CN104610438A - Cotton stress response associated protein GhGeBP and coding gene and application thereof - Google Patents

Abstract

The invention discloses an upland cotton GhGeBP protein, its coding gene and application. The protein GhGeBP 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 of SEQ ID No. 2 in the sequence listing A protein derived from 1) whose sequence has undergone substitution and/or deletion and/or addition of one or several amino acid residues and which is relevant to plant stress response. The protein and its coding gene of the invention can be used to breed high salt-tolerant cotton varieties (lines), and have good application prospects.

Description

Cotton stress response-related protein GhGeBP and its coding gene and application

technical field

The invention belongs to the fields of agricultural plant genetic improvement technology and plant biotechnology application, and specifically relates to a cotton stress response-related gene, its encoded protein and its application.

Background technique

GeBP (GL1enhancer binding protein) is a family of transcription factors unique to plants. Arabidopsis contains 23 members, and 5 member genes of this family are found in upland cotton. Julien Curaba et al. (2003) reported that GeBP is mainly expressed in meristems of vegetative organs and young leaf primordia, and acts as a repressor to determine the fate of leaf cell differentiation; Chevalier et al. (2008) showed that GeBP and GeBP-like proteins Encodes a class of non-conserved novel transcription factor proteins containing leucine zipper structure, and this type of protein is involved in Cytokinin pathway, inhibiting the negative regulation of ARR gene on cytokinin response. Perazza et al. (2011) found that CPR5 (CONSTITUTIVE EXPRESSOR OF PATHOGENESIS-RELATEDGENES5) and GeBP/GPLs play opposite roles in regulating cell expansion. However, there is no research report on the role of GeBP in plant stress resistance.

Contents of the invention

The object of the present invention is to provide a protein, its coding gene and its application. The name of the protein provided by the present invention is GhGeBP, which is derived from upland cotton (Gossypium hirsutum).

The protein of the present invention is the following 1) or 2) protein:

1) A protein composed of the amino acid sequence shown in SEQ ID №.2 in the sequence listing;

2) The amino acid residue sequence of SEQ ID №.2 in the sequence listing is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues, and the protein derived from 1) is related to plant stress response.

The amino acid sequence shown in SEQ ID №.2 in the sequence listing consists of 389 amino acid residues.

The GhGeBP protein in the above 1) and 2) can be synthesized artificially, or its coding gene can be synthesized first, and then biologically expressed. The gene encoding the GhGeBP protein in the above 1) and 2) can be deleted by deleting one or several amino acid residue codons from the DNA sequence shown in the 88th-1254th nucleotide of SEQ ID №.1 in the sequence listing, And/or obtained after carrying out a missense mutation of one or several base pairs.

The nucleic acid molecules encoding the GhGeBP protein also belong to the protection scope of the present invention.

The nucleic acid molecule can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as mRNA, hnRNA or tRNA.

Another object of the present invention is to provide the gene encoding the protein.

The coding gene has one of the following nucleotide sequences:

1) The nucleotide sequence at positions 88-1254 of SEQ ID №: 1 in the sequence listing;

2) The polynucleotide sequence of the protein sequence of SEQ ID №: 2 in the coding sequence list;

3) The nucleotide sequence that can hybridize with the DNA sequence defined by SEQ ID №: 1 in the sequence listing under high stringency conditions;

4) A DNA sequence that has more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encodes the same functional protein; specifically, the homology is more than 95%; more specifically, it is 96% More specifically, more than 97%; more specifically, more than 98%; more specifically, more than 99%.

The above-mentioned high stringency conditions can be 6×SSC, 0.5% SDS solution, hybridization at 65°C, and then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS respectively.

Among them, SEQ ID №: 1 in the sequence listing consists of 1378 nucleotides, its open reading frame (ORF) is nucleotides 88-1254 from the 5′ end, and SEQ ID №: 2 in the coding sequence listing The protein shown is the GhGeBP protein of the present invention.

Recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria containing the above nucleic acid molecules also belong to the protection scope of the present invention.

The recombinant vector can be a recombinant expression vector or a recombinant cloning vector.

The recombinant expression vector can be constructed with existing expression vectors. The expression vector can also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyA signal directs the addition of polyA to the 3' end of the pre-mRNA. When using the gene to construct a recombinant expression vector, any enhanced, constitutive, tissue-specific or inducible promoter can be added before its transcription initiation nucleotide, and they can be used alone or with other promoters Used in combination; in addition, when using the gene of the present invention to construct a recombinant expression vector, enhancers, including translation enhancers or transcription enhancers, can also be used. In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes that express enzymes or luminescent compounds that can produce color changes in plants (GUS gene, GFP gene, luciferase Genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

The pair of primers for amplifying the full-length coding gene of the present invention or any fragment thereof also falls within the protection scope of the present invention.

Another object of the present invention is to provide the application of the protein, coding gene and recombinant vector containing the coding gene, expression cassette, transgenic cell line or recombinant bacteria of the present invention in at least one of the following:

1) Regulate plant salt stress response;

2) Regulating plant ABA stress response.

The adjustment of plant salt stress response is to enhance the salt tolerance of plants;

The regulation of plant ABA stress response is to enhance the ABA tolerance of plants.

The plant is a dicotyledon or a monocotyledon; the dicotyledon is specifically Arabidopsis or cotton; the cotton is specifically Gossypium hirsutum.

Another object of the present invention is to provide the application of the protein, coding gene and recombinant vector containing the coding gene, expression cassette, transgenic cell line or host bacteria in the cultivation of transgenic plants according to the present invention.

Specifically, the transgenic plant has at least one of the following traits: 1) enhanced salt tolerance of the plant; 2) enhanced ABA tolerance of the plant.

Specifically, the plant is a dicotyledon or a monocotyledon; the dicotyledon is specifically Arabidopsis or cotton; and the cotton is specifically Gossypium hirsutum.

Another object of the present invention is to provide a method for cultivating transgenic plants, which is to introduce the coding gene described in the present invention into the target plant to obtain transgenic plants.

Compared with the target plant, the transgenic plant has at least one of the following traits: 1) the salt tolerance of the plant is enhanced; 2) the ABA tolerance of the plant is enhanced.

Specifically, the plant is a dicotyledon or a monocotyledon; the dicotyledon is specifically Arabidopsis or cotton; and the cotton is specifically Gossypium hirsutum.

The present invention clones the cotton GhGeBP gene from upland cotton, successfully constructs a plant overexpression vector, and uses the Agrobacterium-mediated inflorescence dipping method to transform the model plant Arabidopsis thaliana. Salt tolerance and ABA tolerance are improved, so the invention enriches the genetic resources for cultivating high salt-tolerant cotton varieties.

Description of drawings

Figure 1 shows the differential expression of GhGeBP gene in different tissue parts of upland cotton "Zhong G5" treated with 150 mM NaCl stress for 0.5 h, where CK is the result of the normal control group, and NaCl is the result of the salt stress treatment group.

Fig. 2 is a diagram of the PCR identification results of some transgenic Arabidopsis plants, wherein CK+ indicates that the plasmid DNA is used as a template, and CK- indicates that the wild-type Arabidopsis DNA is used as a template.

Detailed ways

The experimental methods used in the following examples are conventional methods unless otherwise specified.

The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Seeds of the upland cotton variety "Zhong G5" (Wang Kunbo, Cui Rongxia, Wang Chunying, etc. Main Features of Zhong G5 in New Cotton Materials. China Cotton. 2000, 24.) were provided by the Cotton Research Institute of the Chinese Academy of Agricultural Sciences, and the public can obtain them from China Agricultural University.

Embodiment 1, the preparation of cotton GhGeBP gene

Genomic DNA and total RNA were extracted from G5 hydroponic seedlings of upland cotton (Gossypium hirsutum L.) as materials; the extracted total RNA was reverse transcribed to obtain cDNA; genomic DNA and cDNA were used as templates, and GhG1F and GhG1R were used as templates. Primers for PCR amplification. The primer sequences for the above-mentioned PCR amplification are as follows:

GhG1F: 5'ATTTGCTATTGCCTCCTTCCCTGTT3'

GhG1R: 5'GAGCTACAGATACCCCCCATGATTG3'

The PCR reaction system is 50 μl, and the reaction system is: 2 μl template (cDNA or genomic DNA), 1 μl primer (10 μM), 5 μl 10×LA buffer, 8 μl dNTP (2.5 mM each), 0.5 μl LA-Taq enzyme, 34.5 μl ddH ₂ O .

The PCR reaction program was: 94°C for 5 min, 1 cycle; 94°C for 30 s, 60°C for 30 s, 72°C for 90 s (2 min), 30 cycles; 72°C for 10 min, 4°C∞, 1 cycle.

The obtained PCR amplification products were subjected to 1% agarose gel electrophoresis, and the fragments were recovered, connected to pMD18-T vector (TAKARA, D101A), transformed into Escherichia coli competent DH5α strain, and incubated overnight at 37°C in the dark. After blue-white screening, single clones were picked, positive clones were verified by colony PCR, and the positive clones were sent for sequencing. The sequencing results showed that the fragment amplified using genomic DNA as a template was the same size as the fragment amplified using cDNA as a template. By analyzing the sequencing results and comparing the cDNA sequence with the genomic DNA sequence, it was found that the gene had no introns; the above PCR The amplified nucleic acid fragment has the nucleotide sequence of SEQ ID №: 1 in the sequence listing, with a total of 1378 bp, of which the coding region is 1167 bp long (excluding the terminator). The coding region sequence is shown in SEQ ID №: 1 in the sequence listing As shown in nucleotides 88-1254, the amino acid sequence shown in SEQ ID №: 2 in the coding sequence list has a total of 389 amino acid residues, a molecular weight of 43KD, and an isoelectric point of 5.03. The fragment having the nucleotide sequence of positions 88-1254 in SEQ ID No. 1 in the sequence listing is named GhGeBP.

Embodiment 2, GhGeBP gene functional verification

(1) Expression analysis of GhGeBP gene under salt stress treatment

G5 hydroponic seedlings in planting cotton, Hoagland nutrient solution hydroponic. When the seedlings grow to the stage of three leaves and one heart, they are treated with 150mM NaCl solution, and the control is ordinary Hoagland nutrient solution. The roots, stems and leaves of the 150mM NaCl solution for 0.5h and the control are respectively taken, quick-frozen in liquid nitrogen, and stored at -70°C , for RNA extraction. 3 copies of each material.

Cotton total RNA was extracted by improved CTAB method, cDNA was obtained by reverse transcription, and RT-PCR analysis was carried out with GhG3F and GhG3R as primers and GhActin as internal reference gene. The primer sequences are as follows:

GhG3F: 5'GGAGGCGAAATGGAGGAAATTGC3'

GhG3R: 5'GAGCTACAGATACCCCCCATGATTG3'

RT-PCR analysis results are shown in Figure 1. The results showed that the GhGeBP gene was expressed in roots, stems, leaves and other tissues, and the expression of GhGeBP gene in each tissue was significantly up-regulated under the salt stress treatment for 0.5 h.

(2) Using GhGeBP gene to breed salt stress-tolerant plants

1. Preparation of plant expression vector pBI121-GhGeBP

Primers GhG1F and GhG1R were introduced into restriction endonuclease sites respectively, named GhG2F and GhG2R, and the sequences of primers GhG2F and GhG2R were as follows:

GhG2F: 5'GCTCTAGAGCATTTGCTATTGCCTCCTTCCCTGTT3'Xba Ⅰ

GhG2R: 5'CGAGCTCGGAGCTACAGATACCCCCCATGATTG3'Sac Ⅰ

Using the above-designed primers containing restriction sites, the cDNA of G5 hydroponic seedlings in the upland cotton cultivar was used as a template for PCR amplification. Ligate the amplified target fragment with the correct sequencing site to the plant expression vector pBI121 (pBI121 is from the China Plasmid Vector Strain Cell Line Gene Collection Center); the ligation product is transformed into Escherichia coli DH5α, cultured overnight at 37°C; pick Shaking monoclonal bacteria, sequencing to verify the correctness of the sequence. The foreign gene inserted in the resulting plasmid has the nucleotide sequence shown in SEQ ID No. 1 in the sequence table, and the plasmid is named pBI121-GhGeBP.

2. Acquisition of recombinant Agrobacterium

The plasmid pBI121-GhGeBP was transformed into competent cells of Agrobacterium tumefaciens EHA105 (purchased from Beijing Tianenze Gene Technology Co., Ltd.) by freeze-thaw method. Select culture in YEP solid culture containing 50 μg/ml kanamycin sulfate and 50 μg/ml rifampicin at 28°C; identify positive single clones by colony PCR (using primers GhG1F and GhG1R in Example 1), and The correctly identified Agrobacterium, that is, the recombinant Agrobacterium containing the recombinant plant expression vector pBI121-GhGeBP, was named EHA105/pBI121-GhGeBP.

3. Obtaining transgenic Arabidopsis

1) Transform Arabidopsis thaliana by inflorescence dipping method

a) Preparation of Agrobacterium infection solution

Take the recombinant Agrobacterium tumefaciens EHA105/pBI121-GhGeBP and inoculate it in 5ml YEP liquid medium (containing 50μg/ml kanamycin sulfate and 50μg/ml rifampicin), shake the bacteria overnight, and transfer the bottle to 500ml YEP the next day In liquid medium, culture at 28°C until OD600 is 1.6-2.0; centrifuge to collect the bacteria, use MS solution (MS basic medium components: macroelements, trace elements, iron salts, vitamin B5, 5% sucrose, 40μl/500ml SilwetL -77) Suspend until OD600 is 0.8-1.0 to obtain Agrobacterium infection liquid.

b) Preparation of target plant Arabidopsis

Colombian ecotype Arabidopsis Col-0 (purchased from Salk Institute Genomic Analysis Laboratory) was transplanted for about 4 weeks. After bolting, the head of the main inflorescence without cauline leaves was removed to promote the growth of its lateral inflorescences. Lateral inflorescences begin to flower and are ready for transformation.

c) Conversion

Put the whole Arabidopsis thaliana plant in step 2) upside down together with the flowerpot in a container containing 250ml of the Agrobacterium infection solution prepared in step 1) for transformation; put the Arabidopsis plant sideways on the tray, and use black plastic Cover it with cloth, uncover the plastic cloth one day later, place the flower pot upright, carry out normal light cultivation, and collect mature T1 generation seeds.

d) Screening of resistant seedlings

After the seeds of the T1 generation were sterilized, spread them on MS solid medium (containing 50 μg/ml kanamycin sulfate), vernalize at 4°C for 2-3 days, culture at 21°C for 10 days, then select resistant plants and transplant them into the soil. The T2 generation seeds were harvested per plant. Plant and screen T2 generation seeds according to the same method, transplant 3 T2 generation strains with a segregation ratio of resistance of 3:1, and harvest the T3 generation seeds on each individual plant in the T2 generation strains, and randomly select 16 The seeds of the T3 generation lines were screened for resistance in the same way, and six homozygous transgenic lines that no longer produced resistance segregation in the T3 generation were obtained. The plants or seeds of the homozygous transgenic line of the T3 generation were used for mass propagation for subsequent PCR identification and phenotypic identification.

The T1 generation represents the seeds obtained by selfing of the transformed contemporary plants and the plants grown from it; the T2 generation represents the seeds obtained from the selfing of the T1 generation and the plants grown by it; the T3 generation represents the seeds and plants grown from the selfing of the T2 generation the plants grown from it.

2) PCR identification of transgenic Arabidopsis plants

Genomic DNA was extracted from leaves of homozygous transgenic lines that no longer segregated resistance in the T3 generation, and PCR amplification was performed with primers KanF and KanR, and the predicted product size was 500 bp. The sequences of primers KanF and KanR are as follows:

KanF: 5'-CACTGAAGCGGGAAGGGACT-3'

KanR: 5'-CGATACCGTAAAGCACGAGGAA-3'

The PCR identification results showed that all the selected T3 transgenic Arabidopsis plants were positive; the PCR identification results of some plants are shown in Fig. 2 .

3) Salt tolerance identification of transgenic Arabidopsis plants

The seeds of T3 homozygous transgenic lines (TL1-TL6) and wild-type Arabidopsis Col-0 (WT) identified as positive were sterilized with 0.5% NaClO solution, and plated on MS solid medium plates respectively. Vernalization was performed at 4°C for 3 days, and then transferred to the greenhouse. After 4 days of light cultivation at 21°C, the seedlings were transferred to plants containing different concentrations (0, 100, 150, 200 mM) of NaCl and different concentrations (1, 75, 100, 125 μM) Culture in MS solid medium of ABA for 10 days, weigh the fresh weight of a single plant (including roots, stems and leaves), and set up 3 replicates for each strain, with 20-40 seedlings per replicate.

The salt tolerance identification results of transgenic Arabidopsis plants are expressed in the form of mean ± standard deviation, as shown in Table 1 and Table 2.

Table 1. Fresh weight per plant of transgenic Arabidopsis plants after 10 days of salt stress (unit: mg)

Note: * in Table 1 indicates that compared with the WT plants under the same concentration of NaCl stress, the results are significantly different at p<0.05; ** indicates that compared with the WT plants under the same concentration of NaCl stress, the results are at p <0.01 The difference is extremely significant; # in the table means the comparison of the fresh weight of the same strain under different NaCl stress conditions, and the difference reaches a significant level at p<0.05; ## means the fresh weight of the same strain under different NaCl stress conditions Comparison, the results were extremely significant at p<0.01.

The results in Table 1 were compared using Duncan's method for multiple comparisons. The results showed that under normal medium conditions, there was no significant difference in fresh weight between transgenic lines and WT plants; however, with the increase of NaCl concentration, there was a significant (very significant) difference in fresh weight between transgenic lines and WT plants. It shows that the salt tolerance of Arabidopsis transgenic with GhGeBP gene is improved.

The phenotypes of the same transgenic line were different under different concentrations of NaCl stress. Comparing the fresh weight of the six transgenic lines, it was found that the plant growth of G2121 and G2131 lines was less affected by NaCl.

Table 2. Fresh weight per plant of transgenic Arabidopsis plants after 10 days of ABA stress (unit: mg)

Note: * in Table 2 indicates that compared with the WT plants under the same concentration of ABA stress, the results are significantly different at p<0.05; ** indicates that compared with the WT plants under the same concentration of ABA stress, the results are at p<0.01 The difference is extremely significant; # in the table indicates that the fresh weight of the same strain is compared under different ABA stress conditions, and the result is significantly different at p<0.05; ## indicates that the fresh weight of the same strain is compared under different ABA stress conditions, and the result is in p<0.01 The difference is extremely significant.

The results in Table 2 were compared using the Duncan method for multiple comparisons. The results showed that under normal medium conditions, there was no significant difference in fresh weight between transgenic lines and WT plants; however, with the increase of ABA concentration, there was a significant (very significant) difference in fresh weight between transgenic lines and WT plants. It shows that the transgenic Arabidopsis can improve the tolerance to ABA stress.

The performance of the same transgenic line was different under different concentrations of ABA stress. Compared with the six transgenic lines, the plant growth of the G2103, G2119 and G2131 lines was not affected by ABA stress, while the G2121 transgenic line was not affected by the presence of ABA. With increased growth, the line can be further investigated for its response mechanism.

Claims (10)

1. A protein, which is the protein of the following 1) or 2): 1) A protein composed of the amino acid sequence shown in SEQ ID №.2 in the sequence listing; 2) The amino acid residue sequence of SEQ ID №.2 in the sequence listing is subjected to the substitution and/or deletion and/or addition of one or several amino acid residues, and the protein derived from 1) is related to plant stress response. 2. the coding gene of protein described in claim 1. 3. The coding gene according to claim 2, characterized in that: the coding gene has one of the following nucleotide sequences: 1) The nucleotide sequence at positions 88-1254 of SEQ ID №: 1 in the sequence listing; 2) The polynucleotide sequence of the protein sequence of SEQ ID №: 2 in the coding sequence list; 3) The nucleotide sequence that can hybridize with the DNA sequence defined by SEQ ID №: 1 in the sequence listing under high stringency conditions; 4) A DNA sequence that has more than 90% homology with the DNA sequence defined in 1) or 2) or 3) and encodes the same functional protein; specifically, the homology is more than 95%; more specifically, it is 96% More specifically, more than 97%; more specifically, more than 98%; more specifically, more than 99%. 4. A recombinant vector, an expression cassette, a transgenic cell line or a host bacterium containing the coding gene of claim 2 or 3; the recombinant vector is specifically a recombinant expression vector or a recombinant cloning vector. 5. A pair of primers for amplifying the full length of the coding gene or any fragment thereof according to claim 2 or 3. 6. The protein of claim 1 and the encoding gene of any one of claims 2-3 and the recombinant vector, expression cassette, transgenic cell line or host bacteria of claim 4 in at least one of the following: 1) Regulate plant salt stress response; 2) Regulating plant ABA stress response. 7. The application according to claim 6, characterized in that: The adjustment of plant salt stress response is to enhance the salt tolerance of plants; The regulation of plant ABA stress response is to enhance the ABA tolerance of plants. 8. the protein described in claim 1 and the arbitrary described coding gene of claim 2-3 and the application of recombinant vector, expression cassette, transgenic cell line or host bacterium described in claim 4 in cultivating transgenic plants; Specifically Yes, the transgenic plant has at least one of the following traits: 1) enhanced salt tolerance of the plant; 2) enhanced ABA tolerance of the plant. 9. A method for cultivating a transgenic plant, comprising introducing the coding gene described in any one of claims 2-3 into a target plant to obtain a transgenic plant; compared with the target plant, the transgenic plant has at least one of the following traits : 1) Enhanced salt tolerance of plants; 2) Enhanced ABA tolerance of plants. 10. The method according to claim 9, characterized in that: the target plant is a dicotyledon or a monocotyledon; and the dicotyledon is specifically Arabidopsis or cotton.