ggt?cca?gga?ttt?aca?gga?ggg?ggt?ata?ctc????1632Gly?Thr?Thr?Val?Leu?Lys?Gly?Pro?Gly?Phe?Thr?Gly?Gly?Gly?Ile?Leu
530?????????????????535?????????????????540cga?aga?aca?act?aat?ggc?aca?ttt?gga?acg?tta?aga?gta?acg?gtt?aat????1680Arg?Arg?Thr?Thr?Asn?Gly?Thr?Phe?Gly?Thr?Leu?Arg?Val?Thr?Val?Asn545?????????????????550?????????????????555?????????????????560tca?cca?tta?aca?caa?caa?tat?cgc?cta?aga?gtt?cgt?ttt?gcc?tca?aca????1728Ser?Pro?Leu?Thr?Gln?Gln?Tyr?Arg?Leu?Arg?Val?Arg?Phe?Ala?Ser?Thr
565?????????????????570?????????????????575gga?aat?ttc?agt?ata?agg?gta?ctc?cgt?gga?ggg?gtt?tct?atc?ggt?gat????1776Gly?Asn?Phe?Ser?Ile?Arg?Val?Leu?Arg?Gly?Gly?Val?Ser?Ile?Gly?Asp
580?????????????????585?????????????????590gtt?aga?tta?ggg?agc?aca?atg?aac?aga?ggg?cag?gaa?cta?act?tac?gaa????1824Val?Arg?Leu?Gly?Ser?Thr?Met?Asn?Arg?Gly?Gln?Glu?Leu?Thr?Tyr?Glu
595?????????????????600?????????????????605tcc?ttt?ttc?aca?aga?gag?ttt?act?act?act?ggt?ccg?ttc?aat?ccg?cct????1872Ser?Phe?Phe?Thr?Arg?Glu?Phe?Thr?Thr?Thr?Gly?Pro?Phe?Asn?Pro?Pro
610?????????????????615?????????????????620ttt?aca?ttt?aca?caa?gct?caa?gag?att?cta?aca?gtg?aat?gca?gaa?ggt????1920Phe?Thr?Phe?Thr?Gln?Ala?Gln?Glu?Ile?Leu?Thr?Val?Asn?Ala?Glu?Gly625?????????????????630?????????????????635?????????????????640gtt?agc?acc?ggt?ggt?gaa?tat?tat?ata?gat?aga?att?gaa?att?gtc?cct????1968Val?Ser?Thr?Gly?Gly?Glu?Tyr?Tyr?Ile?Asp?Arg?Ile?Glu?Ile?Val?Pro
645?????????????????650?????????????????655gtg?aat?ccg?gca?cga?gaa?gcg?gaa?gag?gat?tta?gaa?gcg?gcg?aag?aaa???2016Val?Asn?Pro?Ala?Arg?Glu?Ala?Glu?Glu?Asp?Leu?Glu?Ala?Ala?Lys?Lys
660?????????????????665?????????????????670gcg?gtg?gcg?agc?ttg?ttt?aca?cgt?aca?agg?gac?gga?tta?cag?gta?aat????2064Ala?Val?Ala?Ser?Leu?Phe?Thr?Arg?Thr?Arg?Asp?Gly?Leu?Gln?Val?Asn
675?????????????????680?????????????????685gtg?aca?gat?tat?caa?gtg?gac?caa?gcg?gca?aat?tta?gtg?rca?tgc?tta???2112Val?Thr?Asp?Tyr?Gln?Val?Asp?Gln?Ala?Ala?Asn?Leu?Val?Ser?Cys?Leu
690?????????????????695?????????????????700tcc?gat?gaa?caa?tat?ggg?cat?gac?aaa?aag?atg?tta?ttg?gaa?gcg?gta???2160Ser?Asp?Glu?Gln?Tyr?Gly?His?Asp?Lys?Lys?Met?Leu?Leu?Glu?Ala?Val705?????????????????710?????????????????715?????????????????720aga?gcg?gca?aaa?cgc?ctc?agc?cgc?gaa?cgc?aac?tta?ctt?caa?gat?cca???2208Arg?Ala?Ala?Lys?Arg?Leu?Ser?Arg?Glu?Arg?Asn?Leu?Leu?Gln?Asp?Pro
725?????????????????730?????????????????735gat?ttt?aat?aca?atc?aat?agt?aca?gaa?gag?aat?ggc?tgg?aag?gca?agt???2256Asp?Phe?Asn?Thr?Ile?Asn?Ser?Thr?Glu?Glu?Asn?Gly?Trp?Lys?Ala?Ser
740?????????????????745?????????????????750aac?ggt?gtt?act?att?agc?gag?ggc?ggt?cca?ttc?ttt?aaa?ggt?cgt?gca???2304Asn?Gly?Val?Thr?Ile?Ser?Glu?Gly?Gly?Pro?Phe?Phe?Lys?Gly?Arg?Ala
755?????????????????760?????????????????765ctt?cag?tta?gca?agc?gca?aga?gaa?aat?tat?cca?aca?tac?att?tat?caa????2352Leu?Gln?Leu?Ala?Ser?Ala?Arg?Glu?Asn?Tyr?Pro?Thr?Tyr?Ile?Tyr?Gln
770?????????????????775?????????????????780aaa?gta?gat?gca?tcg?gtg?tta?aag?cct?tat?aca?cgc?tat?aga?cta?gat????2400Lys?Val?Asp?Ala?Ser?Val?Leu?Lys?Pro?Tyr?Thr?Arg?Tyr?Arg?Leu?Asp785?????????????????790?????????????????795?????????????????800gga?ttt?gtg?aag?agt?agt?caa?gat?tta?gaa?att?gat?ctc?atc?cac?cat????2448Gly?Phe?Val?Lys?Ser?Ser?Gln?Asp?Leu?Glu?Ile?Asp?Leu?Ile?His?His
805?????????????????810?????????????????815cat?aaa?gtc?cat?ctt?gta?aaa?aat?gta?cca?gat?aat?tta?gta?tct?gat????2496His?Lys?Val?His?Leu?Val?Lys?Asn?Val?Pro?Asp?Asn?Leu?Val?Ser?Asp
820?????????????????825?????????????????830act?tac?tca?gat?ggt?tct?tgc?agc?gga?atc?aac?cgt?tgt?gat?gaa?cag????2544Thr?Tyr?Ser?Asp?Gly?Ser?Cys?Ser?Gly?Ile?Asn?Arg?Cys?Asp?Glu?Gln
835?????????????????840?????????????????845cat?cag?gta?gat?atg?cag?cta?gat?gcg?gag?cat?cat?cca?atg?gat?tgc????2592His?Gln?Val?Asp?Met?Gln?Leu?Asp?Ala?Glu?His?His?Pro?Met?Asp?Cys
850?????????????????855?????????????????860tgt?gaa?gcg?gct?caa?aca?cat?gag?ttt?tct?tcc?tat?att?aat?aca?ggg????2640Cys?Glu?Ala?Ala?Gln?Thr?His?Glu?Phe?Ser?Ser?Tyr?Ile?Asn?Thr?Gly865?????????????????870?????????????????875?????????????????880gat?cta?aat?gca?agt?gta?gat?cag?ggc?att?tgg?gtt?gta?tta?aaa?gtt????2688Asp?Leu?Asn?Ala?Ser?Val?Asp?Gln?Gly?Ile?Trp?Val?Val?Leu?Lys?Val
885?????????????????890?????????????????895cga?aca?aca?gat?ggg?tat?gcg?acg?tta?gga?aat?ctt?gaa?ttg?gta?gag????2736Arg?Thr?Thr?Asp?Gly?Tyr?Ala?Thr?Leu?Gly?Asn?Leu?Glu?Leu?Val?Glu
900?????????????????905?????????????????910gtt?ggg?cca?tta?tcg?ggt?gaa?tct?cta?gaa?cgg?gaa?caa?aga?gat?aat????2784Val?Gly?Pro?Leu?Ser?Gly?Glu?Ser?Leu?Glu?Arg?Glu?Gln?Arg?Asp?Asn
915?????????????????920?????????????????925gcg?aaa?tgg?aat?gca?gag?cta?gga?aga?aaa?cgt?gca?gaa?ata?gat?cgt????2832Ala?Lys?Trp?Asn?Ala?Glu?Leu?Gly?Arg?Lys?Arg?Ala?Glu?Ile?Asp?Arg
930?????????????????935?????????????????940gtg?tat?tta?gct?gcg?aaa?caa?gca?att?aat?cat?ctg?ttt?gta?gac?tat????2880Val?Tyr?Leu?Ala?Ala?Lys?Gln?Ala?Ile?Asn?His?Leu?Phe?Val?Asp?Tyr945?????????????????950?????????????????955?????????????????960caa?gat?caa?caa?tta?aat?cca?gaa?att?ggg?cta?gca?gaa?att?aat?gaa????2928Gln?Asp?Gln?Gln?Leu?Asn?Pro?Glu?Ile?Gly?Leu?Ala?Glu?Ile?Asn?Glu
965?????????????????970?????????????????975gct?tca?aat?ctt?gta?gag?tca?att?tcg?ggt?gta?tat?agt?gat?aca?cta????2976Ala?Ser?Asn?Leu?Val?Glu?Ser?Ile?Ser?Gly?Val?Tyr?Ser?Asp?Thr?Leu
980?????????????????985?????????????????990tta?cag?att?cct?ggg?att?aac?tac?gaa?att?tac?aca?gag?tta?tcc?gat????3024Leu?Gln?Ile?Pro?Gly?Ile?Asn?Tyr?Glu?Ile?Tyr?Thr?Glu?Leu?Ser?Asp
995????????????????1000????????????????1005cgc?tta?caa?caa?gca?tcg?tat?ctg?tat?acg?tct?aga?aat?gcg?gtg?caa????3072Arg?Leu?Gln?Gln?Ala?Ser?Tyr?Leu?Tyr?Thr?Ser?Arg?Asn?Ala?Val?Gln???1010????????????????1015????????????????1020aat?gga?gac?ttt?aac?agt?ggt?cta?gat?agt?tgg?aat?aca?act?atg?gat????3120Asn?Gly?Asp?Phe?Asn?Ser?Gly?Leu?Asp?Ser?Trp?Asn?Thr?Thr?Met?Asp1025???????????????1030????????????????1035????????????????1040gca?tcg?gtt?cag?caa?gat?ggc?aat?atg?cat?ttc?tta?gtt?ctt?tcg?cat????3168Ala?Ser?Val?Gln?Gln?Asp?Gly?Asn?Met?His?Phe?Leu?Val?Leu?Ser?His
1045????????????????1050????????????????1055tgg?gat?gca?caa?gtt?tcc?caa?caa?ttg?aga?gta?aat?ccg?aat?tgt?aag????3216Trp?Asp?Ala?Gln?Val?Ser?Gln?Gln?Leu?Arg?Val?Asn?Pro?Asn?Cys?Lys
1060????????????????1065????????????????1070tat?gtc?tta?cgt?gtg?aca?gca?aga?aaa?gta?gga?ggc?gga?gat?gga?tac????3264Tyr?Val?Leu?Arg?Val?Thr?Ala?Arg?Lys?Val?Gly?Gly?Gly?Asp?Gly?Tyr
1075????????????????1080????????????????1085gtc?aca?atc?cga?gat?ggc?gct?cat?cac?caa?gaa?act?ctt?aca?ttt?aat????3312Val?Thr?Ile?Arg?Asp?Gly?Ala?His?His?Gln?Glu?Thr?Leu?Thr?Phe?Asn???1090????????????????1095????????????????1100gca?tgt?gac?tac?gat?gta?aat?ggt?acg?tat?gtc?aat?gac?aat?tcg?tat????3360Ala?Cys?Asp?Tyr?Asp?Val?Asn?Gly?Thr?Tyr?Val?Asn?Asp?Asn?Ser?Tyr1105???????????????1110????????????????1115????????????????1120ata?aca?gaa?gaa?gtg?gta?ttc?tac?cca?gag?aca?aaa?cat?atg?tgg?gta????3408Ile?Thr?Glu?Glu?Val?Val?Phe?Tyr?Pro?Glu?Thr?Lys?His?Met?Trp?Val
1125????????????????1130????????????????1135gag?gtg?agt?gaa?tcc?gaa?ggt?tca?ttc?tat?ata?gac?agt?att?gag?ttt????3456Glu?Val?Ser?Glu?Ser?Glu?Gly?Ser?Phe?Tyr?Ile?Asp?Ser?Ile?Glu?Phe
1140????????????????1145????????????????1150att?gaa?aca?caa?gag?tag????????????????????????????????????????????3474Ile?Glu?Thr?Gln?Glu
1155＜210〉2＜211〉1157＜212〉PRT＜213〉bacillus thuringiensis＜400〉2Met Asn Arg Asn Asn Gln Asn Glu Tyr Glu Ile Ile Asp Ala Pro His, 15 10 15Cys Gly Cys Pro Ser Asp Asp Asp Val Arg Tyr Pro Leu Ala Ser Asp
20??????????????????25??????????????????30Pro?Asn?Ala?Ala?Leu?Gln?Asn?Met?Asn?Tyr?Lys?Asp?Tyr?Leu?Gln?Met
35??????????????????40??????????????????45Thr?Asp?Glu?Asp?Tyr?Thr?Asp?Ser?Tyr?Ile?Asn?Pro?Ser?Leu?Ser?Ile
50??????????????????55??????????????????60Ser?Gly?Arg?Asp?Ala?Val?Gln?Thr?Ala?Leu?Thr?Val?Val?Gly?Arg?Ile?65??????????????????70??????????????????75??????????????????80Leu?Gly?Ala?Leu?Gly?Val?Pro?Phe?Ser?Gly?Gln?Ile?Val?Ser?Phe?Tyr
85??????????????????90??????????????????95Gln?Phe?Leu?Leu?Asn?Thr?Leu?Trp?Pro?Val?Asn?Asp?Thr?Ala?Ile?Trp
100?????????????????105?????????????????110Glu?Ala?Phe?Met?Arg?Gln?Val?Glu?Glu?Leu?Val?Asn?Gln?Gln?Ile?Thr
115?????????????????120?????????????????125Glu?Phe?Ala?Arg?Asn?Gln?Ala?Leu?Ala?Arg?Leu?Gln?Gly?Leu?Gly?Asp
130?????????????????135?????????????????140Ser?Phe?Asn?Val?Tyr?Gln?Arg?Ser?Leu?Gln?Asn?Trp?Leu?Ala?Asp?Arg145?????????????????150?????????????????155?????????????????160Asn?Asp?Thr?Arg?Asn?Leu?Ser?Val?Val?Arg?Ala?Gln?Phe?Ile?Ala?Leu
165?????????????????170?????????????????175Asp?Leu?Asp?Phe?Val?Asn?Ala?Ile?Pro?Leu?Phe?Ala?Val?Asn?Gly?Gln
180?????????????????185?????????????????190Gln?Val?Pro?Leu?Leu?Ser?Val?Tyr?Ala?Gln?Ala?Val?Asn?Leu?His?Leu
195?????????????????200?????????????????205Leu?Leu?Leu?Lys?Asp?Ala?Ser?Leu?Phe?Gly?Glu?Gly?Trp?Gly?Phe?Thr
210?????????????????215?????????????????220Gln?Gly?Glu?Ile?Ser?Thr?Tyr?Tyr?Asp?Arg?Gln?Leu?Glu?Leu?Thr?Ala225?????????????????230?????????????????235?????????????????240Lys?Tyr?Thr?Asn?Tyr?Cys?Glu?Thr?Trp?Tyr?Asn?Thr?Gly?Leu?Asp?Arg
245?????????????????250?????????????????255Leu?Arg?Gly?Thr?Asn?Thr?Glu?Ser?Trp?Leu?Arg?Tyr?His?Gln?Phe?Arg
260?????????????????265?????????????????270Arg?Glu?Met?Thr?Leu?Val?Val?Leu?Asp?Val?Val?Ala?Leu?Phe?Pro?Tyr
275?????????????????280?????????????????285Tyr?Asp?Val?Arg?Leu?Tyr?Pro?Thr?Gly?Ser?Asn?Pro?Gln?Leu?Thr?Arg
290?????????????????295?????????????????300Glu?Val?Tyr?Thr?Asp?Pro?Ile?Val?Phe?Ash?Pro?Pro?Ala?Asn?Val?Gly305?????????????????310?????????????????315?????????????????320Leu?Cys?Arg?Arg?Trp?Gly?Thr?Asn?Pro?Tyr?Asn?Thr?Phe?Ser?Glu?Leu
325?????????????????330?????????????????335Glu?Asn?Ala?Phe?Ile?Arg?Pro?Pro?His?Leu?Phe?Asp?Arg?Leu?Asn?Ser
340?????????????????345?????????????????350Leu?Thr?Ile?Ser?Ser?Asn?Arg?Phe?Pro?Val?Ser?Ser?Asn?Phe?Met?Asp
355?????????????????360?????????????????365Tyr?Trp?Ser?Gly?His?Thr?Leu?Arg?Arg?Ser?Tyr?Leu?Asn?Asp?Ser?Ala
370?????????????????375?????????????????380Val?Gln?Glu?Asp?Ser?Tyr?Gly?Leu?Ile?Thr?Thr?Thr?Arg?Ala?Thr?Ile385?????????????????390?????????????????395?????????????????400Asn?Pro?Gly?Val?Asp?Gly?Thr?Asn?Arg?Ile?Glu?Ser?Thr?Ala?Val?Asp
405?????????????????410?????????????????415Phe?Arg?Ser?Ala?Leu?Ile?Gly?Ile?Tyr?Gly?Val?Asn?Arg?Ala?Ser?Phe
420?????????????????425?????????????????430Val?Pro?Gly?Gly?Leu?Phe?Asn?Gly?Thr?Thr?Ser?Pro?Ala?Asn?Gly?Gly
435?????????????????440?????????????????445Cys?Arg?Asp?Leu?Tyr?Asp?Thr?Asn?Asp?Glu?Leu?Pro?Pro?Asp?Glu?Ser
450?????????????????455?????????????????460Thr?Gly?Ser?Ser?Thr?His?Arg?Leu?Ser?His?Val?Thr?Phe?Phe?Ser?Phe465?????????????????470?????????????????475?????????????????480Gln?Thr?Asn?Gln?Ala?Gly?Ser?Ile?Ala?Asn?Ala?Gly?Ser?Val?Pro?Thr
485?????????????????490?????????????????495Tyr?Val?Trp?Thr?Arg?Arg?Asp?Val?Asp?Leu?Asn?Asn?Thr?Ile?Thr?Pro
500?????????????????505?????????????????510Asn?Arg?Ile?Thr?Gln?Leu?Pro?Leu?Val?Lys?Ala?Ser?Ala?Pro?Val?Ser
515?????????????????520?????????????????525Gly?Thr?Thr?Val?Leu?Lys?Gly?Pro?Gly?Phe?Thr?Gly?Gly?Gly?Ile?Leu
530?????????????????535?????????????????540Arg?Arg?Thr?Thr?Asn?Gly?Thr?Phe?Gly?Thr?Leu?Arg?Val?Thr?Val?Asn545?????????????????550?????????????????555?????????????????560Ser?Pro?Leu?Thr?Gln?Gln?Tyr?Arg?Leu?Arg?Val?Arg?Phe?Ala?Ser?Thr
565?????????????????570?????????????????575Gly?Asn?Phe?Ser?Ile?Arg?Val?Leu?Arg?Gly?Gly?Val?Ser?Ile?Gly?Asp
580?????????????????585?????????????????590Val?Arg?Leu?Gly?Ser?Thr?Met?Asn?Arg?Gly?Gln?Glu?Leu?Thr?Tyr?Glu
595?????????????????600?????????????????605Ser?Phe?Phe?Thr?Arg?Glu?Phe?Thr?Thr?Thr?Gly?Pro?Phe?Asn?Pro?Pro
610?????????????????615?????????????????620Phe?Thr?Phe?Thr?Gln?Ala?Gln?Glu?Ile?Leu?Thr?Val?Asn?Ala?Glu?Gly625?????????????????630?????????????????635?????????????????640Val?Ser?Thr?Gly?Gly?Glu?Tyr?Tyr?Ile?Asp?Arg?Ile?Glu?Ile?Val?Pro
645?????????????????650?????????????????655Val?Asn?Pro?Ala?Arg?Glu?Ala?Glu?Glu?Asp?Leu?Glu?Ala?Ala?Lys?Lys
660?????????????????665?????????????????670Ala?Val?Ala?Ser?Leu?Phe?Thr?Arg?Thr?Arg?Asp?Gly?Leu?Gln?Val?Asn
675?????????????????680?????????????????685Val?Thr?Asp?Tyr?Gln?Val?Asp?Gln?Ala?Ala?Asn?Leu?Val?Ser?Cys?Leu
690?????????????????695?????????????????700Ser?Asp?Glu?Gln?Tyr?Gly?His?Asp?Lys?Lys?Met?Leu?Leu?Glu?Ala?Val705?????????????????710?????????????????715?????????????????720Arg?Ala?Ala?Lys?Arg?Leu?Ser?Arg?Glu?Arg?Asn?Leu?Leu?Gln?Asp?Pro
725?????????????????730?????????????????735Asp?Phe?Asn?Thr?Ile?Asn?Ser?Thr?Glu?Glu?Asn?Gly?Trp?Lys?Ala?Ser
740?????????????????745?????????????????750Asn?Gly?Val?Thr?Ile?Ser?Glu?Gly?Gly?Pro?Phe?Phe?Lys?Gly?Arg?Ala
755?????????????????760?????????????????765Leu?Gln?Leu?Ala?Ser?Ala?Arg?Glu?Asn?Tyr?Pro?Thr?Tyr?Ile?Tyr?Gln
770?????????????????775?????????????????780Lys?Val?Asp?Ala?Ser?Val?Leu?Lys?Pro?Tyr?Thr?Arg?Tyr?Arg?Leu?Asp785?????????????????790?????????????????795?????????????????800Gly?Phe?Val?Lys?Ser?Ser?Gln?Asp?Leu?Glu?Ile?Asp?Leu?Ile?His?His
805?????????????????810?????????????????815His?Lys?Val?His?Leu?Val?Lys?Asn?Val?Pro?Asp?Asn?Leu?Val?Ser?Asp
820?????????????????825?????????????????830Thr?Tyr?Ser?Asp?Gly?Ser?Cys?Ser?Gly?Ile?Asn?Arg?Cys?Asp?Glu?Gln
835?????????????????840?????????????????845His?Gln?Val?Asp?Met?Gln?Leu?Asp?Ala?Glu?His?His?Pro?Met?Asp?Cys
850?????????????????855?????????????????860Cys?Glu?Ala?Ala?Gln?Thr?His?Glu?Phe?Ser?Ser?Tyr?Ile?Asn?Thr?Gly865?????????????????870?????????????????875?????????????????880Asp?Leu?Asn?Ala?Ser?Val?Asp?Gln?Gly?Ile?Trp?Val?Val?Leu?Lys?Val
885?????????????????890?????????????????895Arg?Thr?Thr?Asp?Gly?Tyr?Ala?Thr?Leu?Gly?Asn?Leu?Glu?Leu?Val?Glu
900?????????????????905?????????????????910Val?Gly?Pro?Leu?Ser?Gly?Glu?Ser?Leu?Glu?Arg?Glu?Gln?Arg?Asp?Asn
915?????????????????920?????????????????925Ala?Lys?Trp?Asn?Ala?Glu?Leu?Gly?Arg?Lys?Arg?Ala?Glu?Ile?Asp?Arg
930?????????????????935?????????????????940Val?Tyr?Leu?Ala?Ala?Lys?Gln?Ala?Ile?Asn?His?Leu?Phe?Val?Asp?Tyr945?????????????????950?????????????????955?????????????????960Gln?Asp?Gln?Gln?Leu?Asn?Pro?Glu?Ile?Gly?Leu?Ala?Glu?Ile?Asn?Glu
965?????????????????970?????????????????975Ala?Ser?Asn?Leu?Val?Glu?Ser?Ile?Ser?Gly?Val?Tyr?Ser?Asp?Thr?Leu
980?????????????????985?????????????????990Leu?Gln?Ile?Pro?Gly?Ile?Asn?Tyr?Glu?Ile?Tyr?Thr?Glu?Leu?Ser?Asp
995????????????????1000????????????????1005Arg?Leu?Gln?Gln?Ala?Ser?Tyr?Leu?Tyr?Thr?Ser?Arg?Asn?Ala?Val?Gln???1010????????????????1015????????????????1020Asn?Gly?Asp?Phe?Asn?Ser?Gly?Leu?Asp?Ser?Trp?Asn?Thr?Thr?Met?Asp025????????????????1030????????????????1035????????????????1040Ala?Ser?Val?Gln?Gln?Asp?Gly?Asn?Met?His?Phe?Leu?Val?Leu?Ser?His
1045????????????????1050????????????????1055Trp?Asp?Ala?Gln?Val?Ser?Gln?Gln?Leu?Arg?Val?Asn?Pro?Asn?Cys?Lys
1060????????????????1065????????????????1070Tyr?Val?Leu?Arg?Val?Thr?Ala?Arg?Lys?Val?Gly?Gly?Gly?Asp?Gly?Tyr
1075????????????????1080????????????????1085Val?Thr?Ile?Arg?Asp?Gly?Ala?His?His?Gln?Glu?Thr?Leu?Thr?Phe?Asn???1090????????????????1095????????????????1100Ala?Cys?Asp?Tyr?Asp?Val?Asn?Gly?Thr?Tyr?Val?Asn?Asp?Asn?Ser?Tyr105????????????????1110????????????????1115????????????????1120Ile?Thr?Glu?Glu?Val?Val?Phe?Tyr?Pro?Glu?Thr?Lys?His?Met?Trp?Val
1125????????????????1130????????????????1135Glu?Val?Ser?Glu?Ser?Glu?Gly?Ser?Phe?Tyr?Ile?Asp?Ser?Ile?Glu?Phe
1140????????????????1145????????????????1150Ile?Glu?Thr?Gln?Glu
1155＜210〉explanation 3＜211〉1878＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence: adorned Cry9C protein DNA＜220 of encoding〉＜221〉CDS＜222〉(1) .. (1878)＜400〉3atg gct gac tac ctg cag atg acc gac gag gac tac acc gac agc tac 48Met Ala Asp Tyr Leu Gln Met Thr Asp Glu Asp Tyr Thr Asp Ser Tyr 15 10 15atc aac ccc agc ctg agc atc agc ggt cgc gac gcc gtg cag acc gct 96Ile Asn Pro Ser Leu Ser Ile Ser Gly Arg Asp Ala Val Gln Thr Ala
20??????????????????25??????????????????30ctg?acc?gtg?gtg?ggt?cgc?atc?ctg?ggt?gcc?ctg?ggc?gtg?ccc?ttc?agc????144Leu?Thr?Val?Val?Gly?Arg?Ile?Leu?Gly?Ala?Leu?Gly?Val?Pro?Phe?Ser
35??????????????????40??????????????????45ggt?cag?atc?gtg?agc?ttc?tac?cag?ttc?ctg?ctg?aac?acc?ctg?tgg?cca????192Gly?Gln?Ile?Val?Ser?Phe?Tyr?Gln?Phe?Leu?Leu?Asn?Thr?Leu?Trp?Pro
50??????????????????55??????????????????60gtg?aac?gac?acc?gcc?atc?tgg?gaa?gct?ttc?atg?cgc?cag?gtg?gag?gag????240Val?Asn?Asp?Thr?Ala?Ile?Trp?Glu?Ala?Phe?Met?Arg?Gln?Val?Glu?Glu?65??????????????????70??????????????????75??????????????????80ctg?gtg?aac?cag?cag?atc?acc?gag?ttc?gct?cgc?aac?cag?gcc?ctg?gct????288Leu?Val?Asn?Gln?Gln?Ile?Thr?Glu?Phe?Ala?Arg?Asn?Gln?Ala?Leu?Ala
85??????????????????90??????????????????95cgc?ctg?cag?ggc?ctg?ggc?gac?agc?ttc?aac?gtg?tac?cag?cgc?agc?ctg????336Arg?Leu?Gln?Gly?Leu?Gly?Asp?Ser?Phe?Asn?Val?Tyr?Gln?Arg?Ser?Leu
100????????????????105?????????????????110cag?aac?tgg?ctg?gcc?gac?cgc?aac?gac?acc?aag?aac?ctg?agc?gtg?gtg????384Gln?Asn?Trp?Leu?Ala?Asp?Arg?Asn?Asp?Thr?Lys?Asn?Leu?Ser?Val?Val
115?????????????????120?????????????????125agg?gcc?cag?ttc?atc?gcc?ctg?gac?ctg?gac?ttc?gtg?aac?gcc?atc?ccc????432Arg?Ala?Gln?Phe?Ile?Ala?Leu?Asp?Leu?Asp?Phe?Val?Asn?Ala?Ile?Pro
130?????????????????135?????????????????140ctg?ttc?gcc?gtg?aac?ggc?cag?cag?gtg?ccc?ctg?ctg?agc?gtg?tac?gcc????480Leu?Phe?Ala?Val?Asn?Gly?Gln?Gln?Val?Pro?Leu?Leu?Ser?Val?Tyr?Ala145?????????????????150?????????????????155?????????????????160cag?gcc?gtg?aac?ctg?cac?ctg?ctg?ctg?ctg?aag?gat?gca?tcc?ctg?ttc????528Gln?Ala?Val?Asn?Leu?His?Leu?Leu?Leu?Leu?Lys?Asp?Ala?Ser?Leu?Phe
165?????????????????170?????????????????175ggc?gag?ggc?tgg?ggc?ttc?acc?cag?ggc?gag?atc?agc?acc?tac?tac?gac????576Gly?Glu?Gly?Trp?Gly?Phe?Thr?Gln?Gly?Glu?Ile?Ser?Thr?Tyr?Tyr?Asp
180?????????????????185?????????????????190cgc?cag?ctc?gag?ctg?acc?gcc?aag?tac?acc?aac?tac?tgc?gag?acc?tgg????624Arg?Gln?Leu?Glu?Leu?Thr?Ala?Lys?Tyr?Thr?Asn?Tyr?Cys?Glu?Thr?Trp
195?????????????????200?????????????????205tac?aac?acc?ggt?ctg?gac?cgc?ctg?agg?ggc?acc?aac?acc?gag?agc?tgg????672Tyr?Asn?Thr?Gly?Leu?Asp?Arg?Leu?Arg?Gly?Thr?Asn?Th?Glu?Ser?Trp
210?????????????????215?????????????????220ctg?cgc?tac?cac?cag?ttc?cgc?agg?gag?atg?acc?ctg?gtg?gtg?ctg?gac????720Leu?Arg?Tyr?His?Gln?Phe?Arg?Arg?Glu?Met?Thr?Leu?Val?Val?Leu?Asp225?????????????????230?????????????????235?????????????????240gtg?gtg?gcc?ctg?ttc?ccc?tac?tac?gac?gtg?cgc?ctg?tac?ccc?acc?ggc????768Val?Val?Ala?Leu?Phe?Pro?Tyr?Tyr?Asp?Val?Arg?Leu?Tyr?Pro?Thr?Gly
245?????????????????250?????????????????255agc?aac?ccc?cag?ctg?aca?cgt?gag?gtg?tac?acc?gac?ccc?atc?gtg?ttc????816Ser?Asn?Pro?Gln?Leu?Thr?Arg?Glu?Val?Tyr?Thr?Asp?Pro?Ile?Val?Phe
260?????????????????265?????????????????270aac?cca?cca?gcc?aac?gtg?ggc?ctg?tgc?cgc?agg?tgg?ggc?acc?aac?ccc????864Asn?Pro?Pro?Ala?Asn?Val?Gly?Leu?Cys?Arg?Arg?Trp?Gly?Thr?Asn?Pro
275?????????????????280?????????????????285tac?aac?acc?ttc?agc?gag?ctg?gag?aac?gcc?ttc?atc?agg?cca?ccc?cac????912Tyr?Asn?Thr?Phe?Ser?Glu?Leu?Glu?Asn?Ala?Phe?Ile?Arg?Pro?Pro?His
290?????????????????295?????????????????300ctg?ttc?gac?cgc?ctg?aac?agc?ctg?acc?atc?agc?agc?aat?cga?ttc?ccc????960Leu?Phe?Asp?Arg?Leu?Asn?Ser?Leu?Thr?Ile?Ser?Ser?Asn?Arg?Phe?Pro305?????????????????310?????????????????315?????????????????320gtg?agc?agc?aac?ttc?atg?gac?tac?tgg?agc?ggt?cac?acc?ctg?cgc?agg????1008Val?Ser?Ser?Asn?Phe?Met?Asp?Tyr?Trp?Ser?Gly?His?Thr?Leu?Arg?Arg
325?????????????????330?????????????????335agc?tac?ctg?aac?gac?agc?gcc?gtg?cag?gag?gac?agc?tac?ggc?ctg?atc????1056Ser?Tyr?Leu?Asn?Asp?Ser?Ala?Val?Gln?Glu?Asp?Ser?Tyr?Gly?Leu?Ile
340?????????????????345?????????????????350acc?acc?acc?agg?gcc?acc?atc?aac?cca?ggc?gtg?gac?ggc?acc?aac?cgc????1104Thr?Thr?Thr?Arg?Ala?Thr?Ile?Ash?Pro?Gly?Val?Asp?Gly?Thr?Asn?Arg
355?????????????????360?????????????????365atc?gag?agc?acc?gct?gtg?gac?ttc?cgc?agc?gct?ctg?atc?ggc?atc?tac????1152Ile?Glu?Ser?Thr?Ala?Val?Asp?Phe?Arg?Ser?Ala?Leu?Ile?Gly?Ile?Tyr
370?????????????????375?????????????????380ggc?gtg?aac?agg?gcc?agc?ttc?gtg?cca?ggt?ggc?ctg?ttc?aac?ggc?acc????1200Gly?Val?Asn?Arg?Ala?Ser?Phe?Val?Pro?Gly?Gly?Leu?Phe?Asn?Gly?Thr385?????????????????390?????????????????395?????????????????400acc?agc?cca?gcc?aac?ggt?ggc?tgc?cga?gat?ctg?tac?gac?acc?aac?gac????1248Thr?Ser?Pro?Ala?Asn?Gly?Gly?Cys?Arg?Asp?Leu?Tyr?Asp?Thr?Asn?Asp
405?????????????????410?????????????????415gag?ctg?cca?ccc?gac?gag?agc?acc?ggc?agc?agc?acc?cac?cgc?ctg?agc????1296Glu?Leu?Pro?Pro?Asp?Glu?Ser?Thr?Gly?Ser?Ser?Thr?His?Arg?Leu?Ser
420?????????????????425?????????????????430cac?gtc?acc?ttc?ttc?agc?ttc?cag?acc?aac?cag?gct?ggc?agc?atc?gcc????1344His?Val?Thr?Phe?Phe?Ser?Phe?Gln?Thr?Asn?Gln?Ala?Gly?Ser?Ile?Ala
435?????????????????440?????????????????445aac?gct?ggc?agc?gtg?ccc?acc?tac?gtg?tgg?acc?agg?agg?gac?gtg?gac????1392Asn?Ala?Gly?Ser?Val?Pro?Thr?Tyr?Val?Trp?Thr?Arg?Arg?Asp?Val?Asp
450?????????????????455?????????????????460ctg?aac?aac?acc?atc?acc?ccc?aac?cgc?atc?acc?cag?ctg?ccc?ctg?gtg????1440Leu?Asn?Asn?Thr?Ile?Thr?Pro?Asn?Arg?Ile?Thr?Gln?Leu?Pro?Leu?Val465?????????????????470?????????????????475?????????????????480aag?gcc?agc?gct?ccc?gtg?agc?ggc?acc?acc?gtg?ctg?aag?ggt?cca?ggc????1488Lys?Ala?Ser?Ala?Pro?Val?Ser?Gly?Thr?Thr?Val?Leu?Lys?Gly?Pro?Gly
485?????????????????490?????????????????495ttc?acc?ggt?ggc?ggt?ata?ctg?cgc?agg?acc?acc?aac?ggc?acc?ttc?ggc????1536Phe?Thr?Gly?Gly?Gly?Ile?Leu?Arg?Arg?Thr?Thr?Asn?Gly?Thr?Phe?Gly
500?????????????????505?????????????????510acc?ctg?cgc?gtg?acc?gtg?aat?tcc?cca?ctg?acc?cag?cag?tac?cgc?ctg????1584Thr?Leu?Arg?Val?Thr?Val?Asn?Ser?Pro?Leu?Thr?Gln?Gln?Tyr?Arg?Leu
515?????????????????520?????????????????525cgc?gtg?cgc?ttc?gcc?agc?acc?ggc?aac?ttc?agc?atc?cgc?gtg?ctg?agg????1632Arg?Val?Arg?Phe?Ala?Ser?Thr?Gly?Asn?Phe?Ser?Ile?Arg?Val?Leu?Arg
530?????????????????535?????????????????540ggt?ggc?gtg?agc?atc?ggc?gac?gtg?cgc?ctg?ggc?agc?acc?atg?aac?agg????1680Gly?Gly?Val?Ser?Ile?Gly?Asp?Val?Arg?Leu?Gly?Ser?Thr?Met?Asn?Arg545?????????????????550?????????????????555?????????????????560ggc?cag?gag?ctg?acc?tac?gag?agc?ttc?ttc?acc?cgc?gag?ttc?acc?acc????1728Gly?Gln?Glu?Leu?Thr?Tyr?Glu?Ser?Phe?Phe?Thr?Arg?Glu?Phe?Thr?Thr
565?????????????????570?????????????????575acc?ggt?ccc?ttc?aac?cca?ccc?ttc?acc?ttc?acc?cag?gcc?cag?gag?atc????1776Thr?Gly?Pro?Phe?Asn?Pro?Pro?Phe?Thr?Phe?Thr?Gln?Ala?Gln?Glu?Ile
580?????????????????585?????????????????590ctg?acc?gtg?aac?gcc?gag?ggc?gtg?agc?acc?ggt?ggc?gag?tac?tac?atc????1824Leu?Thr?Val?Asn?Ala?Glu?Gly?Val?Ser?Thr?Gly?Gly?Glu?Tyr?Tyr?Ile
595?????????????????600?????????????????605gac?cgc?atc?gag?atc?gtg?ccc?gtg?aac?cca?gct?cgc?gag?gcc?gag?gag????1872Asp?Arg?Ile?Glu?Ile?Val?Pro?Val?Asn?Pro?Ala?Arg?Glu?Ala?Glu?Glu
610 615 620gac tga 1878Asp625＜210〉4＜211〉625＜212〉PRT＜213〉artificial sequence＜400〉4Met Ala Asp Tyr Leu Gln Met Thr Asp Glu Asp Tyr Thr Asp Ser Tyr, 15 10 15Ile Asn Pro Ser Leu Ser Ile Ser Gly Arg Asp Ala Val Gln Thr Ala
20??????????????????25??????????????????30Leu?Thr?Val?Val?Gly?Arg?Ile?Leu?Gly?Ala?Leu?Gly?Val?Pro?Phe?Ser
35??????????????????40??????????????????45Gly?Gln?Ile?Val?Ser?Phe?Tyr?Gln?Phe?Leu?Leu?Asn?Thr?Leu?Trp?Pro
50??????????????????55??????????????????60Val?Asn?Asp?Thr?Ala?Ile?Trp?Glu?Ala?Phe?Met?Arg?Gln?Val?Glu?Glu?65??????????????????70??????????????????75??????????????????80Leu?Val?Asn?Gln?Gln?Ile?Thr?Glu?Phe?Ala?Arg?Asn?Gln?Ala?Leu?Ala
85??????????????????90??????????????????95Arg?Leu?Gln?Gly?Leu?Gly?Asp?Ser?Phe?Asn?Val?Tyr?Gln?Arg?Ser?Leu
100?????????????????105?????????????????110Gln?Asn?Trp?Leu?Ala?Asp?Arg?Asn?Asp?Thr?Lys?Asn?Leu?Ser?Val?Val
115?????????????????120?????????????????125Arg?Ala?Gln?Phe?Ile?Ala?Leu?Asp?Leu?Asp?Phe?Val?Asn?Ala?Ile?Pro
130?????????????????135?????????????????140Leu?Phe?Ala?Val?Asn?Gly?Gln?Gln?Val?Pro?Leu?Leu?Ser?Val?Tyr?Ala145?????????????????150?????????????????155?????????????????160Gln?Ala?Val?Asn?Leu?His?Leu?Leu?Leu?Leu?Lys?Asp?Ala?Ser?Leu?Phe
165?????????????????170?????????????????175Gly?Glu?Gly?Trp?Gly?Phe?Thr?Gln?Gly?Glu?Ile?Ser?Thr?Tyr?Tyr?Asp
180?????????????????185?????????????????190Arg?Gln?Leu?Glu?Leu?Thr?Ala?Lys?Tyr?Thr?Asn?Tyr?Cys?Glu?Thr?Trp
195?????????????????200?????????????????205Tyr?Asn?Thr?Gly?Leu?Asp?Arg?Leu?Arg?Gly?Thr?Asn?Thr?Glu?Ser?Trp
210?????????????????215?????????????????220Leu?Arg?Tyr?His?Gln?Phe?Arg?Arg?Glu?Met?Thr?Leu?Val?Val?Leu?Asp225?????????????????230?????????????????235?????????????????240Val?Val?Ala?Leu?Phe?Pro?Tyr?Tyr?Asp?Val?Arg?Leu?Tyr?Pro?Thr?Gly
245?????????????????250?????????????????255Ser?Asn?Pro?Gln?Leu?Thr?Arg?Glu?Val?Tyr?Thr?Asp?Pro?Ile?Val?Phe
260?????????????????265?????????????????270Asn?Pro?Pro?Ala?Asn?Val?Gly?Leu?Cys?Arg?Arg?Trp?Gly?Thr?Asn?Pro
275?????????????????280?????????????????285Tyr?Asn?Thr?Phe?Ser?Glu?Leu?Glu?Asn?Ala?Phe?Ile?Arg?Pro?Pro?His
290?????????????????295?????????????????300Leu?Phe?Asp?Arg?Leu?Asn?Ser?Leu?Thr?Ile?Ser?Ser?Asn?Arg?Phe?Pro305?????????????????310?????????????????315?????????????????320Val?Set?Ser?Asn?Phe?Met?Asp?Tyr?Trp?Ser?Gly?His?Thr?Leu?Arg?Arg
325?????????????????330?????????????????335Ser?Tyr?Leu?Asn?Asp?Ser?Ala?Val?Gln?Glu?Asp?Ser?Tyr?Gly?Leu?Ile
340?????????????????345?????????????????350Thr?Thr?Thr?Arg?Ala?Thr?Ile?Asn?Pro?Gly?Val?Asp?Gly?Thr?Asn?Arg
355?????????????????360?????????????????365Ile?Glu?Ser?Thr?Ala?Val?Asp?Phe?Arg?Ser?Ala?Leu?Ile?Gly?Ile?Tyr
370?????????????????375?????????????????380Gly?Val?Asn?Arg?Ala?Ser?Phe?Val?Pro?Gly?Gly?Leu?Phe?Asn?Gly?Thr385?????????????????390?????????????????395?????????????????400Thr?Ser?Pro?Ala?Asn?Gly?Gly?Cys?Arg?Asp?Leu?Tyr?Asp?Thr?Asn?Asp
405?????????????????410?????????????????415Glu?Leu?Pro?Pro?Asp?Glu?Ser?Thr?Gly?Ser?Ser?Thr?His?Arg?Leu?Ser
420?????????????????425?????????????????430His?Val?Thr?Phe?Phe?Ser?Phe?Gln?Thr?Asn?Gln?Ala?Gly?Ser?Ile?Ala
435?????????????????440?????????????????445Asn?Ala?Gly?Ser?Val?Pro?Thr?Tyr?Val?Trp?Thr?Arg?Arg?Asp?Val?Asp
450?????????????????455?????????????????460Leu?Asn?Asn?Thr?Ile?Thr?Pro?Asn?Arg?Ile?Thr?Gln?Leu?Pro?Leu?Val465?????????????????470?????????????????475?????????????????480Lys?Ala?Ser?Ala?Pro?Val?Ser?Gly?Thr?Thr?Val?Leu?Lys?Gly?Pro?Gly
485?????????????????490?????????????????495Phe?Thr?Gly?Gly?Gly?Ile?Leu?Arg?Arg?Thr?Thr?Ash?Gly?Thr?Phe?Gly
500?????????????????505?????????????????510Thr?Leu?Arg?Val?Thr?Val?Asn?Ser?Pro?Leu?Thr?Gln?Gln?Tyr?Arg?Leu
515?????????????????520?????????????????525Arg?Val?Arg?Phe?Ala?Ser?Thr?Gly?Asn?Phe?Ser?Ile?Arg?Val?Leu?Arg
530?????????????????535?????????????????540Gly?Gly?Val?Ser?Ile?Gly?Asp?Val?Arg?Leu?Gly?Ser?Thr?Met?Asn?Arg545?????????????????550?????????????????555?????????????????560Gly?Gln?Glu?Leu?Thr?Tyr?Glu?Ser?Phe?Phe?Thr?Arg?Glu?Phe?Thr?Thr
565?????????????????570?????????????????575Thr?Gly?Pro?Phe?Asn?Pro?Pro?Phe?Thr?Phe?Thr?Gln?Ala?Gln?Glu?Ile
580?????????????????585?????????????????590Leu?Thr?Val?Asn?Ala?Glu?Gly?Val?Ser?Thr?Gly?Gly?Glu?Tyr?Tyr?Ile
595?????????????????600?????????????????605Asp?Arg?Ile?Glu?Ile?Val?Pro?Val?Asn?Pro?Ala?Arg?Glu?Ala?Glu?Glu
610?????????????????615?????????????????620Asp625