CN110607309A - Protein capable of enhancing drought resistance of plants and coding gene and application thereof - Google Patents

Abstract

The invention provides a gene capable of enhancing plant drought resistance, a protein coded by the gene and application of high-drought-resistance corn obtained by using a transgenic technology. The invention provides a gene capable of enhancing plant drought resistance, a protein coded by the gene and application thereof in culturing high-drought-resistance corn, wherein the gene is introduced into a corn plant by an agrobacterium-mediated method to obtain transgenic corn; compared with the untransformed corn variety, the transgenic corn plant has stronger drought resistance.

Description

Protein capable of enhancing drought resistance of plants and coding gene and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and relates to a protein capable of enhancing plant drought resistance, and a coding gene and application thereof.

Background

Corn (Oryza sativa L.) is an important food crop. Corn production is a large water demand process, and therefore is very sensitive to water shortage. The main corn planting periods (4-9 months in northeast, 3-10 months in Huang-Huai region, 3-11 months in Yangtze river basin, and 1-12 months in south China and southwest region) in China are often influenced by drought in different degrees. It was preliminarily estimated that crop drought areas and drought areas grow at rates of 408.8 and 209.0 million hectares per 10 years. According to the forecast of agricultural policy research center of Chinese academy of agricultural sciences, China needs 2.04 hundred million tons of corn in 2020, the production capacity is 1.84 million tons, the yield per hectare is about 7.8 tons, 2100 million tons of corn are imported annually to meet the increasing demand of food and animal husbandry, wherein the yield increase of more than 40 percent is realized by breeding and popularization of new varieties, and the drought tolerance of the corn needs to be improved to meet the demand of China on the corn.

In the traditional corn resistance breeding, phenotype selection is carried out on plants through resistance identification, the time consumption is long, the limitation of environmental conditions is easy, errors are easily caused by identification results, and the selection efficiency is low. Moreover, the genetic background of each corn variety in China is single, the problem of stress resistance still exists, and the breeding of drought-tolerant corn strains through conventional hybridization is very difficult, so that the drought-tolerant corn strains have no reliable drought-tolerant germplasm resource materials, have no credible and stable breeding character evaluation indexes, and are caused by inherent defects (randomness and blindness) of the conventional breeding technology. Therefore, it is difficult to achieve the coordinated aggregation of drought tolerance traits and other agronomic traits in the same variety or at least the breeding schedule thereof, etc. to meet the production requirements.

Transgenic breeding technology has become an irreplaceable breeding strategy in maize breeding. The most important characteristics are that the transgenic line has reliable transgenic screening marker, can be directionally operated and improve important characters, has relatively high breeding speed, and more importantly can realize the directional polymerization of the characters. Since 1996, the only transgenic maize varieties that were internationally commercialized or applied for commercialization were insect-resistant transgenic Bt genes, herbicide-resistant maize, and insect-resistant maize. To date, a transgenic "drought-resistant maize variety" has not been developed worldwide.

Disclosure of Invention

The invention aims to provide a gene capable of enhancing the drought resistance of plants, wherein the cDNA sequence of the gene is shown in SEQ ID NO.1, and the expression quantity of the gene is obviously increased when the gene is induced at the early stage of drought stress. The coded amino acid sequence is shown as SEQID NO. 2.

Another purpose of the invention is to provide the application of the gene capable of enhancing the drought resistance of the plant in improving the drought resistance of corn or other plants.

The gene capable of enhancing the drought resistance of the plant provided by the invention is used for improving the drought resistance of the corn, and a target gene is introduced into the corn to obtain a transgenic corn plant.

Wherein the target gene is introduced into the corn through a recombinant eukaryotic expression vector which takes chloramphenicol resistance and herbicide resistance Basta as selection markers.

Wherein the eukaryotic expression vector is plasmid pGSA 1252.

Wherein, the cDNA sequence of the target gene is cloned to the restriction site of Nco1/BamH1 at the downstream of a3 XCaMV 35S strong promoter of the plasmid pGSA1252 to obtain a recombinant plasmid pGSA1252: RA33G 4.

The method for introducing RA33G4 into corn comprises the following steps:

(1) introducing the recombinant plasmid pGSA1252: RA33G4 into agrobacterium tumefaciens; (2) preparing the agrobacterium tumefaciens introduced with the plasmid into a bacterial liquid with OD600 of 0.5 and containing 100 mu M acetosyringone;

(3) slightly wounding mature embryo of corn with tip of scalpel, sterilizing with 75% alcohol, transferring seed into the bacterial liquid of step (2), adding 20ml MS liquid culture medium and 6 μ l 50mg/ml acetosyringone, and culturing at 28 deg.C and 100rpm on shaking table for 18-20 h;

(4) taking out mature corn seeds, transferring the mature corn seeds to a co-culture medium containing MS, 30g/L sucrose, 100 mu M acetosyringone, 7g/L agar and pH6.0, and carrying out dark co-culture at 28 ℃ for 3 days;

(5) after the mature corn seeds are washed clean, the mature corn seeds are transferred to an antibacterial and screening culture medium containing MS, 0.1mg/L IAA, 250mg/L antibiotic Cef, 20ppm Basta, 30g/L cane sugar and 7g/L agar with the pH value of 6.0 for culturing for one week; after one week of culture, opening a bottle cap, culturing the resistant seedlings at 28 ℃ for 16h under illumination for 3 days, transferring to sterilized soil, and culturing at 28 ℃ for 16h under illumination to obtain herbicide Basta resistant corn seedlings;

(6) after the herbicide Basta resistant corn seedlings are transplanted into the pot soil and normally grow for 7 days at 28 ℃, 40ppm Basta is sprayed on the leaf surfaces, and the corn plants which still can survive are high drought resistance transgenic corn plants.

The invention has the advantages of

The invention provides a gene capable of enhancing plant drought resistance, a protein coded by the gene and application thereof in culturing high-drought-resistance corn, wherein the gene is introduced into a corn plant by an agrobacterium-mediated method to obtain transgenic corn; compared with the untransformed corn variety, the transgenic corn plant has stronger drought resistance.

Drawings

FIG. 1 is an electrophoresis diagram of the PCR method for identifying transgenic maize plant material.

FIG. 2 is a blot of Southern hybridization to identify transgenic maize plant material.

FIG. 3 shows the result of identifying the drought resistance of inbred homozygous T4 generation of transgenic corn plant.

Detailed Description

The following examples further illustrate the present invention but are not to be construed as limiting the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

EXAMPLE 1 preparation of transgenic drought-resistant maize

The corn seed material is as follows:

(1) designing a primer for cloning the gene by referring to the sequence of SEQ ID NO.1 and using software, and cloning from a cDNA library of corn to obtain the full length of the cDNA gene;

(2) cloning the cDNA sequence of a target gene to a commercial plasmid pGSA1252 at the downstream Nco1/BamH1 restriction enzyme site of a3 XCaMV 35S strong promoter to obtain a recombinant plasmid pGSA1252, wherein RA33G 4; the commercial plasmid pGSA1252 carries a chloramphenicol resistant gene and a herbicide resistant Basta selection marker gene Bar;

(3) preparing the agrobacterium tumefaciens introduced with the plasmid into a bacterial liquid with OD600 of 0.5 and containing 100 mu M acetosyringone;

(4) slightly wounding mature embryo of corn with tip of scalpel, sterilizing with 75% alcohol, transferring seed into the bacterial liquid of step (2), adding 20ml MS liquid culture medium and 6 μ l 50mg/ml acetosyringone, and culturing at 28 deg.C and 100rpm on shaking table for 18-20 h;

(5) taking out mature corn seeds, transferring the mature corn seeds to a co-culture medium containing MS, 30g/L sucrose, 100 mu M acetosyringone, 7g/L agar and pH6.0, and carrying out dark co-culture at 28 ℃ for 3 days;

(6) after the mature corn seeds are washed clean, the mature corn seeds are transferred to an antibacterial and screening culture medium containing MS, 0.1mg/L IAA, 250mg/L antibiotic Cef, 20ppm Basta, 30g/L cane sugar and 7g/L agar with the pH value of 6.0 for culturing for one week; after one week of culture, opening a bottle cap, culturing the resistant seedlings at 28 ℃ for 16h under illumination for 3 days, transferring to sterilized soil, and culturing at 28 ℃ for 16h under illumination to obtain herbicide Basta resistant corn seedlings;

(7) after the herbicide Basta resistant corn seedlings are transplanted into the pot soil and normally grow for 7 days at 28 ℃, 40ppm Basta is sprayed on the leaf surfaces, and the corn plants which still can survive are high drought resistance transgenic corn plants.

Example 2 molecular characterization of highly drought-resistant transgenic maize plant material

1. Identification by PCR method

Extracting genome DNA of plant materials, and amplifying fragments of a Basta gene and a 35S-RA33G4 gene by using PCR, wherein the sequence of a primer for amplifying the Basta gene is as follows: Bar-F5'-GCACCATCGTCAACCACTACATCG-3' and Bar-R5 '-AAATCTCGGTGACGGGCAGGAC 3', the primer sequence for amplifying the 35S-RA33G4 gene is as follows: 35S-F5 '-CGTCTTCAAAGCAAGTGGATTG-3' and RA33G 4-R5'-GCGGTACCGTGACAGATGATGCATGGGG-3'. Wherein the primer pair of the 35S-RA33G4 gene amplifies a partial segment comprising two genes of 35S and RA33G 4. The specific PCR process and conditions were performed according to conventional methods, and the PCR products were subjected to agarose gel electrophoresis (FIG. 1), where M is 100bpmarker in FIG. 1; 1: a positive plasmid; 2: a non-transgenic plant; 3: RNA is not reverse transcribed; 4-11: transgenic plants; as can be seen from the figure, the samples in lanes 4-11 amplified two bands, which confirmed the transgenic plants.

2. Southern hybridization assay

After the positive corn plant DNA detected by PCR is digested by restriction enzyme HindIII, the membrane is transferred and hybridized according to the method of molecular cloning experimental instruction. The probe is prepared by PCR amplification of two pairs of primers (Bar-F and Bar-R; 35S-RA33G4) by taking pGSA1252 as a template and RA33G4 as a marker, the marker of the probe adopts a digoxin marker kit and a method thereof, the obtained hybridization diagram is shown in figure 2, M in figure 2: 1kb marker; 1: negative CK (HindIII enzyme cuts the total DNA of wild corn); 2: positive CK (HindIII single digested expression vector pGSA1252:: RA33G 4); 3-10: positive transgenic plants (HindIII digested transgenic corn total DNA). As can be seen from FIG. 2, the transgenic lines of 3-8 and 10 were already homozygous lines.

Example 3 identification of the drought resistance of inbred homozygous T4 generation of transgenic maize plants

Through the experiment of the example 1, 6-10 transgenic seedlings of each variety of yellow morning four (HZ4), Chang 7-2(C7-2), Ye 478(Y478) and Zheng 58(Z58) of the maize inbred line of China are obtained, and the T4 generation maize seeds are obtained through 4 generations of inbreeding breeding and breeding in a laboratory. Corn seeds of T4 generation were surface-sterilized in 75% alcohol for 20min, then dressed in 50% carbondazolwettable powder wettable powder (to prevent seed-borne and soil-borne diseases), after which the seeds were sown in pot soil as described above. 3-4 seeds were sown per pot, placed in a greenhouse for cultivation and managed as indicated above. Greenhouse conditions: keeping the temperature at 30 +/-0.5 ℃, setting the light cycle at 12-h natural light/12-h dark, and controlling the air humidity at 50-60%. When the seedlings grow to the trefoil stage, adopting unwatered natural soil to be dry, observing and recording the phenotype of the plants and analyzing related parameters continuously for 10-15 days when the water content in the soil below 10cm of the surface of the pot soil is 0 and the water content in the soil below 10cm reaches 50 percent (measured by a soil moisture meter). Non-transgenic plants were set as parallel controls and phenotype was referenced to the controls, mainly based on the degree of wilting of the leaves and the mortality of the plants. The experimental results are shown in figure 3, the transgenic plants have no obvious difference under the condition of sufficient water supply, the transgenic plants show obvious drought resistance under the condition of medium drought stress, and the drought resistance among four transgenic varieties under severe stress is obviously superior to that of non-transgenic plants.

Sequence listing

<110> Guangxi university

<120> protein capable of enhancing drought resistance of plants, and coding gene and application thereof

<130> ZYWS

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<170> SIPOSequenceListing 1.0

<210> 1

<211> 430

<212> DNA

<213> protein capable of enhancing plant drought resistance, and coding gene and application thereof (Oryza sativa L.)

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agcgaaagga gagaaggaat ccgatccatc caagccaagc caaggcagag aagaagaaga 60

agtggaggaa gaagaagcat cggcagcatg tcggagggga ctgccaactg cgtggacatc 120

ctgatcgcca tcatcctgcc tccgctgggg gtgttcctca agtacgggtg cggccacgag 180

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tacgccatca ccaagaacaa ctagctagtc atccttgccg acgacgatcc atgtctctgt 300

atctgcgtct gttcatatgc ctgatgccga cttgtgctgt atgtagtgct tgaattcagt 360

cgtgttttgc taccgtaccc accccatgca tcatctgtca ccgcgacgca ataatcatag 420

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Met Ser Glu Gly Thr Ala Asn Cys Val Asp Ile Leu Ile Ala Ile Ile

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Leu Pro Pro Leu Gly Val Phe Leu Lys Tyr Gly Cys Gly His Glu Phe

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Trp Ile Cys Leu Leu Leu Thr Phe Leu Gly Tyr Ile Pro Gly Ile Ile

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Tyr Ala Ile Tyr Ala Ile Thr Lys Asn Asn

50 55

Claims (8)

1. A gene capable of enhancing the drought resistance of a plant, which is characterized in that: the cDNA sequence of the gene is shown in SEQ ID NO. 1.

2. The gene of claim 1 encoding a protein characterized by: the amino acid sequence of the protein is shown as SEQ ID NO. 2.

3. Use of the gene of claim 1 to increase drought resistance in maize or other plants.

4. A method for obtaining high drought resistance corn by utilizing a transgenic technology is characterized by comprising the following steps: introducing the gene of claim 1 into maize to obtain a transgenic maize plant.

5. The method of claim 4, wherein: the gene is introduced into the corn through a recombinant eukaryotic expression vector taking chloramphenicol resistance and herbicide resistance Basta as selection markers.

6. The method of claim 4, wherein: the eukaryotic expression vector is plasmid pGSA 1252.

7. The method of claim 6, wherein: the cDNA sequence of the target gene is cloned to the restriction site of Nco1/BamH1 at the downstream of a3 XCaMV 35S strong promoter of plasmid pGSA1252, and the recombinant plasmid pGSA1252 is obtained, namely RA33G 4.

8. The method of claim 7, wherein the recombinant plasmid pGSA1252: RA33G4 is introduced into maize comprising the steps of:

(1) introducing the recombinant plasmid pGSA1252: RA33G4 into agrobacterium tumefaciens;

(2) preparing the agrobacterium tumefaciens introduced with the plasmid into a bacterial liquid with OD600 of 0.5 and containing 100 mu M acetosyringone;

(3) slightly wounding mature embryo of corn with tip of scalpel, sterilizing with 75% alcohol, transferring seed into the bacterial liquid of step (2), adding 20ml MS liquid culture medium and 6 μ l 50mg/ml acetosyringone, and culturing at 28 deg.C and 100rpm on shaking table for 18-20 h;

(4) taking out mature corn seeds, transferring the mature corn seeds to a co-culture medium containing MS, 30g/L sucrose, 100 mu M acetosyringone, 7g/L agar and pH6.0, and carrying out dark co-culture at 28 ℃ for 3 days;

(5) after the mature corn seeds are washed clean, the mature corn seeds are transferred to an antibacterial and screening culture medium containing MS, 0.1mg/L IAA, 250mg/L antibiotic Cef, 20ppm Basta, 30g/L cane sugar and 7g/L agar with the pH value of 6.0 for culturing for one week; after one week of culture, opening a bottle cap, culturing the resistant seedlings at 28 ℃ for 16h under illumination for 3 days, transferring to sterilized soil, and culturing at 28 ℃ for 16h under illumination to obtain herbicide Basta resistant corn seedlings;

(6) after the herbicide Basta resistant corn seedlings are transplanted into the pot soil and normally grow for 7 days at 28 ℃, 40ppm Basta is sprayed on the leaf surfaces, and the corn plants which still can survive are high drought resistance transgenic corn plants.