Application of peanut AhP5CS2 gene in improving plant stress resistance

Abstract

The invention discloses the application of peanut AhP5CS2 gene in improving plant stress resistance, belonging to the technical field of improving plant stress resistance. The nucleic acid sequence of the peanut AhP5CS2 gene in the invention is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO:2. The gene is used to construct a plant expression vector and transform tobacco, which can significantly improve the stress resistance of tobacco in the germination stage, seedling stage and seedling stage. Therefore, the peanut AhP5CS2 gene has potential application value in improving plant stress resistance.

Description

Technical Field

[0001] The invention belongs to the technical field of improving plant stress resistance, and particularly relates to an application of a peanut AhP5CS2 gene in improving plant stress resistance. Background Art

[0002] With the increase in population and the deterioration of the environment, the area of ​​cultivated land is decreasing day by day, and the productivity level of conventional cultivated land is facing bottlenecks. By improving the overall improvement technology capabilities of arid and semi-arid areas, and significantly improving the productivity level and agricultural production efficiency in arid and semi-arid areas, it is of great significance to increase the reserve of cultivated land resources and ensure national food security. Peanuts are mainly grown in arid and semi-arid subtropical areas. Drought causes peanut production to decrease by more than 20% each year, and the global direct economic losses are as high as 3 billion US dollars. Although peanuts are resistant to barrenness, drought and strong adaptability, to ensure the stable and increased production of peanuts in arid and semi-arid areas, it is necessary to start from improving variety characteristics and screening highly resistant varieties.

[0003] Seed germination is a complex and changing process that is affected by various environmental conditions. Among them, drought has the greatest impact on the germination of peanut seeds, and the quality of germination greatly affects the construction of the crop population in the later stage. Improving the stress resistance of crop varieties through stress breeding and genetic transformation technology is two effective ways to improve the germination rate and drought resistance of peanuts in arid and semi-arid areas. However, traditional conventional hybrid breeding in stress breeding has a long cycle, low efficiency, and slow development; and although the mutation frequency of mutagenesis breeding has been greatly increased, its beneficial mutation frequency is low, the mutation direction is difficult to grasp, and the chemical mutagen is highly toxic and has a residual effect. Genetic transformation to improve crop quality has directional efficiency, but at present, peanut drought resistance genes have been less explored, and the results of transgenic peanuts are seriously insufficient; therefore, screening peanut drought resistance genes during seed germination, analyzing their stress resistance molecular mechanisms, and cultivating new drought-resistant and stress-resistant varieties are effective ways to rationally utilize the temperature and light resources in arid and semi-arid areas. Summary of the invention

[0004] In view of the problems existing in the prior art, the object of the present invention is to provide an application of peanut AhP5CS2 gene in improving plant stress resistance.

[0005] In order to achieve the above object, the present invention adopts the following technical solution:

[0006] The invention discloses an application of peanut AhP5CS2 gene in improving plant stress resistance. The nucleic acid sequence of the peanut AhP5CS2 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded by the peanut AhP5CS2 gene is shown in SEQ ID NO:2.

[0007] Based on the above scheme, the stress resistance is at least one of resistance to drought stress, resistance to oxidative stress, and resistance to high salt stress.

[0008] In a specific embodiment, the stress resistance is resistance to drought stress; specifically, under drought stress, the germination rate of the plant in the germination period can be faster, the germination rate can be improved, the root length in the seedling stage can be longer, the wilting degree in the seedling stage can be lower, the growth can be better, and the drought resistance can be stronger.

[0009] A recombinant vector, a recombinant bacterium, an expression cassette and a transgenic cell line containing a peanut AhP5CS2 gene, wherein the nucleic acid sequence of the peanut AhP5CS2 gene is shown in SEQ ID NO:1.

[0010] A method for improving plant stress resistance is to construct the coding region sequence of the peanut AhP5CS2 gene into a plant expression vector, transform plant cells, and express it in plants to improve plant stress resistance; the nucleic acid sequence of the peanut AhP5CS2 gene is shown in SEQ ID NO: 1.

[0011] On the basis of the above scheme, the method for improving plant stress resistance can improve the stress resistance of plants in the germination stage, seedling stage and seedling stage.

[0012] In a specific embodiment, the improving plant stress resistance is achieved by increasing the proline and soluble sugar content in the plant.

[0013] The peanut AhP5CS2 gene is used for increasing the proline and / or soluble sugar content in plants. The nucleic acid sequence of the peanut AhP5CS2 gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the peanut AhP5CS2 gene is shown in SEQ ID NO: 2.

[0014] A method for increasing the content of proline and / or soluble sugar in plants is to construct the coding region sequence of the peanut AhP5CS2 gene into a plant expression vector, transform plant cells, and express it in the plant to increase the content of proline and / or soluble sugar in the plant; the nucleic acid sequence of the peanut AhP5CS2 gene is shown in SEQ ID NO: 1.

[0015] Advantages of the technical solution of the present invention

[0016] The present invention connects the full-length cDNA of the peanut AhP5CS2 gene to an expression vector initiated by an overexpression vector, and transforms tobacco by the Agrobacterium-mediated leaf disc method. It is found that the overexpression of the gene can cause the model plant tobacco to show drought-resistant phenotypes during the germination and seedling stages, which are specifically manifested as faster germination and higher germination rate; longer seedling root length, and more drought-resistant seedlings. The proline and soluble sugar content of AhP5CS2 transgenic tobacco increases rapidly under drought stress, which can effectively alleviate the damage caused by drought stress to plants. Therefore, the peanut AhP5CS2 gene has potential application value in improving plant stress resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is the electrophoresis diagram of the cloned target gene AhP5CS2;

[0018] Figure 2 This is the result of restriction enzyme digestion of AhP5CS2-pMD19-T simple vector;

[0019] Figure 3 This is the result of restriction digestion of the target gene connected to the pBI121 expression vector plasmid;

[0020] Figure 4 RT-PCR identification of the expression level of AhP5CS2 in the positive seedlings of the transgenic lines (lane 1 is the wild type WT, and the other lanes are the transgenic lines);

[0021] Figure 5 The results are for drought resistance of different tobacco strains during the germination period;

[0022] Figure 6 The results are for drought resistance of different tobacco strains at the seedling stage;

[0023] Figure 7 The results of drought resistance of different tobacco strains at the seedling stage;

[0024] Figure 8 Figure 2 shows the proline and soluble sugar contents in different tobacco strains. DETAILED DESCRIPTION

[0025] The terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art.

[0026] The present invention is further described in detail below in conjunction with specific examples and with reference to data. The following examples are only intended to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0027] pMD19-T simple cloning vector was purchased from Takara T-Vector pMD TM 19(Simple); Code No. 3271;

[0028] Escherichia coli DH5α strain was purchased from Solarbio; Code No. C1100;

[0029] The pBI121 empty vector with 35S was purchased from MKbio; Code No. MF3721-5UG;

[0030] Agrobacterium LBA4404 was purchased from Tolo Bio; Code No. CC96302.

[0031] In the following examples, the Agrobacterium-mediated leaf disc method was used to introduce the expression vector into the model plant tobacco; in practical applications, other methods such as the vacuum infection method of germinated seeds may also be used for the introduction method.

[0032] In the following examples, the extraction and purification of peanut RNA can be carried out using Tiangen RNA prep Pure polysaccharide and polyphenol plant total RNA extraction kit (DP441), or directly extracted using the Trizol method; the synthesis of the first-chain cDNA can be carried out using Tiangen FastKing one-step genomic cDNA first-chain synthesis premix reagent (KR118), or existing processes or other kits. However, both of the above preferably use the specific method described in the present invention. Then, the first-chain cDNA is used as a template, and the specific primers corresponding to AhP5CS2 are used to perform Phusion high-fidelity enzyme amplification using the obtained cDNA as a template.

[0033] Example 1 Cloning of peanut AhP5CS2 gene

[0034] The peanut AhP5CS2 gene (Arachis hypogaea delta1-pyrroline-5-carboxylatesynthase) encodes Δ1-pyrroline-5-carboxylate synthase, which is a key enzyme in the synthesis of proline from the glutamate pathway. Its CDS sequence (5'→3') is 2103 bp (the sequence is shown in SEQ ID NO: 1), encoding a synthase of 700 amino acids (the sequence is shown in SEQ ID NO: 2).

[0035] SEQ ID NO: 1:

[0036]

[0037] SEQ ID NO:2

[0038] MQPPVKNGFNVFLGNSTEWRLSNGPAYLNSPTAYIEPESVTLDPSRAFLKDVKRLIVKVGTAVVTRSDGRLALGRLGALCEQLKELNSRGYEVILVTSGAVGLGRQRLRYRRLANSSFSDLQKPQGEFDGKACAAVGQSSLMALYDTMFSQLDVTSSQLLVNDGFFRDAGFRKQL SDTVHSLLNLRVIPIFNENDAVSTRKAPYEDSSGIFWDNDSLAGLLALELKADLLVLLSDVEGLYSGPPSDPNSKLIDTYVKEKHQKEITFGDKSRLGRGGMTAKVNAAVCAAYAGTPVIITSGYATDNIIRVLQGDRIGTVFHKDAHLWAHIKQVSAREMAISARDCSRRLQSL NSEERRKILLAVADALENNLSLIMEENGVDIADAELAGLEKSLISHCWACEICSNLADKLTLERISCPLGVVLVIFESRPDALVQIAALAVRSGGLLLKGGKEAKRSNAALHKVITSAIPEKVGDKLIGLVTSREEIPDLLKLDDVIDLVVPRGSNKLVSQIKESTKIPVLGHS DGICCHVYVDKAANIDMAKQIVKDAKIDYPAACNAMETLLVHKDLSETGGLNQLVAELTKEGVELYGGPRASGLLNITKTSSFHKDSHTECIVTEDPEVAETFLRRVDSAAVFHNASTRFCDGARFGLGAEVVGISTSRIHARGPVGVEGILRGSGQVVDGDRGVTYTHKELPIIA

[0039] The cloning method of the peanut AhP5CS2 gene is as follows:

[0040] (I) Extraction of total RNA from peanut

[0041] Use Tiangen RNA prep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (DP441) to extract total RNA from peanuts. The specific steps are as follows:

[0042] 1. Peanut seeds were germinated for 5 days, and then ground into powder in liquid nitrogen. 0.1 g was taken into an RNase-Free centrifuge tube, and 500 μL RL (β-mercaptoethanol should be added in advance) was added, vortexed and mixed, and placed at room temperature for 1-3 minutes.

[0043] 2. Centrifuge at 12,000 rpm for 2 min;

[0044] 3. Transfer the supernatant solution to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12,000 rpm for 2 minutes, and carefully pipette the supernatant in the collection tube into an RNase-Free centrifuge tube, trying to avoid the pipette tip from contacting the cell debris precipitate in the collection tube;

[0045] 4. Slowly add 0.4 times the volume of the supernatant anhydrous ethanol, mix well, transfer all the solution into the adsorption column CR3, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0046] 5. Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 30-60 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0047] 6. Add 80 μL of DNase I working solution to the center of adsorption column CR3 and leave at room temperature for 15 minutes;

[0048] 7. Add 350 μL of deproteinized solution RW1 to the adsorption column CR3, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube;

[0049] 8. Add 500 μL of rinsing solution RW (ethanol needs to be added in advance) to the adsorption column CR3, centrifuge at 12,000 rpm for 30 seconds, pour out the waste liquid in the collection tube, put the adsorption column CR3 back into the collection tube, and repeat the above steps once;

[0050] 9. Centrifuge at 12,000 rpm for 2 minutes and discard the waste liquid. Place the adsorption column CR3 at room temperature for 5-10 minutes to completely dry the residual rinse liquid in the adsorption material;

[0051] 10. Place the adsorption column CR3 in a new RNase-Free centrifuge tube, add 50 μL RNase-Free ddH2O to the middle part of the adsorption membrane, place at 37°C for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes to obtain RNA extraction solution.

[0052] (ii) Reverse transcription of the extracted peanut total RNA into cDNA

[0053] Use Tiangen FastKing One-step Genomic cDNA First-Strand Synthesis Premix (KR118) for reverse transcription:

[0054] Take 1 μg of total RNA, add 4 μL of 5×FastKing-RT SuperMix, add water to 20 μL, react at 42°C for 15 min, and inactivate at 95°C for 3 min.

[0055] (III) Cloning of AhP5CS2 gene

[0056] The peanut cDNA prepared above was used as a template and the following primer pairs were used for PCR amplification of the peanut AhP5CS2 gene.

[0057] Primer pair sequences for cloning AhP5CS2 gene:

[0058] Upstream primer 5'- TCTAGA ATGCAGCCTCCCGTCAAAAATGG-3' (SEQ ID NO: 3);

[0059] Downstream primer 5'- GTCGAC TGCTATGATTGGAAGTTCCTTGTG-3' (SEQ ID NO: 4);

[0060] The underlined parts are restriction sites, the restriction site of the upstream primer is XbaI, and the restriction site of the downstream primer is SalI.

[0061] Using Novozyme Max Super-Fidelity DNA Polymerase high-fidelity enzyme was used for amplification, and the reaction system was: 2×Phanta Max Buffer 25μL, dNTP Mix (10mM) 1μL, upstream primer 2μL, downstream primer 2μL, Phanta Max Super-Fidelity DNA Polymerase 1μL, cDNA template 1μL, and water was added to 50μL.

[0062] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 sec, annealing at 55°C for 15 sec, extension at 72°C for 90 sec, for a total of 35 cycles; post-extension at 72°C for 5 min.

[0063] After the reaction is completed, agarose gel electrophoresis is performed and the target band is detected. Figure 1 As shown, the gel was cut and gel recovery was performed according to the common agarose gel DNA recovery kit (DP209) of Tiangen Company.

[0064] The ends of the products amplified by high-fidelity enzymes are blunt-ended and need to be tailed. The reaction system is as follows: 2 μL of 10× reaction buffer, 1.5 μL of dNTP, 0.2 μL of Taq enzyme, and the gel recovery product is filled to 20 μL. React at 72°C for 30 minutes.

[0065] Take 4 μL of the tailed product and perform TA ligation with the pMD19-T simple cloning vector, incubate overnight at 16°C, transform E. coli DH5α strain by heat shock method, and grow overnight on LB plate containing ampicillin. Pick a single white colony into LB culture medium, incubate at 37°C for 4-6 hours, and perform bacterial liquid PCR. Select positive colonies and incubate overnight in LB liquid medium containing ampicillin.

[0066] The plasmid DNA of the above positive bacteria was extracted using the Tiangen Plasmid Extraction Kit (DP103), and sequencing was completed by Sangon Biotechnology (Shanghai) Co., Ltd. The plasmid with correct sequencing was named AhP5CS2-pMD19-Tsimple for standby use.

[0067] Example 2 Overexpression vector containing peanut AhP5CS2 gene

[0068] The overexpression vector containing the peanut AhP5CS2 gene was constructed by the following method:

[0069] AhP5CS2-pMD19-T simple and the empty vector pBI121 with 35S were digested with XbaI and SalI at 37°C for one hour, and then subjected to agarose gel electrophoresis. The correct bands were cut out and recovered from the gel. The digestion results of AhP5CS2-pMD19-T simple were as follows: Figure 2 As shown. The target fragment obtained after restriction digestion was connected to the pBI121 vector after restriction digestion using Thermo's T4 DNA ligase overnight. The ligation product was transformed into the DH5α strain and grown overnight on an LB plate containing kanamycin. A single white colony was picked up and added to the LB culture medium. After culturing at 37°C for 4-6 hours, a bacterial liquid PCR was performed. Positive colonies were selected and cultured overnight in an LB liquid culture medium containing kanamycin. Plasmid DNA extraction: Plasmid DNA was extracted using the Tiangen Plasmid Extraction Kit (DP103), and XbaI and SalI were used for restriction digestion and identification, as shown in the figure. Figure 3 As shown, the overexpression vector of 35S::AhP5CS2 was constructed.

[0070] The above description uses the pBI121 empty vector as an example, and other suitable expression vectors may also be selected according to the type of transformation target.

[0071] Example 3 Obtaining transgenic tobacco overexpressing AhP5CS2

[0072] The method for transforming tobacco with the 35S:AhP5CS2 overexpression vector constructed in Example 2 comprises the following steps:

[0073] (I) Obtaining Agrobacterium containing 35S::AhP5CS2:

[0074] 1. Thaw the competent cells of Agrobacterium tumefaciens (LBA4404) on ice, add 3 μL 35S::AhP5CS2 plasmid, then place on ice for 30 min, freeze in liquid nitrogen for 1 min, and place in a 37°C water bath for 5 min;

[0075] 2. Add 1 mL of YEP medium and culture at 28°C with shaking at 200 rpm until the logarithmic growth phase;

[0076] 3. Centrifuge at 8000 rpm for 2 minutes, discard the supernatant, spread the precipitate on a YEP plate containing kanamycin and rifampicin, and invert it in a 28°C incubator for 2-3 days;

[0077] 4. Pick a single colony into YEP culture medium, culture at 28°C for 4-6 hours, then perform PCR on the culture medium, select 3-4 positive colonies, add about 20 mL of YEP (kanamycin plus rifampicin) culture medium, and culture overnight to obtain Agrobacterium culture medium containing 35S::AhP5CS2.

[0078] (II) Agrobacterium-mediated transformation of tobacco leaves

[0079] 1. Move the common tobacco plants that have grown to 2-3 true leaves into small pots for cultivation until the leaves are relatively strong;

[0080] 2. Take healthy common tobacco leaves, rinse them with water 2-3 times, disinfect them with 70% ethanol for 30 seconds, disinfect them with 0.1% mercuric chloride (HgCl2) for 10 minutes, then rinse them with sterile water for 5-6 times, and absorb the liquid on the surface of the leaves with sterile filter paper;

[0081] 3. Cut the leaves into small pieces of 0.5×0.5 cm, and then place them in MS differentiation medium (MS medium + 0.1 mg / L NAA + 1.5 mg / L 6-BA), and pre-culture them in a 25°C long-day (16 h light / 8 h dark) incubator for about 2 days;

[0082] 4. Pipette 50 μL of Agrobacterium tumefaciens containing 35S::AhP5CS2 and inoculate it into YEP medium (containing kanamycin and rifampicin), shake culture at 28°C and 200 rpm for 24 h until the logarithmic growth phase, centrifuge at 8000 rpm for 5 min, discard the supernatant and suspend the precipitate with MS liquid medium (MS basal medium, Qingdao Hibo Biotechnology, HB8469), and adjust OD600 to 0.4-0.6;

[0083] 5. Soak the pre-cultured tobacco leaves with the resuspended Agrobacterium bacterial solution for 5-10 minutes, remove the excess bacterial solution with sterile filter paper, transfer the leaves to MS solid culture medium, and co-culture at 25°C in the dark for 2 days;

[0084] 6. Rinse the co-cultured explants 2-3 times with sterile water containing carbenicillin (250 mg / L), then rinse once with MS liquid medium containing carbenicillin (250 mg / L), dry the liquid with sterile filter paper, transfer to MS selection medium (MS medium + 0.1 mg / L NAA + 1.5 mg / L 6-BA + 50 mg / L kanamycin + 250 mg / L carbenicillin), and culture in a 25°C long-day incubator. Change the medium every 15 days or so;

[0085] 7. When the buds grow to about 1 cm, use sterile tweezers to move them into MS rooting medium (MS medium + 0.1 mg / L IAA + 50 mg / L kanamycin + 250 mg / L carbenicillin) to promote rooting;

[0086] 8. Transfer the regenerated seedlings to soil and carry out routine management in a long-day greenhouse at 25℃.

[0087] (III) Screening of positive transgenic tobacco seedlings overexpressing AhP5CS2

[0088] 1. When the transformed plants grow to 8 weeks old, take leaves to extract genome, perform PCR identification, and harvest positive transgenic plants to obtain T1 generation.

[0089] 2. T2 was obtained by T1 generation reproduction, RNA was extracted from leaves, and transgenic strains were detected by RT-PCR. Strains with high expression levels were selected for reproduction and further experiments.

[0090] like Figure 4 ,After RT-PCR detection, a total of 5 AhP5CS2 overexpression transgenic lines were obtained, and the transgenic lines OE1, OE2 and OE3 with different AhP5CS2 expression levels were selected for subsequent studies.

[0091] Example 4 Application of AhP5CS2 gene in improving tobacco drought resistance

[0092] (I) Application of AhP5CS2 gene in improving drought resistance of tobacco during germination period

[0093] The transgenic tobacco positive strains OE1, OE2 and OE3 obtained in Example 3 were used as test materials, and non-transgenic wild-type tobacco was used as a control (WT). Tobacco seeds were disinfected with 70% ethanol for 5 minutes, centrifuged and the supernatant was discarded, then disinfected with 2.6% sodium hypochlorite for 10 minutes, centrifuged and the clean supernatant was discarded, and finally rinsed with sterile ddH2O for 5-6 times, and the sterilized sterile seeds were neatly spread on MS solid culture medium containing different concentrations of PEG. 4℃ dark treatment for 3 days, placed in a 25℃ long-day incubator for 10 days, and germination was counted every 12 hours. Seeds breaking through the seed coat were considered to be germinated, and the tobacco seed germination rate was counted every day. All experiments were repeated 3 times.

[0094] The test results are as follows Figure 5 As shown, Figure 5 A in the middle refers to the germination of seeds of different tobacco strains on MS solid medium containing different concentrations of PEG; Figure 5 BE refers to the germination of seeds of different tobacco strains over time on MS solid culture medium containing different concentrations of PEG. Figure 5 Both A and B indicate that the transgenic positive plants have a drought-resistant phenotype during the germination period. In MS medium, there was no difference in the germination rate between transgenic tobacco seeds and the control, but when grown on a medium with a higher concentration of PEG, there was a significant difference: under 10% PEG conditions, the germination rate of WT was about 89.70%, the germination rates of OE1 and OE2 reached 100% and 98.37%, respectively, and OE3 reached 93.43%; under 15% PEG conditions, the germination rate of WT was about 79.33%, the germination rates of OE1, OE2 and OE3 reached 92.27%, 90.57% and 83.83%, respectively. The germination rates of the seeds of the two transgenic positive lines OE1 and OE2 were significantly higher than those of the control, increasing the germination rates by 16.30% and 14.16%, respectively. The drought resistance of OE3 was lower than that of OE1 and OE2, and the difference with WT was small, which was due to the lower expression level of AhP5CS2 in it than that of OE1 and OE2. In summary, AhP5CS2 can improve the germination rate of tobacco seeds under drought stress, and the degree of improvement is related to the expression level of AhP5CS2.

[0095] (II) Application of AhP5CS2 gene in improving drought resistance of tobacco seedlings

[0096] The transgenic tobacco positive strains OE1, OE2 and OE3 obtained in Example 3 were used as test materials, and non-transgenic wild-type tobacco was used as a control (WT). Tobacco seeds were disinfected with 70% ethanol and 2.6% sodium hypochlorite, and neatly dotted on MS solid culture medium containing different concentrations of PEG. They were treated in the dark at 4°C for 3 days, placed in a long-day incubator at 25°C and cultured upright for 10 days, and the root lengths of tobacco strains on different culture dishes were counted. All experiments were repeated 3 times.

[0097] The test results are as follows Figure 6 As shown in Table 1, Figure 6 A refers to the phenotype of seedlings of different tobacco strains on MS solid medium containing different concentrations of PEG; B refers to the root length of seedlings of different tobacco strains on MS solid medium containing different concentrations of PEG. Figure 6 As shown in Figures A, B and Table 1, the growth of transgenic tobacco seeds in MS medium was basically the same as that of the control, but under the condition of 10% PEG, the root length of OE1 and OE2 was 22.32% and 17.81% higher than that of the control; under the condition of 15% PEG, the root length of OE1 and OE2 was 33.90% and 32.61% higher than that of the control; the transgenic line OE3 only increased by 8.88% (10% PEG) and 7.73% (15% PEG), and its drought resistance was lower than that of OE1 and OE2. The above results show that the AhP5CS2 gene can improve the drought resistance of tobacco seedlings.

[0098] Table 1

[0099]

[0100] (III) Application of AhP5CS2 gene in improving drought resistance of tobacco seedlings

[0101] The transgenic tobacco positive strains OE1, OE2 and OE3 obtained in Example 3 were used as test materials, and non-transgenic wild-type tobacco was used as a control (WT). Transgenic tobacco and wild-type tobacco seeds were sown in culture pots and cultured at 25°C for 10 days. Tobacco seedlings with good growth were transferred to culture pots, one plant per pot, and grown for 40 days. Transgenic and wild-type tobacco with healthy growth and consistent growth were selected for drought treatment for 10 days.

[0102] Observe the growth of each tobacco strain. Figure 7 As shown, transgenic positive plants have a drought-resistant phenotype at the seedling stage. Under normal conditions, transgenic and non-transgenic tobacco grow in the same way, but after 10 days of drought treatment, non-transgenic tobacco wilts severely, but transgenic tobacco wilts less, with the degree of wilting WT>OE3>OE2>OE1.

[0103] Effects of drought stress on proline and soluble sugar in transgenic tobacco lines

[0104] The transgenic tobacco positive strains OE1, OE2 and OE3 obtained in Example 3 were used as test materials, and non-transgenic wild-type tobacco was used as a control (WT). Tobacco seeds were disinfected with 70% ethanol and sodium hypochlorite, and then evenly spread on MS solid culture medium containing different concentrations of PEG. They were treated in the dark at 4°C for 3 days, placed in a long-day incubator at 25°C and cultured upright for 10 days, and the materials were taken for the determination of proline and soluble sugar content.

[0105] The proline content was determined by:

[0106] 1. Prepare different concentrations of proline standard solution, boil in water for 30 minutes, take out and cool to room temperature, add toluene and mix well, extract the red substance, measure its OD value at 520nm, and draw a standard curve;

[0107] 2. Take 0.1g of tobacco seedlings from different treatment culture dishes in a mortar, add 3% sulfosalicylic acid and grind them into a homogenate, then transfer them into a stoppered test tube;

[0108] 3. Place the test tube in a boiling water bath for 15 minutes and filter;

[0109] 4. Take out 2 mL of the filtrate and transfer it into a stoppered test tube. Add 2 mL of distilled water, 4 mL of acidic ninhydrin and 2 mL of glacial acetic acid in sequence, mix and shake well, and then boil in a boiling water bath for 2 hours.

[0110] 5. After 2 hours, take out the filtrate, cool it to room temperature, add 4 mL of toluene respectively, shake well and extract;

[0111] 6. After standing for 10 minutes, aspirate the supernatant and measure the absorbance at 520 nm.

[0112] The method for determining the soluble sugar content is: using the soluble sugar determination kit (KT-1-Y) of Suzhou Keming Biotechnology Co., Ltd. to determine the soluble sugar in tobacco.

[0113] The test results are as follows Figure 8 As shown in Table 2, Figure 8 Figures A, B and Table 2 all indicate that proline and soluble sugars accumulated rapidly in transgenic positive tobacco plants under drought stress, which were significantly higher than those in the control. The differences in transgenic lines OE1 and OE2 were more significant than those in OE3, indicating that transgenic tobacco alleviated the damage caused by drought stress and osmotic stress to plants by increasing the content of proline and soluble sugars, thereby enhancing plant drought resistance.

[0114] Table 2

[0115]

[0116] The above is only a preferred embodiment of the present invention, and does not limit the present invention in other forms. Any technician familiar with the profession may use the above disclosed technical content to change or modify it into an equivalent embodiment with equivalent changes. However, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the technical solution of the present invention still belongs to the protection scope of the technical solution of the present invention.

Claims

1. Peanuts AhP5CS2 The application of the gene in improving plant stress resistance is characterized in that: The peanut AhP5CS2 The nucleic acid sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2; the stress resistance is resistance to drought stress; and the plant is tobacco.

2. Contains Peanuts AhP5CS2 A recombinant vector, recombinant bacteria, expression cassette or transgenic cell line of a gene, characterized in that: The peanut AhP5CS2 The nucleic acid sequence of the gene is shown in SEQ ID NO:

1.

3. A method for improving plant stress resistance, characterized in that: Peanuts AhP5CS2 The coding region sequence of the gene is constructed into a plant expression vector, and the plant cells are transformed to express it in the plant to improve the stress resistance of the plant; the peanut AhP5CS2 The nucleic acid sequence of the gene is shown in SEQ ID NO: 1; the stress resistance is resistance to drought stress; and the plant is tobacco.

4. Peanuts AhP5CS2 The use of a gene for increasing the content of proline and soluble sugar in plants under drought stress conditions, characterized in that: The peanut AhP5CS2 The nucleic acid sequence of the gene is shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO: 2; and the plant is tobacco.

5. A method for increasing the content of proline and soluble sugar in plants under drought stress conditions, characterized in that: Peanuts AhP5CS2 The coding region sequence of the gene is constructed into a plant expression vector, and the plant cells are transformed to make it expressed in the plant to increase the proline and soluble sugar content in the plant; the peanut AhP5CS2 The nucleic acid sequence of the gene is shown in SEQ ID NO: 1; the plant is tobacco.

Images