Peanut AhBADH1 gene and its application

Abstract

The invention discloses a peanut AhBADH1 gene and its application, and belongs to the field of gene engineering technology. The AhBADH1 gene of the invention has a sequence as shown in SEQ ID NO: 1, and is isolated from peanut; the gene can improve the salt tolerance of plants in the germination and seedling stages, and plays a role in the synthesis and regulation of betaine and proline; the proline and betaine content in plants transformed with the AhBADH1 gene will increase rapidly under salt stress, thereby effectively alleviating the damage caused by salt stress to plants. Therefore, the AhBADH1 gene of the invention has potential application value in terms of plant salt tolerance.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to peanut AhBADH1 gene and application thereof. Background Art

[0002] Salt stress can cause high osmotic potential and ion toxicity (Na + and Cl - ), resulting in weakened seed vitality, reduced germination, prolonged germination time, or even complete inhibition. Planting crops in saline-alkali land usually faces this technical difficulty. Improving the stress resistance of crop varieties through stress breeding and genetic transformation technology is currently the two effective ways to alleviate salt stress and increase the emergence rate. However, traditional conventional hybrid breeding has a long cycle, low efficiency, and slow development; and although the mutation frequency of induced breeding is greatly increased, the frequency of beneficial mutations is low, the direction of mutation is difficult to grasp, and the toxicity of chemical mutagens is relatively large and has a residual effect. Genetic transformation to improve crop quality is directional and efficient, but the current salt-tolerant genes are rarely mined, and the results of transgenic technology are low; therefore, the development of new stress-resistant genes can provide genetic resources and technical support for the use of transgenic technology to directional improve crop emergence and stress resistance traits. Summary of the invention

[0003] The present invention isolates and obtains AhBADH1 gene from peanut, and its CDS sequence 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 has been proven to improve the salt tolerance of plants during the germination and seedling stages, and plays a role in the regulation of the synthesis of betaine and proline. Based on the above content, the present invention proposes the following technical solutions:

[0004] Application of AhBADH1 gene in improving plant salt tolerance.

[0005] In the above applications, the AhBADH1 gene can be prepared into a recombinant expression vector, an expression cassette, a transgenic cell line, a recombinant bacterium or a recombinant virus to improve the salt tolerance of plants.

[0006] A method for improving the salt tolerance of plants is to construct the AhBADH1 gene into an expression vector to form a recombinant expression vector, then transform the recombinant expression vector into a strain to obtain a recombinant strain carrying the AhBADH1 gene, then infect plant leaves with the recombinant strain to make the plant carry the AhBADH1 gene, and finally regulate the salt tolerance of the plant through the expression of the AhBADH1 gene. Through this method, a transgenic plant with a tolerance degree under salt stress greater than that of the wild type of the target plant can be finally obtained.

[0007] The above recombinant expression vector, in addition to the AhBADH1 gene, also includes the following cloning vectors, but is not limited to the following vectors: binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment; for example, pROKII, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc.;

[0008] The above-mentioned recombinant expression vector may also contain the 3' non-translated region of the foreign gene, i.e., a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylation to the 3' end of the mRNA precursor, such as the non-translated region transcribed at the 3' end of Agrobacterium crown gall induction (Ti) plasmid genes (such as rouge synthase Nos gene) and plant genes (such as soybean storage protein gene) all have similar functions;

[0009] When the AhBADH1 gene is used to construct a recombinant expression vector, any enhancing promoter, such as the cauliflower mosaic virus (CAMV) 35S promoter, the corn ubiquitin promoter (Ubiquitin), a constitutive promoter, or a tissue-specific expression promoter, such as a seed-specific expression promoter, or an inducible promoter, can be added before the transcription start nucleotide; these promoters can be used alone or in combination with other plant promoters;

[0010] In addition, when the AhBADH1 gene is used to construct a recombinant expression vector, an enhancer may be used, including a translation enhancer or a transcription enhancer. The enhancer region may be the ATG start codon or the start codon of the adjacent region, etc., but must be the same as the reading frame of the coding sequence to ensure the correct translation of the entire sequence.

[0011] The sources of the above-mentioned translation control signals and start codons are wide-ranging and can be natural or synthetic; the translation start region can be derived from the transcription start region or the structural gene; in order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (selective marker genes such as GUS gene and luciferase gene), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methatrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. If the safety of transgenic plants is considered, no selective marker gene can be added and the transformed plants can be directly screened by phenotypic traits.

[0012] The plant expression vector carrying the AhBADH1 gene of the present invention can be used to transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, etc., and the transformed plant tissues can be cultivated into plants. The transformed plant host can be monocotyledonous plants such as wheat, corn, and rice, or dicotyledonous plants such as tobacco, Arabidopsis, soybean, rapeseed, poplar, and alfalfa.

[0013] The strain used to infect plants can be selected from Agrobacterium tumefaciens (such as GV3101, LBA4404 and EHA105), Agrobacterium rhizogenes and the like.

[0014] Specifically, the AhBADH1 gene improves the salt tolerance of plants during the germination period. Furthermore, under salt stress, plants transformed with the AhBADH1 gene have faster germination rate and higher germination rate during the germination period compared with the wild type.

[0015] Specifically, the AhBADH1 gene improves the salt tolerance of plants at the seedling stage. Further, under salt stress, plants transformed with the AhBADH1 gene have longer roots, higher fresh weight, better growth, and better salt tolerance at the seedling stage compared with wild-type plants.

[0016] The present invention also provides the use of the AhBADH1 gene in regulating betaine synthesis in plants. Further, the "regulation" means that under salt stress, the expression level of betaine in plants transformed with the AhBADH1 gene is significantly increased compared with the wild type.

[0017] The present invention also provides the use of the AhBADH1 gene in regulating proline synthesis in plants. Further, the "regulation" means that under salt stress, the expression level of proline in plants transformed with the AhBADH1 gene is significantly increased compared with the wild type.

[0018] In further research, we found that under normal conditions, betaine does not affect seed germination, but under salt stress conditions, betaine can increase the germination rate of seeds. Based on this, the present invention provides the use of betaine in increasing the germination rate of plant seeds under salt stress. Specifically, the concentration of betaine is selected from 0 to 15 mM, preferably 10 mM.

[0019] The beneficial effects of the present invention are:

[0020] The present invention found in the salt stress response test that the AhBADH1 gene isolated from peanut can improve the salt tolerance of plants in the germination and seedling stages, and plays a role in the synthesis and regulation of betaine and proline. The proline and betaine content in plants transformed with the AhBADH1 gene will increase rapidly under salt stress, thereby effectively alleviating the damage caused by salt stress to plants. The present invention aims to provide gene resources for the targeted improvement of crop emergence and stress resistance traits using transgenic technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Figure 1 is a vector construction diagram; wherein, band A is a schematic diagram of the cloned target gene AhBADH1, which is 1512 bp in size; band B is a schematic diagram of the restriction results of the positive clone plasmid obtained by connecting the target gene to the pMD19-T1simple cloning vector; and band C is a schematic diagram of the restriction results of the target gene connected to the pBI121 expression vector plasmid;

[0022] Figure 2 For identification of positive transgenic lines; Lane 1 is the wild type WT, and the remaining lanes are transgenic lines;

[0023] Figure 3 Figure 1 is the phenotype of the germination period; Figure A is the phenotype of the transgenic line 35S:AhBADH1 and the wild type WT grown on culture dishes with different NaCl concentrations for 10 days; Figure B is the statistical graph of the germination rate corresponding to Figure A;

[0024] Figure 4 Figure 1 is the seedling phenotype; Figure A is the phenotype of the transgenic line 35S:AhBADH1 and the wild type WT germinated on culture dishes with different NaCl concentrations for 21 days; Figure B is the root length statistics corresponding to Figure A; Figure C is the fresh weight statistics corresponding to Figure A;

[0025] Figure 5Figure 1 is a graph of betaine and proline content; Figure A is the betaine content of the 21-day transgenic line 35S:AhBADH1 and the wild-type WT at different NaCl concentrations; Figure B is the proline content of the 21-day transgenic line 35S:AhBADH1 and the wild-type WT at different NaCl concentrations;

[0026] Figure 6 Figure 3 shows the effect of exogenous application of betaine on peanut seed germination under salt stress; Figure A shows the germination rate of peanut seeds under 0 mM NaCl with different concentrations of betaine; Figure B shows the germination rate of peanut seeds under 50 mM NaCl with different concentrations of betaine; Figure C shows the germination rate of peanut seeds under 100 mM NaCl with different concentrations of betaine; Figure D shows the germination rate of peanut seeds under 150 mM NaCl with different concentrations of betaine. DETAILED DESCRIPTION

[0027] The present invention uses peanut seeds (Tifrunner variety) as experimental materials, starts from transcriptomics analysis, searches for key regulatory factors that respond to high salt stress, and identifies a total of 256 differentially expressed genes. Among them, the Arachis hypogaea BetaineAldehyde Dehydrogenase 1 (arahy.59SDUF) gene encodes betaine aldehyde dehydrogenase (a key enzyme for synthesizing betaine), which is significantly upregulated by 50 times by salt induction. The present invention names it AhBADH1. The AhBADH1 gene was found from the peanut genome database website Peanutbase, and its CDS sequence is 1512bp, as shown in SEQ ID NO:1; a dehydrogenase encoding 503 amino acids, and its amino acid sequence is shown in SEQ ID NO:2.

[0028] SEQ ID NO: 1:

[0029]

[0030] SEQ ID NO:2:

[0031] MAISTPRRELFIDGEWKAPLLNNRIPIINPSTELIIGDIPAATKEDVELAVDAARRALSRNKGKDWSTASGSLRATYLRAIAAKITEKKDELAKLEATDNGKPLDEAAADMDDVIGCFSYAELA EGLDAKQKAHVSLPMENFKSYVIKEPIGVVALITPWNYPLLMAAWKVGAALAAGCTAVLKPSELASVTCLELAEICKEVGLPAGVLNVITGLGTEAGAPLASHPDVDKISFTGSSATGSKIMQAAA LQVKPVSLELGGKSPIIVFDDVDIDKVAEWTMLGCFFTNGQICSATSRLIVHENIATKFLNRLVEWAKNIKVSDPLEDGCKLGAIVSEAQYQKVLKFISTAKSEGATILTGGSRPENLKKGYFVEP TIITDVSTSMQIWREEVFGPVLCAKTFSSEEEAIELANDTHYGLGSAVMSNDPERCERISKAVQAGIVWINCSQPTFIQAPWGGIKRSGFGRELGEWGLDNFLSVKQVTKYISDEQWGWYKCPSKM

[0032] The steps for constructing Arabidopsis transgenic lines using AhBADH1 gene are as follows:

[0033] (I) Construction of recombinant expression vector

[0034] Primers were designed based on the CDS sequence of the peanut AhBADH1 gene. The primer sequences are as follows:

[0035] Upstream primer 5'- GGTACC ATGGCGATCTCAACACCAAGAAGAG-3' (SEQ ID NO: 3);

[0036] Downstream primer 5'- GTCGAC TTACATCTTTGAAGGACACTTATACCAGCC-3' (SEQ ID NO: 4).

[0037] In the above primer sequences, the underlined parts are restriction sites, the restriction site of the upstream primer is BamHI, and the restriction site of the downstream primer is SalI.

[0038] 1. RNA extraction

[0039] Take 0.1g of fresh peanut tissue material and grind it into powder in liquid nitrogen, add 450μL RL (β-mercaptoethanol needs to be added in advance), vortex and mix, and place at room temperature for 1-3min. Transfer the solution to the filter column CS (the filter column CS is placed in the collection tube), centrifuge at 12000rpm for 2-5min, carefully pipette the supernatant in the collection tube into the RNase-Free centrifuge tube, and try to avoid the tip from contacting the cell debris precipitate in the collection tube. Slowly add 0.5 times the volume of supernatant anhydrous ethanol, mix well, transfer all the solution to the adsorption column CR3, centrifuge at 12000rpm for 30-60s, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. Add 350μL of deproteinization solution RW1 to the adsorption column CR3, centrifuge at 12000rpm for 30-60s, pour out the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. Add 80 μL of DNase I working solution to the center of adsorption column CR3 and place at room temperature for 15 minutes. Add 350 μL of deproteinization solution RW1 to adsorption column CR3, centrifuge at 12000rpm for 30-60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. Add 500 μL of rinse solution RW (ethanol needs to be added in advance) to adsorption column CR3, let stand at room temperature for 2 minutes, centrifuge at 12000rpm for 30-60 seconds, discard the waste liquid in the collection tube, and put the adsorption column CR3 back into the collection tube. Repeat the above steps once. Centrifuge at 12000rpm for 2 minutes and discard the waste liquid. Place the adsorption column CR3 at room temperature for 5-10 minutes to thoroughly dry the residual rinse solution in the adsorption material. 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 12000 rpm for 2 minutes to obtain RNA extraction solution.

[0040] 2. Synthesis of the first strand of reverse transcribed cDNA

[0041] RNA concentration was determined and then purified using Novozyme III All-in-one RT SuperMix Perfectfor qPCR for reverse transcription. Take 1μg of total RNA, add 4μL 5×All-in-one qRT SuperMix, 1μL EnzymeMix, add water to 20μL, react at 50℃ for 15min, and inactivate at 85℃ for 5s.

[0042] 3. Cloning of AhBADH1 gene

[0043] Using Novozyme Max Super-Fidelity DNA Polymerase high-fidelity enzyme was used for amplification, and the reaction system was: 25 μL 2×Phanta Max Buffer, 1 μL dNTP Mix (10 mM each), 2 μL upstream primer, 2 μL downstream primer, 1 μL Phanta Max Super-Fidelity DNA Polymerase, 1 μL cDNA template, and water was added to 50 μL.

[0044] The reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, for a total of 35 cycles; post-extension at 72°C for 5 min.

[0045] After the reaction is completed, agarose gel electrophoresis is performed and the target band is detected. Figure 1 As shown in A, the gel was cut and gel recovered. The gel recovery method was carried out according to the ordinary agarose gel DNA recovery kit (DP209) of Tiangen Company. The ends of the products amplified by high-fidelity enzymes are blunt ends and need to be tailed. The reaction system is as follows: 1.5μL 10× reaction buffer, 1.2μL dNTP, 0.15μL Taq enzyme, and 15μL of gel recovery product was added. Reaction at 72℃ for 30min. The sequence was completed by Sangon Biotechnology (Shanghai) Co., Ltd.

[0046] 4. Obtaining positive plasmid

[0047] Take 4 μL of the tailed product and connect it with the pMD19-T simple cloning vector, connect 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, culture at 37°C for 4 to 6 hours, perform bacterial liquid PCR, select positive colonies and culture overnight in LB liquid culture medium. Use Tiangen plasmid extraction kit to extract plasmid DNA and obtain AhBADH1-pMD19-T simple vector plasmid.

[0048] 5. Construction of expression vector

[0049] AhBADH1-pMD19-T simple and the empty vector pBI121 with 35S were digested with BamHI and SalI at 37℃ for 1h, and then subjected to agarose gel electrophoresis. The correct bands were removed and recovered from the gel. Figure 1As shown in B. Thermo's T4 DNA ligase was used for overnight ligation, and the ligation product was transformed into DH5α strain and grown overnight on LB plates containing kanamycin. A single white colony was picked and added to LB culture medium. After culturing at 37°C for 4 to 6 hours, PCR was performed on the culture medium. Positive colonies were selected and cultured in LB liquid medium overnight. Plasmid DNA was extracted using the Tiangen Plasmid Extraction Kit, and BamHI and SalI enzymes were used for identification. Figure 1 As shown in C. The overexpression vector of 35S:AhBADH1 was constructed.

[0050] (II) Transformation of Agrobacterium

[0051] Take 3 μL of plasmid and transform Agrobacterium EHA105 by electroporation method. The operation is as follows:

[0052] The electrode spacing is 0.1 cm. After sterilization, insert the electroporation cuvettes into crushed ice, compact the ice surface, and let it stand in the ice for 5 minutes to fully cool the electroporation cup. Take the Agrobacterium competent state stored at -70℃ and insert it into ice for 5-10 minutes. After it melts, add 3μL plasmid DNA and immediately insert it into ice. In the clean bench, use a sterile pipette to quickly transfer the competent state-plasmid mixture to the electroporation cup, cover the cup lid, and keep the empty tube for standby use. Start the electroporator and set the electroporation parameters: C=25μF, PC=200ohm, V=2400V. Wipe off the moisture on the outside of the electroporation cup with a paper towel, and quickly put the electroporation cup into the electroporation tank for the electroporation step. After the electroporation is completed, quickly insert the electroporation cup into ice, add 700μLYEP and transfer it to the original competent empty tube, and shake and culture at 28℃, 150-200rpm for 2-3h. Centrifuge and discard the supernatant, take the precipitate and spread it on the YEP plate containing kanamycin and rifampicin, and invert it in a 28℃ incubator for 2-3 days. Pick a single colony to the YEP culture medium, culture it at 28℃ for 4-6 hours, and then perform PCR on the culture medium. Select 3-4 positive colonies, add about 20mL of YEP (kanamycin plus rifampicin) to the culture medium, and culture it overnight. Obtain the Agrobacterium culture containing AhBADH1.

[0053] (III) Agrobacterium-mediated inflorescence infection and screening of positive transgenic Arabidopsis seedlings overexpressing AhBADH1

[0054] 1. Infection of Arabidopsis inflorescence

[0055] Infection solution formula (10mL system): sucrose 0.5g; Silwet (toxic, protect from light) 3.0μL; ddH2O dilute to 10mL.

[0056] Infection steps:

[0057] Pipette 50μL AhBADH1 Agrobacterium culture solution into a conical flask containing 50mL YEP culture solution, and culture overnight in a 28℃ incubator with shaking. Centrifuge at 5000rpm for 5min at room temperature, collect the bacteria 2-3 times, and resuspend the bacteria in 10mL infection solution. At around 10-11 am, immerse the opened flowers in the infection solution for 6-7s. Keep in the dark and wrap them in a wet state for 24h. After an interval of 5-7d, perform a second infection to improve the infection efficiency.

[0058] 2. Screening of transgenic positive seedlings

[0059] The seeds of Arabidopsis thaliana after infection are the T0 generation. The T0 generation seeds are scattered on a culture medium containing kanamycin and cultured, and the green seedlings are selected as potential positive strains. PCR identification is performed, and individual plants are harvested to obtain the T1 generation. The T1 generation seeds are scattered on a culture medium containing kanamycin and cultured, and the green seedlings in the strain with a ratio of green seedlings to yellow seedlings of approximately 3:1 are selected for reproduction, and individual plants are harvested to obtain the T2 generation. The T2 generation seeds are scattered on a culture medium containing kanamycin, and strains with all green seedlings are selected for reproduction, thus obtaining homozygous transgenic plants, extracting the genome of the transgenic strain, detecting the transgenic strain with RT-PCR, and selecting strains with high expression levels for further experiments. The experimental results are as follows. Figure 2 As shown, a total of 5 AhBADH1 overexpressing transgenic lines were obtained. RT-PCR detection showed that the expression levels of transgenic materials OE1, OE2, OE3 and OE5 were higher than those of the control, among which OE3 and OE5 had higher expression levels and were used as subsequent experimental materials.

[0060] Other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those of ordinary skill in the art. 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.

[0061] Example 1

[0062] Transgenic lines have salt tolerance phenotype:

[0063] 1. Germination phenotype of transgenic Arabidopsis thaliana overexpressing AhBADH1

[0064] Through RT-PCR detection and analysis, transgenic positive lines OE3 and OE5 with high AhBADH1 expression were selected as experimental materials. Arabidopsis seeds were disinfected with freshly prepared 70% ethanol for 5 minutes, centrifuged and discarded the supernatant, then disinfected with 2.6% sodium hypochlorite for 10 minutes, centrifuged and discarded the clean supernatant, and finally rinsed with sterile ddH2O containing Tween-20 for 5 to 6 times, and the sterilized sterile seeds were neatly spread on 1 / 2MS solid culture medium containing different concentrations of NaCl. Treated in the dark at 4℃ for 3 days, placed in a long-day incubator at 22℃ for 10 days, and counted every 12 hours for germination. Seeds breaking through the seed coat were considered to have germinated, and the germination rate of Arabidopsis seeds was counted every day. All experiments were repeated three times.

[0065] The experimental results are as follows Figure 3 As shown:

[0066] Figure 3 A. Figure 3 B can indicate that the transgenic positive plants have a salt-tolerant phenotype during the germination period. In 1 / 2MS medium, there was no difference in the germination rate between transgenic Arabidopsis seeds and the control, but when grown on a medium containing NaCl, there was a significant difference: under 150mM NaCl conditions, the germination rate of WT was about 82%, and the germination rates of OE3 and OE5 reached 98% and 91%, respectively; under 200mM NaCl conditions, the germination rate of WT was about 54%, and the germination rates of OE3 and OE5 reached 84% and 66%, respectively. The seed germination rates of the two overexpression lines were significantly higher than those of the control, and OE3 had a higher salt tolerance than OE5, germinating faster and with a higher germination rate, showing a higher tolerance to salt stress.

[0067] 2. Seedling phenotype of transgenic Arabidopsis thaliana overexpressing AhBADH1

[0068] The transgenic Arabidopsis seeds overexpressing AhBADH1 were sterilized with 70% ethanol and sodium hypochlorite, and then neatly dotted on 1 / 2MS solid medium containing different concentrations of NaCl. They were kept in the dark at 4°C for 3 days, placed in a long-day incubator at 22°C and cultured upright for 21 days. The root length and fresh weight of the Arabidopsis strains on different culture dishes were counted. All experiments were repeated 3 times.

[0069] The experimental results are shown in Table 1 and Figure 4 As shown:

[0070] Table 1 Figure 4 A. Figure 4 B. Figure 4C can indicate that the transgenic positive plants have a salt-tolerant phenotype at the seedling stage. In 1 / 2MS medium, the growth of transgenic Arabidopsis seeds was basically the same as that of the control, but under 150mM NaCl conditions, the root length of OE3 and OE5 was 65.12% and 68.11% higher than that of the control, and the fresh weight was 12.81% and 9.92% higher than that of the control; under 200mM NaCl conditions, the root length of OE3 and OE5 was 110.52% and 46.32% higher than that of the control, and the fresh weight was 10.02% and 5.30% higher than that of the control; the salt tolerance of the transgenic line OE3 at the seedling stage was significantly higher than that of OE5.

[0071] Table 1

[0072]

[0073] Example 2

[0074] Salt stress induces betaine and proline accumulation in transgenic lines:

[0075] The transgenic Arabidopsis seeds overexpressing AhBADH1 were sterilized with 70% ethanol and sodium hypochlorite, and then evenly spread on 1 / 2MS solid medium containing different concentrations of NaCl. They were kept in the dark at 4°C for 3 days, placed in a long-day incubator at 22°C and cultured vertically for 21 days, and then the samples were used for the determination of betaine and proline content.

[0076] 1. Determination of betaine content

[0077] First, prepare different concentrations of betaine standard solution, measure the OD value at 525nm, and draw a standard curve. Take 0.1g of Arabidopsis seedlings from different treatment dishes and place them in a mortar with distilled water and grind them into a homogenate. Place them in a 23℃ shaker for 20h, centrifuge for 15min, take the supernatant and adjust the pH to 1.0 with concentrated hydrochloric acid. Store in a 4℃ refrigerator for 15min, add 5mL of 15mg / mL Reinecke salt, and let stand at 4℃ for 1h. Centrifuge at 10000rpm for 15min, discard the supernatant, add 5mL of ether, shake well, and centrifuge again. Let stand for 10min to allow the ether to evaporate completely, and add 5mL of 70% acetone. Use 70% acetone as a blank control and measure the absorbance value at 525nm.

[0078] 2. Determination of proline content

[0079] First, prepare different concentrations of proline standard solution, boil in water bath for 30 minutes, take out and cool to room temperature, add toluene to mix and extract the red substance, measure the OD value at 520nm, and draw a standard curve. Take 0.1g of Arabidopsis seedlings on different treatment dishes in a mortar, add 3% sulfosalicylic acid to fully grind into a homogenate, and transfer to a stoppered test tube. Put the test tube in a boiling water bath to react for 15 minutes and filter. Take out 2mL of the filtrate and transfer it to a stoppered test tube, add 2mL of distilled water, 4mL of acidic ninhydrin and 2mL of glacial acetic acid in turn, mix and shake, and then boil in water bath for 2h. After 2h, take out the filtrate, cool to room temperature, add 4mL of toluene respectively, shake and extract. After standing for 10min, aspirate the supernatant and measure the absorbance value at 520nm.

[0080] The experimental results are shown in Table 2 and Figure 5 As shown:

[0081] Table 2 Figure 5 A. Figure 5 B can indicate that betaine and proline accumulated rapidly in transgenic positive plants under salt stress, which was significantly higher than that in the control. The difference between transgenic line OE3 and OE5 was more significant. These two osmotic regulating substances can effectively alleviate the damage caused by salt stress to plants and enhance plant salt tolerance.

[0082] Table 2

[0083]

[0084]

[0085] Example 3

[0086] External application of betaine improves the germination rate of peanut seeds under salt stress:

[0087] Peanut seeds (Tifrunner variety) were germinated on filter paper plates containing 1 / 10 hogland culture medium with different concentrations of betaine (0mM, 5mM, 10mM, 15mM) and different concentrations of NaCl (0mM, 50mM, 100mM, 150mM) for 10 days, and the germination rate was counted. Germination was considered when 3mm white tip was exposed. All experiments were repeated three times.

[0088] Germination rate (%) = (number of germinated seeds / total number of peanut seeds tested) × 100%.

[0089] The experimental results are as follows Figure 6 As shown:

[0090] Under normal conditions, betaine does not affect peanut seed germination ( Figure 6A), with the increase of salt concentration, the degree of inhibition of peanut seed germination is more significant, but betaine can promote peanut seed germination and improve the germination rate under different salt concentrations, and the promotion effect shows a trend of first increasing and then decreasing. Among them, 10mM betaine is the most suitable betaine concentration for improving the salt tolerance of peanut during germination period, and the differences between different treatments under 100mM NaCl and 150mM NaCl salt concentrations are more significant ( Figure 6 B. Figure 6 C. Figure 6 D). 10 mM betaine can increase the germination rate of peanut seeds by 7.50% and 16.03% under 100 mM NaCl and 150 mM NaCl, respectively.

[0091] 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. A method for improving the salt tolerance of Arabidopsis thaliana, characterized in that: Yes AhBADH1 The gene is constructed into an expression vector to form a recombinant expression vector, and then the recombinant expression vector is transformed into a strain to obtain a strain carrying the gene. AhBADH1 The recombinant strain of the gene was then used to infect plant leaves, making Arabidopsis thaliana carry the gene AhBADH1 genes, and ultimately through AhBADH1 Gene expression to improve salt tolerance in Arabidopsis; Said AhBADH1 The nucleic acid sequence of the gene is shown in SEQ ID NO:

1.

2. The method according to claim 1, characterized in that The strain is selected from Agrobacterium tumefaciens or Agrobacterium rhizogenes.

3. The method according to claim 1, characterized in that The recombinant expression vector comprises the following cloning vectors: binary Agrobacterium vectors or vectors used for plant microprojectile bombardment.

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