CN103183732A - Cotton Gh FPP1 protein as well as coding gene and application thereof - Google Patents

Abstract

Translated fromChinese

本发明公开了一种棉花Gh FPF1蛋白及其编码基因与应用。该蛋白Gh FPF1，是具有下述氨基酸残基序列之一的蛋白质：1)序列表中的SEQ ID №.2的氨基酸残基序列；2)将序列表中的SEQ ID №.2的氨基酸残基序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与植物光合效率相关的由1)衍生的蛋白质。本发明的蛋白和其编码基因可用来培育生育期短的棉花，为转基因植物的培育奠定了基础。The invention discloses a cotton Gh FPF1 protein, its coding gene and application. The protein Gh FPF1 is a protein having one of the following amino acid residue sequences: 1) the amino acid residue sequence of SEQ ID No. 2 in the sequence listing; 2) the amino acid residue sequence of SEQ ID No. 2 in the sequence listing A protein derived from 1) whose base sequence has undergone substitution and/or deletion and/or addition of one or several amino acid residues and is related to plant photosynthetic efficiency. The protein and its coding gene of the invention can be used to breed cotton with a short growth period, which lays a foundation for the cultivation of transgenic plants.

Description

One grow cotton Gh FPF1 albumen and encoding gene and application

Technical field

The present invention relates to grow cotton albumen and encoding gene and an application.

Background technology

The popularization of short season cotton is to solve the important channel that China's grain and cotton is striven ground, guaranteed grain security.Precocious type is one of important character of cotton variety, and the florescence is one of important indicator of weighing the ripe property of cotton.

Flower is the secondary organ of plant, the over-ground part of higher plant is from stem end meristematic tissue (shoot apical meristem, SAM), namely be positioned at the undifferentiated minicell of a group on stem top, stem end meristematic tissue and then develop into stem, leaf, inflorescence meristem and floral meristem.The decision process of blooming is first stage that the plant reproductive growth starts, and is the basis that flower is made a start and floral organ forms, so the decision process of blooming is directly controlled the morning and evening of crop growth phase.Therefore clone the cotton genes involved of blooming, it is carried out expression analysis and transgenosis functional verification, the genetic resources of high-quality is provided for the short season cotton breeding.

Summary of the invention

An object of the present invention is to provide a kind of albumen, name is called Gh FPF1, derives from cotton (Gossypium spp).

Albumen of the present invention is following 1) or 2) albumen:

1) protein of the composition of the aminoacid sequence shown in the SEQ ID № .2 in the sequence table;

2) with the amino acid residue sequence of the SEQ ID № .2 in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with flowering of plant by 1) protein of deriving.

The aminoacid sequence shown in the SEQ ID № .2 is made up of 109 amino-acid residues in the sequence table.

Above-mentioned 1) and 2) but in Gh FPF1 albumen synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 1) and 2) in the encoding gene of Gh FPF1 albumen can be by the dna sequence dna shown in the 8-334 position Nucleotide of SEQ ID № .1 in the sequence table be lacked the codon of one or several amino-acid residue, and/or carry out obtaining after the missense mutation of one or several base pair.

The nucleic acid molecule of described Gh FPF1 albumen of encoding also belongs to protection scope of the present invention.

Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule also can be RNA, as mRNA, hnRNA or tRNA etc.

Another purpose of the present invention provides the encoding gene of described albumen.

Described encoding gene has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: the nucleotide sequence of 1 8-334 position;

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 2 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.

The rigorous condition of above-mentioned height can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of hybridization down, is used 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Wherein, the SEQ ID № in the sequence table: 1 is made up of 401 Nucleotide, and its open reading frame (ORF) is from 5 ' terminal 8-334 position Nucleotide, SEQ ID № in the code sequence tabulation: the protein shown in 2, i.e. Gh FPF1 albumen of the present invention.

The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.

Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.

Described recombinant expression vector can be used existing expression vector establishment.Described 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.When using described gene constructed recombinant expression vector, can add any enhancement type, composing type, organizing specific type or inducible promoter before its transcription initiation Nucleotide, they can use separately or be used in combination with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, as be added in the plant to express and to produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.), have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (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 primer of encoding gene total length of the present invention or its any fragment of increasing is to also belonging to the scope of protection of the invention.

Another object of the present invention provide albumen of the present invention, encoding gene and contain the recombinant vectors of described encoding gene, expression cassette, transgenic cell line or the reorganization bacterium following 1)-3) application at least a:

1) regulates plant breeding time;

2) regulate the flowering of plant time;

3) regulate plant lotus throne number of sheets amount;

4) regulate axis leave quantity.

Described adjusting plant be breeding time make plant breeding time in advance;

Described adjusting flowering of plant is for promoting flowering of plant;

Described adjusting plant lotus throne number of sheets amount is for reducing plant lotus throne number of sheets amount;

Described adjusting axis leave quantity is for reducing axis leave quantity;

Described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

Recombinant vectors, expression cassette, transgenic cell line or host bacterium that also purpose of the present invention provides albumen of the present invention, encoding gene and contains described encoding gene are being cultivated the application of cultivating in the transgenic plant.

Concrete, described transgenic plant have following at least a proterties: 1) breeding time is in advance; 2) flowering time in advance; 3) lotus throne number of sheets amount reduces; 4) stem leaf quantity reduces.

Concrete described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

Further object of the present invention provides a kind of method of cultivating transgenic plant, is encoding gene of the present invention is imported the purpose plant, obtains transgenic plant.

Described transgenic plant are compared with described purpose plant, have following at least a proterties: 1) breeding time in advance; 2) flowering time in advance; 3) lotus throne number of sheets amount reduces; 4) stem leaf quantity reduces.

Concrete, described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

This research clones cotton Gh FPF1 gene from upland cotton, success makes up plant and crosses expression vector, adopt agriculture bacillus mediated inflorescence dip method transformation mode plant Arabidopis thaliana, compared with the control, transgenic arabidopsis was bloomed 5.1 days in advance than non-transgenic Arabidopis thaliana, the lotus throne leaf lacks 2.7 with the sum of stem leaf than the wild-type Arabidopis thaliana simultaneously, therefore utilizes this gene to can be used to cultivate short cotton breeding time, for the cultivation of transgenic plant is laid a good foundation.

Description of drawings

Fig. 1 is for changeing the evaluation figure of Gh FPF1 gene Arabidopis thaliana on the dna molecular level, and wherein numbering 1 is that blank, 2 is for wild-type Arabidopis thaliana, 3 positive contrasts (recombinant plasmid), 4-10 are respectively each strain of transgenic arabidopsis.

Fig. 2 is T₃In generation, changeed the evaluation figure of Gh FPF1 gene Arabidopis thaliana on the RNA molecular level, and WT is the contrast of wild-type Arabidopis thaliana, and COL3, COL7, COL4 are respectively each strain system of transgenic arabidopsis.

Fig. 3 is wild-type and changes Gh FPF1 gene T₃For the phenotype comparison diagram of Arabidopis thaliana growth during 3 weeks, wherein A represents the wild-type Arabidopis thaliana, and B represents transgenic arabidopsis.

Fig. 4 is wild-type and changes Gh FPF1 gene T₃For the phenotype comparison diagram of Arabidopis thaliana growth during 4 weeks, wherein A represents the wild-type Arabidopis thaliana, and B represents transgenic arabidopsis.

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

The preparation ofembodiment 1, cotton gene Gh FPF1

1, the extraction of RNA

Get middle cotton institute's 36 seeds (available from The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute) immersion and plant in small flower after one day, place the illumination cultivation chamber after one week of growth, round and deposit in-70 ° of refrigerators after a seedling places the liquid nitrogen quick-frozen.Extract the RNA of above-mentioned materials.

2, the preparation of cDNA

The RNA reverse transcription thatstep 1 is prepared is cDNA.

3, the amplification of gene

Primer sequence is:

Upstream primer P1 5 '-AGAGAAAATGAGCGGTCCTTG-3 '

Downstream primer P2 5 '-GCCCGAACATGGTGATTAAG-3 '

Primer and the preparation-obtained middle cotton 36cDNA of institute ofstep 2 with above-mentioned design are template, carry out pcr amplification.

Pcr amplification product is checked order.Sequencing result shows, above-mentioned pcr amplification obtains having SEQ ID № in the sequence table: 1 nucleotide sequence, be total to 401bp, head of district 327bp wherein encodes, this coding region sequence is as SEQ ID №: in 1 shown in the Nucleotide of 8-334 position, SEQ ID № in the code sequence tabulation: the aminoacid sequence shown in 2, totally 109 amino-acid residues.This had SEQ ID № in the sequence table: the fragment called after Gh FPF1 of the nucleotide sequence of 8-334 position in 1.

The functional verification ofembodiment 2, cotton gene Gh FPF1

(1), the structure of expression vector

(1) has the acquisition of the target gene fragment of specific restriction enzyme site

Upstream primer: 5 '-CTAGTCTAGAATGAGCGGTCCTTGGTGTTT-3 ' contains restriction enzyme site XbaI (T/CTAGA)

Downstream primer: 5 '-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3 ' contains restriction enzyme site SmaI (CCC/GGG)

With the primer that contains restriction enzyme site of above-mentioned design, with middle cotton the cDNA of 36 seedling be template, carry out pcr amplification.The purpose fragment that has restriction enzyme site that amplification is obtained is connected to pGEM-T Easy cloning vector, transforms DH5 α competent cell, cuts checking and sequencing filters out the correct recombinant vectors of sequence by PCR and enzyme.

(2) structure of pBI121-Gh FPF1 plant expression vector

Recombinant vectors and the pBI121 plasmid of step (1) preparation are used SmaI and XbaI double digestion respectively, and electrophoresis reclaims the big fragment product of target gene fragment and pBI121 carrier; The enzyme of target gene fragment and pBI121 is cut big fragment product to be connected with the T4 ligase enzyme and to spend the night; Connect product transformed into escherichia coli DH5 α, 37 ℃ of overnight incubation; The picking mono-clonal shakes bacterium, the exactness of sequence verification sequence.The sequence of the foreign gene that inserts in the gained plasmid is SEQ ID №: 1 8-337 position Nucleotide, and with this plasmid called after pBI121-Gh FPF1.

(2), change the acquisition of Gh FPF1 gene Arabidopis thaliana

1, the acquisition of reorganization Agrobacterium

Plasmid pBI121-Gh FPF1 is changed in the Agrobacterium LBA4404 competent cell, obtain the bacterium of recombinating.Extract the plasmid of reorganization bacterium and send to order-checking, the reorganization bacterium called after LBA4404/pBI121-Gh FPF1 that contains plasmid pBI121-Gh FPF1 that order-checking is correct.

2, adopt inflorescence dip method arabidopsis thaliana transformation

(1) the reorganization Agrobacterium bacterium liquid 20 μ l with-20 ℃ of preservations are inoculated in the 1ml LB liquid nutrient medium, and the activation of spending the night of 28 ℃, 180rpm shaking culture is got activation bacterium liquid 200 μ l and joined 28 ℃ of 20ml LB liquid nutrient mediums, 180rpm shaking culture

(2) treat that bacterium liquid OD value is about at 1.2 o'clock, the centrifugal bacterium liquid of 3000 commentaries on classics/per minutes is collected thalline

(3) the conversion medium prescription is: 1/2MS (macroelement reduces by half, and other are constant), 5% sucrose, 0.01 μ g/ml benzyladenine (BAP), 0.03%silwetL-77,20mg/L Syringylethanone, KOH adjust pH to 5.7 (Steven et al.1998).

(4) with above-mentioned conversion medium suspension thalline, transfer OD to 0.8 to begin to contaminate

(5) the Arabidopis thaliana inflorescence is placed conversion medium 30-50s, contaminate the back and with preservative film Arabidopis thaliana is wrapped, secretly cultivate to be placed under the normal condition in one day and cultivate.

3, change the evaluation of Gh FPF1 gene Arabidopis thaliana plant

(1) detection of Gh FPF1 gene in the commentaries on classics Gh FPF1 gene Arabidopis thaliana plant

Be planted in the 1/2MS that contains kantlex after the seed disinfection with results and go up (agar concentration 0.6%), after carry out 4 ℃ ofvernalization 3 days, transfer in the culturing room and cultivated about 10 days, two negative plant leaf flavescence, not regrowth, the normal plantlet of transplant of will growing is planted to small flower, after waiting to grow one month, detects in the plant whether change cotton gene GhFPF1 over to.The preparation method of Gh FPF1 gene is identical withembodiment 1 in the plant to be measured, with ddH₂O is blank, the negative contrast of non-transgenic Arabidopis thaliana DNA, and the positive contrast of recon plasmid, the primer sequence is during pcr amplification:

Upstream primer: 5 '-GATGTGATATCTCCACTGACGT-3 '

Downstream primer: 5 '-TCCCCCGGGCATCATTTATCCATAACCATGAAC-3 '

Wherein, upstream primer and expression vector are complementary, and downstream primer and gene are complementary.

Amplified production is carried out electrophoresis detection at 1.5% sepharose, detected result is seen Fig. 1, all can detect the band (gene order adds a bit of sequence about 450bp altogether on the expression vector) of about 450bp in the normal plant of the above-mentioned growth of choosing and the positive controls, and all do not detect corresponding dna molecular fragment in blank and the wild-type Arabidopis thaliana negative control group, illustrate that Gh FPF1 gene has been incorporated in the above-mentioned arabidopsis gene group.

(2) qRT-PCR detects

The T of the positive strain of 7 strains that results steps (1) filter out₀For seed, with seminal propagation to T₃In generation, is with T₃For planting under seed and the wild-type Arabidopis thaliana seed equal conditions, two weeks put in order strain respectively and are sampled to-70 ℃ of preservations.Picked at random three transgenic line COL3, COL4, COL7 carry out qRT-PCR and detect.Extract transgenosis and wild-type Arabidopis thaliana RNA respectively according to the method forembodiment 1, and reverse transcription is cDNA, is template, is confidential reference items with arabidopsis gene UBQ5 with this cDNA, carry out qRT-PCR and detect, the primer sequence that amplification Gh FPF1 gene and UBQ5 internal control gene use is:

Gh FPF1 upstream primer: 5 '-AAACTCAGGTTCGGACCAAAGG-3 '

Downstream primer: 5 '-CCGTCGTATCTTTCCCAACCAA-3 '

UBQ5 upstream primer: 5 '-TAACCCTTGAGGTTGAATCATC-3 '

Downstream primer: 5 '-GTCGATTCCTTCTGGATGTTGT-3 '

The qRT-PCR detected result is seen Fig. 2, and the result shows that Gh FPF1 gene is at different transgenosis T₃For transcribing of difference (height) level all arranged in the plant, but in the wild-type Arabidopis thaliana, do not detect Gh FPF1 gene transcription product.The qRT-PCR detected result has proved that further Gh FPF1 gene has been incorporated into transgenosis T3 in the genome of Arabidopis thaliana and successfully be transcribed into mRNA.

(3), change the phenotype of Gh FPF1 gene Arabidopis thaliana plant

1, with transgenosis T₃For growing and cultivation under Arabidopis thaliana and the wild-type Arabidopis thaliana equal conditions, the phenotype when observing 3 weeks of growth and 4 weeks respectively the results are shown in Figure 3,4.As can be seen from Figure 3, the transgenic arabidopsis bolting is early than the wild-type Arabidopis thaliana; As can be seen from Figure 4, the flowering time of transgenic arabidopsis is early than the wild-type Arabidopis thaliana.This experimental result shows that obviously shift to an earlier date than non-transgenic Arabidopis thaliana the breeding time of transgenic arabidopsis.

2, choose 7 transgenosis T3 for strain system and wild-type and change growing and cultivation under the empty carrier Arabidopis thaliana equal conditions, each strain is 30, adds up flowering time, and lotus throne leaf and stem leaf are counted in the lump, the results are shown in Table 1.In the table 1, WT is the wild-type Arabidopis thaliana, and COL1-COL7 is that 7 T3 that choose are for the transgenic arabidopsis strain.

Table 1

The result shows that transgenic arabidopsis was bloomed 5.1 days in advance than wild-type Arabidopis thaliana, and the lotus throne leaf lacks 2.7 with the sum of stem leaf than the wild-type Arabidopis thaliana; Transgenic arabidopsis was bloomed 5.0 days in advance than changeing the empty carrier Arabidopis thaliana, and the lotus throne leaf lacks 2.6 with the sum of stem leaf than changeing the empty carrier Arabidopis thaliana.The statistics of table 1 further specifies cotton Gh FPF1 gene and have the regulation and control effect of breeding time in Arabidopis thaliana, be embodied as flowering time early, lotus throne leaf and stem leaf quantity reduces.

Claims (10)

1. an albumen is following 1) or 2) albumen:

1) protein of the composition of the aminoacid sequence shown in the SEQ ID № .2 in the sequence table;

2) with the amino acid residue sequence of the SEQ ID № .2 in the sequence table through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with flowering of plant by 1) protein of deriving.

2. the encoding gene of the described albumen of claim 1.

3. encoding gene according to claim 2, it is characterized in that: described encoding gene has one of following nucleotide sequence:

1) SEQ ID № in the sequence table: the nucleotide sequence of 1 8-334 position;

2) SEQ ID № in the code sequence tabulation: the polynucleotide sequence of 2 protein sequences;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 1 dna sequence dna hybridization that limits;

4) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding; Concrete, described homology is more than 95%; Concrete is more than 96% again; Concrete is more than 97% again; Concrete is more than 98% again; Concrete is more than 99% again.

4. the recombinant vectors, expression cassette, transgenic cell line or the host bacterium that contain claim 2 or 3 described encoding genes; Described recombinant vectors is specially recombinant expression vector or recombinant cloning vector.

5. the primer of amplification claim 2 or 3 described encoding gene total lengths or its any fragment is right.

6. the arbitrary described encoding gene of the described albumen of claim 1 and claim 2-3 and the described recombinant vectors of claim 4, expression cassette, transgenic cell line or host bacterium are following 1)-4) application at least a:

1) regulates plant breeding time;

2) regulate the flowering of plant time;

3) regulate plant lotus throne number of sheets amount;

4) regulate axis leave quantity.

7. application according to claim 6 is characterized in that:

Described adjusting plant be breeding time make plant breeding time in advance;

Described adjusting flowering of plant is for promoting flowering of plant;

Described adjusting plant lotus throne number of sheets amount is for reducing plant lotus throne number of sheets amount;

Described adjusting axis leave quantity is for reducing axis leave quantity;

Described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

8. the arbitrary described encoding gene of the described albumen of claim 1 and claim 2-3 and the described recombinant vectors of claim 4, expression cassette, transgenic cell line or the host bacterium application in cultivating transgenic plant; Concrete, described transgenic plant have following at least a proterties: 1) breeding time is in advance; 2) flowering time in advance; 3) lotus throne number of sheets amount reduces; 4) stem leaf quantity reduces; Concrete described plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

9. a method of cultivating transgenic plant is that the arbitrary described encoding gene of claim 2-3 is imported the purpose plant, obtains transgenic plant; Described transgenic plant are compared with described purpose plant, have following at least a proterties: 1) breeding time in advance; 2) flowering time in advance; 3) lotus throne number of sheets amount reduces; 4) stem leaf quantity reduces.

10. method according to claim 9, it is characterized in that: described purpose plant is dicotyledons or monocotyledons; Described dicotyledons is specially Arabidopis thaliana or cotton.

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