CN102373187B - Plant stress tolerance-associated protein and encoding gene and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种植物Δ¹-吡咯琳-5-羧酸合成酶及其编码基因和应用。本发明提供的蛋白质，是如下(a)或(b)的蛋白质：(a)由序列表中序列1所示的氨基酸序列组成的蛋白质；(b)将序列1的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且参与脯氨酸合成的由序列1衍生的蛋白质。本发明提供的蛋白及其编码基因，可以调控植物脯氨酸的合成，受盐和干旱胁迫诱导，参与羊草对盐和干旱胁迫的响应。将本发明的基因导入植物，可以提高植物的抗逆性。本发明提供的蛋白及其编码基因对于培育抗逆性提高的羊草及其他植物新品种具有重要实用价值。The invention discloses a plant Δ¹ -pyrroline-5-carboxylic acid synthetase, its coding gene and application. The protein provided by the present invention is the protein of the following (a) or (b): (a) a protein composed of the amino acid sequence shown in Sequence 1 in the sequence listing; (b) the amino acid sequence of Sequence 1 is processed by one or more A protein derived from Sequence 1 with substitution and/or deletion and/or addition of amino acid residues and involved in proline synthesis. The protein and its coding gene provided by the invention can regulate the synthesis of proline in plants, be induced by salt and drought stress, and participate in the response of Leymus chinensis to salt and drought stress. Introducing the gene of the present invention into plants can improve the stress resistance of the plants. The protein and its coding gene provided by the invention have important practical value for cultivating Leymus chinensis and other new plant varieties with improved stress resistance.

Description

One kind of plant resistance related protein and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant resistance related protein and encoding gene thereof and application.

Background technology

It is to affect the most serious environment stress factor of plant-growth that Drought and salt is coerced, and is the primary limiting factor of China's crop yield and sown area.Proline(Pro) is the osmotic stress material that plant accumulates under environment stress, plays and regulates Premeabilisation of cells pressure, protected protein matter molecule, as the effects such as scavenging agent of active oxygen, to keep the normal growth of plant.Plant accumulates the height of proline(Pro) ability, has determined the power of stress resistance of plant.

In plant materials, synthetic ornithine/arginine and two approach of L-glutamic acid of passing through of proline(Pro), under environment stress, the L-glutamic acid approach is the main path of proline biosynthesis.Δ¹-pyrroles beautiful jade-5-carboxylic acid synthetase (P5CS) is the rate-limiting enzyme in the L-glutamic acid approach, it is a bifunctional enzyme, have Glutamate kinase and paddy ammonia enzyme-γ-semialdehyde deaminase active, thefront 2 steps reaction of catalysis proline synthesis, it again can be by product proline(Pro) feedback inhibition simultaneously.

Sheep's hay has another name called the alkali grass, is one of important constructive species of Eurasia grassland region east meadow steppe.Sheep's hay is the herbage of a kind of high yield, good palatability, and Salt And Alkali Tolerance, but intolerant to waterlogging or severe drought are cultivated the high resistance to cold and diseases sheep's hay, can bring huge economic benefit and ecological benefits undoubtedly.The drought resisting of plant, Salt And Alkali Tolerance proterties are the quantitative characters of controlled by multiple genes, give by the resistance of cross-breeding raising crop and bring very large difficulty, therefore can come by genetically engineered the resistance of Crop Improvement.Increasing the expression amount of plant P5CS gene by transgenosis, thereby improve the ability of plant accumulation proline(Pro), is an effective way of cultivating drought resisting, saline alkali tolerant plant.

Summary of the invention

The purpose of this invention is to provide anti-contrary (drought resisting, salt tolerant) the associated protein Δ of a kind of plant¹-pyrroles beautiful jade-5-carboxylic acid synthetase and encoding gene and application.

Plant stress tolerance-associated protein provided by the invention (LcP5CS1) is a kind of Δ¹-pyrroles beautiful jade-5-carboxylic acid synthetase derives from sheep's hay (Leymus chinensis (Trin.) Tzvel.), is following (a) or protein (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence ofsequence 1 through replacement and/or disappearance and/or the interpolation of one or several amino-acid residue and participate in proline synthesis by the derivative protein ofsequence 1.

Sequence 1 is comprised of 730 amino-acid residues in the sequence table, be the conserved domain of AAK_super family from the 21st the-the 295th amino acids residue of N-terminal, being the conserved domain of ALDH-SF super family from 310-710 of N-terminals, is P5CS Multi-domain sequence from the 22nd the-the 730th amino acids residue of N-terminal.

For the ease of the purifying of LcP5CS1, label as shown in table 1 on the aminoterminal of the protein that can form at the amino acid residue sequence bysequence 1 or carboxyl terminal connect.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the LcP5CS1 synthetic, also can synthesize first its encoding gene, carry out again biological expression and obtain.The encoding gene of LcP5CS1 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The gene of plant stress tolerance-associated protein (LcP5CS1) also belongs to protection scope of the present invention on the coding.

Described gene can be following 1) or 2) or 3) or 4) dna molecular:

1) in thesequence table sequence 2 from the dna molecular shown in the Nucleotide of 5 ' terminal 3-2195 position;

2) dna molecular shown in the sequence in the sequence table 2;

3) can be with 1 under stringent condition) or 2) gene recombination that limits and the gene of encoding said proteins;

4) with 1) or 2) gene that limits has homology more than 90% and the gene of encoding said proteins.

Sequence 2 in the sequence table is by 2197 based compositions, and its open reading frame (ORF) is from 5 ' terminal 3-2195 position Nucleotide.

Above-mentioned stringent condition can be 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

The recombinant vectors that contains above arbitrary described gene also belongs to protection scope of the present invention, such as recombinant expression vector.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.

Described plant expression vector comprises double base agrobacterium vector (such as pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 etc.) and can be used for carrier of plant micropellet bombardment etc.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (such as kermes synthetic enzyme Nos gene), plant gene (storing protein gene such as soybean) 3 ' end to transcribe such as the Agrobacterium crown-gall nodule all has similar functions.

When using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor, constitutive promoter or inducible promoter, such as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, stress induced promoter Rd29A etc., they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (such as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

The expression cassette, transgenic cell line and the recombinant bacterium that contain above arbitrary described gene (LcP5CS1) all belong to protection scope of the present invention.

The total length of amplification said gene or the primer pair of arbitrary fragment also belong within protection scope of the present invention.

In described albumen, described gene, described recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium any one all can be applicable to cultivate plant with adverse resistance.

Another object of the present invention provides a kind of method of cultivating anti-salt, drought-enduring transgenic plant.

The method of the anti-salt of cultivation provided by the present invention, drought-enduring transgenic plant can be with the above-mentioned Δ of coding¹The gene (LcP5CS1) of-pyrroles beautiful jade-5-carboxylic acid synthetase imports in the purpose plant (such as vegetable cell or tissue), obtains the transgenic plant that resistance of reverse is higher than described purpose plant.

Utilize any carrier that can guide foreign gene in plant, to express, with the gene transfered plant cell of encoding said proteins, can obtain transgenic cell line and the transfer-gen plant of anti-salt, drought tolerance enhancing.Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that transforms is cultivated into plant.The plant host that is converted both can be monocotyledons, also can be dicotyledons, as: tobacco, lucerne place, Arabidopis thaliana, paddy rice, wheat, corn, cucumber, tomato, willow, turfgrass etc.

Albumen provided by the invention and encoding gene thereof are the rate-limiting enzymes of plant proline biosynthesis under environment stress.With gene transfered plant of the present invention, thereby the accumulation volume that improves the proline(Pro) of plant improves the resistance of plant, but the particularly expression of arid and this gene of high-salt stress induced strong, by transgenosis means raising crop Δ¹-pyrroles beautiful jade-5-carboxylic acid synthetase expression amount can improve the resistance of crop and careless class, helps to improve crop yield, expand sown area and realize the healthy and sustainable development of grassland ecosystem.Albumen provided by the invention and encoding gene thereof have important practical value for the sheep's hay of cultivating the resistance raising and other neies variety of plant.The present invention helps to understand plant accumulates proline(Pro) under osmotic stress the mechanism of action, for disclosing that Proline Accumulation mechanism keeps to plant that normal growth is grown and the regulatory function of resistance, providing the genetics foundation for improving crop yield with the genetically engineered research that improves quality.

Description of drawings

Fig. 1 is the agarose gel electrophoresis detected result of the total RNA of chinense seedlings;

Fig. 2 is the agarose gel electrophoresis detected result of pcr amplification LcP5CS1 full-length cDNA;

Fig. 3 is the systematic evolution tree of LcP5CS1;

Fig. 4 is the response modes (sxemiquantitative RT-PCR) of LcP5CS1 gene under the salt stress treatment condition;

Fig. 5 is the response modes (sxemiquantitative RT-PCR) of LcP5CS1 gene under arid treatment condition;

Fig. 6 is the accumulation volume of sheep's hay proline(Pro) under salt stress;

Fig. 7 is the accumulation volume of the proline(Pro) of sheep's hay under drought stress;

Fig. 8 is that the PCR of recombinant vectors pSN1301-LcP5CS1 identifies;

Fig. 9 is that the double digestion of recombinant vectors pSN1301-LcP5CS1 is identified;

Figure 10 is Agrobacterium positive transformant PCR qualification result behind the conversion Agrobacterium;

Figure 11 is transgenic arabidopsis PCR qualification result;

Figure 12 is the phenotype that high salt (250mM NaCl) is processed the Arabidopis thaliana and the wild-type Arabidopis thaliana that transformed afterwards LcP5CS1 in 3 days;

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.

Sheep's hay (lucky giving birth to No. one): in September, 2005 is available from Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province

Embodiment 1, sheep's hay plant stress tolerance-associated protein (Δ¹-pyrroles beautiful jade-5-carboxylic acid synthetase) and the acquisition of encoding gene

One, the clone of the sheep's hay plant gene of anti-retrocorrelation LcP5CS1

1, the extraction of vegetable material processing and total RNA

Process 12h sheep's hay (lucky giving birth to No. one) seedling with 250mM NaCl, concrete grammar is:

The normal growth sheep's hay in 8 weeks (lucky giving birth to No. one) seedling is carried out salt stress and processes.

Soak root with the 250mM NaCl aqueous solution, incubated at room temperature, getting and processing 12h sheep's hay (lucky giving birth to No. one) seedling is material, extracts total RNA, carries out 1.2% agarose gel electrophoresis and detects, the result is as shown in Figure 1.The RNA that extracts has two obvious electrophoretic bands, is followed successively by from top to bottom 28S RNA and 18S RNA, shows to have obtained higher, the more completely total RNA of purity.

2, the plant gene cloning of anti-the retrocorrelation

According to this laboratory high-flux sequence result, in conjunction with published plant Δ¹The amino acid residue sequence of-pyrroles beautiful jade-5-carboxylic acid synthetase is sought conservative region, and according to the conservative region primers, concrete primer sequence is as follows:

S1 5′-GAATGGGCAGGGGTGGCAT-3′；

S2 5′-CCTCATTGCAAAGGAAGATCC-3′。

The total RNA that extracts chinense seedlings takestep 1 uses PrimeScript as template^TM1st Strand cDNASynthesized Kit test kit (Takara company) and reference reagent box specification sheets, synthetic the first chain cDNA of counter-rotating.Reaction system and reaction conditions are as follows: Oligo-dT (10pmol/ μ l) 1.0 μ l, dNTP Mixture (10mmol/leach) 1.0 μ l, Total RNA (≤1 μ g) 1.0 μ l, RNase-free water7.0 μ l, 65 ℃ of 5min; Then add 5 * Buffer, 4.0 μ l, RNase Inhibitor (40U/ μ l) 0.5 μ l, PrimeScript RTase (200U/ μ l) 0.5 μ l, 42 ℃ of 45min, 70 ℃ of 15min, with the first synthetic chain cDNA be stored in-20 ℃ for subsequent use.

Take the first chain cDNA of obtaining as template, carry out pcr amplification with the primer pair of S1 and S2 composition; The PCR reaction system is: each 1 μ l (10umol/l) of cDNA template, S1 and S2,10 * Buffer, 2.5 μ l, dNTP Mixture (10mmol/leach) 2 μ l, Taq enzyme 0.25 μ l, ddH₂O 12.25 μ l; Reaction conditions is: 94 ℃ of denaturation 5min of elder generation; Then 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 2min 30s, totally 35 circulations; Last 72 ℃ are extended 10min.

After reaction finishes, pcr amplification product is carried out 1.2% agarose gel electrophoresis detect, the result as shown in Figure 2.Among Fig. 2, swimming lane M is DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), andswimming lane 1 is pcr amplification product.The result shows, obtained the purpose fragment that length is about 2200bp through pcr amplification.Reclaim and purified pcr product, be connected on the PMD-18T carrier (Takara company), connect product and transform bacillus coliDH 5 alpha competent cell (Beijing Quanshijin Biotechnology Co., Ltd), screening positive clone carries out bacterium liquid PCR to be identified, the plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.

Sequencing result shows shown insequence 2 by analysis, this sheet segment length 2197bp, the protein shown in thesequence 1 of code sequence tabulation.With the protein called after LcP5CS1 shown in the sequence 1 (being comprised of 730 amino-acid residues), with the encoding gene called after LcP5CS1 of LcP5CS1, its full-length cDNA is shown in thesequence 2 of sequence table.

The bioinformatic analysis ofembodiment 2, LcP5CS1 and LcP5CS1

One, the structure function of the sequential analysis of LcP5CS1 gene and proteins encoded thereof prediction

Utilize DNAMAN and OMIGA software that the full length cDNA sequence of LcP5CS1 is carried out bioinformatic analysis, this sequence total length 2197bp is ORF from the 3rd the-the 2195th at 5 ' end.Infer that its molecular weight is 78.887kDa, iso-electric point pI value 6.69.Analyze the structural domain of LcP5CS1 with online B1AST instrument (http://blast.ncbi.nlm.nih.gov/Blast.cgi), the result shows, this albumen belongs to P5CS Multi-domain, shows that this albumen is a member in the P5CS family.

Two, homology and the systematic evolution tree analysis of LcP5CS1 and other P5CS proteinoid sequence

(the GeneBank number of logging in is: TaP5CS is respectively AAX35536 to other P5CS aminoacid sequence of having cloned in LcP5CS1 and the plant to utilize online Blast instrument, SbP5CS1ACU65226, SbP5CS2ACU65227,0sP5CS1 BAA19916, ZmP5CS ABI21839, AtP5CS1NP181510, AtP5CS2NP191120, BnP5CSAAAK01360, BnP5CSB AAK01361, TomPR01 AAB67877, TomPR02 Q96480, EcGPK YP539318 carries out the homology analysis comparison, sees Fig. 3.

Result such as Fig. 3 show that the homology of LcP5CS1 and monocotyledons Chinese sorghum SbP5CS1 is respectively 87%; And it is lower with the dicots P5CS homology of other unifacial leaf P5CS.Phylogenetic analysis shows, larger difference has appearred in P5CS proteinoid during evolution, monocotyledonous P5CS is poly-to be a class, and except the TomPR01 of tomato was near with the colibacillary EcGPK homology of prokaryotic organism, it was a class that other dicotyledonous P5CS also gathers in the dicotyledons.

Embodiment 3, the LcP5CS1 expression pattern analysis under the Drought and salt stress conditions

The normal growth sheep's hay in 8 weeks (lucky giving birth to No. one) seedling is carried out different treatment.

First group (salt stress processing): soak root, incubated at room temperature with the 250mM NaCl aqueous solution;

Get respectively the processing material blade ofprocessing 0,1,2,4,8,12,24,48h, be stored in the liquid nitrogen rapidly.

Second group (drought stress processing): stop to supply water incubated at room temperature;

Getting respectively cuts off the water supply processes the chinense seedlings blade of 0,1,2,3,4,5,6d, freezes in liquid nitrogen rapidly.

Extract total RNA of above-mentioned two groups of chinense seedlings of processing, carry out RT-PCR.

LcP5CS1 primer: 1:5 '-GTCGGCACCGCTATTGTC-3 ';

2：5′-GCCAAACTGTCGTTATCCC-3′；

Confidential reference items Actin primer: 1:5 '-TGGACTCTGGTGATGGTGTGAG-3 ';

2：5′-GTGCTAAGGGAGGCAAGGATG-3′。

Confidential reference items reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 30s, 26 circulations.

LcP5CS1 reaction conditions: 94 ℃ of 5min; 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 40s, 30 circulations.

The results are shown in Figure 4, Fig. 5.The result shows, the LcP5CS1 transcriptional level obviously is subjected to the Drought and salt stress-inducing, and under inductive condition, the relative expression quantity of LcP5CS1 gene increases sharply.

The functional verification ofembodiment 4, LcP5CS1 (measuring the content of its product proline(Pro))

Arid, proline content is measured under the salt stress, and determining to coerce at Drought and salt the relation with proline content of increasing of lower LcP5CS1 expression amount, thereby whether the content of checking proline(Pro) is relevant with the expression amount of LcP5CS1.The concrete grammar of arid, salt stress is withembodiment 3.

1. proline(Pro) extracts: get different time points sheep's hay blade behind the 0.05-0.5g Stress treatment, with 3% sulphosalicylic acid solution milling and extracting, the final volume of sulphosalicylic acid is 5ml.Homogenate changes in the glass centrifuge tube, extracts 10min in boiling water bath.After the cooling, with the centrifugal 10min of 3000r/min, get supernatant liquor to be measured.

2. assay

1) standard curve making: at 1-10ug/ml concentration of proline scope production standard curve. each 2ml of the accurate solution of label taking, add 2ml 3% sulphosalicylic acid, 2ml glacial acetic acid and 4ml 2.5% ninhydrin solution, after putting the 60min. cooling that develops the color in the boiling water bath, add 4ml toluene extraction red material. after leaving standstill, get toluene and measure mutually the absorption value at 520nm wavelength place, according to proline(Pro) amount and respective absorption value drawing standard curve.

2) sample determination: get the 2ml supernatant liquor, add 2ml water, add again the colouring reagentss such as glacial acetic acid, develop the color with the typical curve program, extraction and colorimetric.

Result such as Fig. 6,7 show, behind Stress treatment, proline content raises in the plant materials, and increasing after the P5CS expression amount increases of proline content, proof improves the LcP5CS1 expression amount can increase proline content in the plant materials, illustrates that LcP5CS1 is the key enzyme of proline synthesis under the environment stress.

The acquisition ofembodiment 5, LcP5CS1 transgenic arabidopsis

The LcP5CS1 gene that above-described embodiment 1 amplification is obtained enters carrier PMD18-T (TaKaRa company) with the primer LcP5CSSN1301-s that contains KpnI and BamHI (5 '-CGGGATCCATGGGCAGGGGTGGCAT-3 ') and LcP5CSSN1301-r (5 '-GGGGTACCTCATTGCAAAGGAAGATCCT-3 ') rear clone that increases, and the pcr amplification condition is: 94 ℃ of denaturation 5min; Then 94 ℃ of 30s, 58 ℃ of 30s, 72 ℃ of 2min 30s, totally 35 circulations; Last 72 ℃ are extended 10min.Positive bacteria liquid upgrading grain after identifying, with KpnI and BamHI double digestion, be inserted into through between the KpnI and BamHI restriction enzyme site of the plant expression vector pSN1301 (available from CAMBIA company) that same enzyme is cut, with the recombinant vectors called after pSN1301-LcP5CS1 that obtains.With primer LcP5CSSN1301-s and LcP5CSSN1301-r pSN1301-LcP5CS1 being carried out PCR identifies, reaction conditions is the same, the result as shown in Figure 8, swimming lane M is for being DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1,3,4,5,7,8,9,10 is for identifying correct recombinant plasmid.With KpnI and BamHI pSN1301-LcP5CS1 being carried out double digestion identifies, the result as shown in Figure 9, wherein, swimming lane M is DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), cut the band that obtains about a 2000bp through enzyme, prove the purpose fragment exact connect ion enter in the plant expression vector.

Recombinant vectors pSN1301-LcP5CS1 imports to Agrobacterium EHA105 strain (available from U.S. Clontech company, cat. no K1612-1) in, PCR screens positive recombinant, primer and reaction conditions are the same, the result as shown in figure 10, among Figure 10, swimming lane M is DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), swimming lane 1,2,3,5 positive recon PCR results.(Columbia is environmental for the Arabidopis thaliana seed, derive from the world, Fan Ersai research centre, French academy of agricultural sciences Arabidopis thaliana seed bank) with 75% ethanol disinfection 2min, behind the 2%NaClO sterilization 15min, be seeded in the MS substratum, after the illumination cultivation 3 days, seedling is moved to Nutrition Soil: in the soil of vermiculite=1: 1, treat that most of plant is all after the bolting, cut whole main tongue at the inflorescence base portion, remove its apical dominance, approximately grow 4-6 newborn side tongue at the axillalry bud position after 1 week, treat that side tongue inflorescence forms bud and part and blooms or forms 1-2 angle really the time, just can be used for conversion.The 10mL pipe adds YEB liquid nutrient medium 2mL, and adds each 50mg/L of the gentle kantlex of screening rifamycin antibiotic.The single colony inoculation of picking Agrobacterium is in pipe, and 28 ℃ of 200rpm shake bacterium.Transferred night on the same day in the triangular flask that contains the antibiotic YEB liquid nutrient medium of same concentration 50ml, 28 ℃ are shaken bacterium and spend the night.Second day change over to 250ml contain shake in the antibiotic YEB liquid nutrient medium bacterium to 0D 600 about 1.2, change in the centrifuge tube the centrifugal 5min of room temperature 5000rmp over to.Abandon supernatant, precipitation is changed in the distilled water that contains 3% sucrose and 0.1%Selwert (Sigma company), make its 0D600 about 0.8.Wild-type plant inflorescence is soaked in the transformed bacteria liquid 20 seconds, and the preservative film parcel keeps in Dark Place and takes off film, normal maintenance after spending the night.Obtain sterilizing behind the seed, be seeded on the MS screening culture medium of the Totomycin that contains 20mg/L, screen positive transgenic seedling, these seedlings are moved in the soil, grow and after 10 days transfer-gen plant is carried out the PCR detection, reaction conditions and primer are the same, and the result as shown in figure 11, swimming lane M is DL2000Plus dna molecular amount standard (Beijing Quanshijin Biotechnology Co., Ltd), 8 the positive transgenic plant strain PCR qualification results of swimming lane 2,5,6,7,8,9,10,12 for obtaining.The positive transgenic plant selfing that obtains obtains T2 for seed.

The resistance experiment ofembodiment 6, LcP5CS transgenic arabidopsis

((Columbia is environmental in contrast with wild-type Arabidopis thaliana seed, derive from the world, Fan Ersai research centre, French academy of agricultural sciences Arabidopis thaliana seed bank)) and the LcP5CS1 transgenic arabidopsis T2 that obtains ofembodiment 5 be seeded in respectively in the MS substratum for seed, after three days seedling is moved in the soil, after one week of hot-house culture, carry out salt stress and process (250mM NaCl), observe phenotype after three days.

Transgenic arabidopsis does not have death, the left figure of result such as Figure 12 after salt is processed three days as a result; And fully wilting death of wild-type, the right figure of result such as Figure 12.Above-mentioned experimental result shows that LcP5CS1 of the present invention can strengthen the anti-adversity ability of plant, thereby the candidate gene that can be used as plant genetic engineering is applied, and is used for improveing the degeneration-resistant proterties of plant.

Claims (7)

1. protein, the protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene according to claim 2, it is characterized in that: described gene is the dna molecular shown in the sequence 2 in the sequence table.

4. the recombinant expression vector, expression cassette or the recombinant bacterium that contain claim 2 or 3 described genes.

5. the total length primer pair of amplification claim 2 or 3 described genes; Described primer pair is comprised of the dna molecular shown in the sequence 3 in the sequence table and the 4 described dna moleculars of the sequence in the sequence table.

6. the application of any one in the described albumen of claim 1, claim 2 or 3 described genes, the described recombinant expression vector of claim 4, expression cassette or the recombinant bacterium in cultivating plant with adverse resistance; Described anti-contrary be salt tolerant; Described plant is Arabidopis thaliana.

7. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant; Described purpose plant is Arabidopis thaliana; Described resistance of reverse is salt tolerance.

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