CN103451215B - Barbadosnut gene, recombinant plasmid and application thereof for improving salt resistance and drought tolerance of plants - Google Patents

Abstract

The invention belongs to the field of plant genetic engineering and provides a gene JcSnRK2 cloned from Jatropha curcas, a eukaryotic recombinant plasmid containing the gene and a plant transformant. Experiments show that the expression of the JcSnRK2 gene can promote Arabidopsis SnRK2. The root growth of 8-deficient plants can significantly improve their high salt tolerance and drought resistance, reduce the water loss rate of leaves, and stimulate the expression of downstream genes that respond to osmotic stress. Therefore, the JcSnRK2 gene and the eukaryotic recombinant plasmid of the present invention can be used in improving the salt resistance and drought tolerance of plants.

Description

A kind of Jatropha curcas gene, recombinant plasmid and application in improving plant salt resistance and drought tolerance

technical field

The invention belongs to the field of plant genetic engineering, in particular to a gene cloned from Jatropha curcas, a eukaryotic recombinant plasmid containing the gene and application thereof.

Background technique

During the growth and development of plants, they often encounter various environmental stresses, such as drought, salinity, high temperature or cold, oxidation, nutrient deficiency, pathogen invasion, etc., especially the global water shortage and nearly half of the agricultural irrigated land Affected by high-salt stress, crop yields decrease or even cause plant death. In recent years, artificial breeding through genetic engineering has become the main biological means to endow plants with stress tolerance.

SnRK2 (sucrose non-fermenting1-related protein kinase2) is closely related to the adversity stress response of plants. At present, Arabidopsis and rice have relatively comprehensive understanding, each of which has 10 family members. The former Named SnRK2.1～SnRK2.10 (Hrabak EM, et al., 2003, The Arabidopsis CDPK–SnRK superfamily of protein kinases, Plant Physiology, 132, 666–680), the latter named SAPK1～SAPK10 (Kobayashi Y, et al. al., 2004, Differential activation of the rice sucrose nonfermenting1-relatedprotein kinase2family by hyperosmotic stress and abscisic acid, The Plant Cell, 16(5):1163-1177). Recently, there are also analyzes on the function of wheat TaSnRK2.4 and maize ZmSAPK8 genes, and their overexpression in Arabidopsis can improve the various stress resistance of transgenic plants (Mao XG, et al., 2010, TaSnRK2.4, anSNF1 -type serine/threonine protein kinase of wheat (Triticum aestivum L.), confers enhanced multistress tolerance in Arabidopsis, Journal of Experimental Botany, 61(3):683–696; Ying S, et al., 2011, Cloning and characterization of a maize SnRK2protein kinase gene confers enhancedsalt tolerance in transgenic Arabidopsis, Plant Cell Rep, 30:1683–1699). Although the SnRK2 family has received attention and research, it has rarely been studied in woody plants, and it has not been reported in the study of Jatropha curcas L. Screening more SnRK2 genes through cloning technology plays an important role in improving plant resistance and cultivating plants that can still grow normally under high-salt and drought conditions.

Contents of the invention

One of purpose of the present invention is to provide a kind of gene that obtains from Jatropha curcas clone, eukaryotic recombinant plasmid containing said gene and plant transformant, two of purpose of the present invention is described gene and eukaryotic recombinant plasmid in It can be used in improving plant salt resistance and drought tolerance to cultivate plants with high salt and drought resistance.

Technical scheme of the present invention is as follows:

Use the conserved sequence of the existing herbaceous SnRK2 gene to design degenerate primers for amplifying the middle fragment, perform 5'Race and 3'Race according to the obtained middle fragment sequence to obtain the upstream and downstream sequences, and then design specific primers based on the spliced sequence to The full-length gene was obtained by RT-PCR amplification using the total RNA of mad tree leaves as a template, which was named JcSnRK2. Sequence analysis shows that the mRNA of the JcSnRK2 gene contains an open reading frame of 1017 bases, its nucleotide sequence is shown in SEQ ID No: 1 in the sequence listing, and its genomic DNA nucleotide sequence is shown in SEQ ID No in the sequence listing As shown in No: 2, the amino acid sequence of the polypeptide encoded by the JcSnRK2 gene is shown in SEQ ID No: 3 in the sequence listing. Bioinformatics analysis and gene expression pattern analysis showed that JcSnRK2 gene belongs to the SnRK2 family member.

The eukaryotic recombinant plasmid of the present invention is composed of the nucleotide sequence shown in SEQ ID No: 1 in the sequence table and the eukaryotic expression vector, and the nucleotide sequence shown in SEQ ID No: 1 in the sequence table is inserted into the true The Xba I and BamH I double restriction sites of the nuclear expression vector, the eukaryotic expression vector is pBI121, pBI221 or pBin438.

The plant transformant of the present invention is formed by introducing the above-mentioned eukaryotic recombinant plasmid into Arabidopsis thaliana SnRK2.8-deficient plants through the method of Agrobacterium infection inflorescence, and undergoes kanamycin resistance screening, GUS staining and PCR identification to obtain Transgenic Arabidopsis seeds with stable expression of JcSnRK2 gene. Experiments have shown that the expression of JcSnRK2 gene can promote the root growth of Arabidopsis SnRK2.8-deleted plants, significantly improve its high salt tolerance and drought resistance, reduce the water loss rate of leaves, and stimulate the expression of downstream genes that respond to osmotic stress. Therefore, the JcSnRK2 gene and the eukaryotic recombinant plasmid of the present invention can be used in improving the salt resistance and drought tolerance of plants.

The present invention has the following beneficial effects:

1. The present invention has cloned a new Jatropha curcas gene JcSnRK2 by the Race method, constructs a eukaryotic recombinant plasmid with the gene and a eukaryotic expression vector, and provides a new eukaryotic recombination for improving salt resistance and drought tolerance of plants plasmid.

2. The present invention proves through experiments that the eukaryotic recombinant plasmid can improve the ability of plants to withstand high salt and drought, can reduce the water loss rate of leaves, and can stimulate the expression of downstream genes that respond to osmotic stress. Therefore, the recombinant Plasmids are used in artificial resistance breeding to produce plants that are resistant to high salinity and drought.

Description of drawings

Fig. 1 is the agarose gel electrophoresis image of the total RNA of Jatropha curcas leaves in Example 1. In the figure, lane M: molecular weight marker (AL2000DNAMarker, purchased from Adelaide Company), lane 1: total RNA of Jatropha curcas leaves.

Fig. 2 is the electrophoresis diagram of PCR amplification of the middle fragment of JcSnRK2 in Example 1. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), lane 1: amplification result of the middle fragment of JcSnRK2.

3 is an electrophoresis image of the downstream fragment of the 3'Race amplified JcSnRK2 gene in Example 1. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), lane 1: PCR amplification result of the downstream fragment of JcSnRK2.

4 is an electrophoresis image of the upstream fragment of the 5'Race amplified JcSnRK2 gene in Example 1. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), lane 1: PCR amplification result of the upstream fragment of JcSnRK2.

5 is an electrophoresis diagram of the PCR amplification result of the JcSnRK2 gene ORF in Example 1. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), and lanes 1 and 2: PCR amplification results of the JcSnRK2 gene ORF.

6 is the electrophoresis diagram of the colony PCR result of the JcSnRK2 gene ORF in Example 1. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), and lanes 1-10: PCR amplification results of 10 transformants.

Figure 7 is the electrophoresis diagram of the PCR amplification result of JcSnRK2 genomic DNA in Example 1, in the figure, M swimming lane: molecular weight marker (AL15000DNAMarker, purchased from Adelaide Company), and swimming lanes 1 and 2 are the PCR amplification results of JcSnRK2 genomic DNA .

FIG. 8 is a sequence structure analysis diagram of JcSnRK2 genomic DNA in Example 1. FIG.

Fig. 9 is a phylogenetic tree analysis diagram of SnRK2 genes of Jatropha curcas and 6 other plants in Example 2.

FIG. 10 is a phylogenetic tree analysis diagram of Jatropha curcas JcSnRK2 and Arabidopsis SnRK2 protein families in Example 2.

Figure 11 is the amino acid sequence analysis of the polypeptide encoded by the JcSnRK2 gene in Example 2.

FIG. 12 is a graph showing tissue-specific expression analysis of the JcSnRK2 gene in Example 2. FIG.

Fig. 13 is an analysis diagram of the expression of JcSnRK2 gene under ABA treatment in Example 2.

Fig. 14 is an analysis diagram of the expression of the JcSnRK2 gene under salt stress in Example 2.

Fig. 15 is an analysis diagram of the expression of the JcSnRK2 gene under drought stress in Example 2.

Fig. 16 is an analysis diagram of the expression of JcSnRK2 gene under cold stress in Example 2.

Fig. 17 is a schematic diagram of the eukaryotic recombinant plasmid of the present invention.

Figure 18 is the electrophoresis diagram of double enzyme digestion in Example 3. In the figure, lane M: molecular weight marker (AL2000DNAMarker), lane 1: double digestion product of pBI121 plasmid XbaI and BamHI, lane 2: plasmid pBI121; lane 3: pET-JcSnRK2 Plasmid; Lane 4: XbaI and BamHI double digestion product of pET-JcSnRK2 plasmid.

Figure 19 is the PCR electrophoresis image of the pBI121-JcSnRK2 Agrobacterium colony transferred in Example 3. In the figure, lane M: molecular weight marker (AL2000 DNA Marker), lane 1-7: amplification results of 7 transformants.

Figure 20 is the GUS staining diagram of Arabidopsis thaliana transfected with JcSnRK2 gene in Example 3, wherein Figures A, B, and C are the GUS staining diagrams of seedlings, siliques and flowers, respectively.

Fig. 21 is the PCR verification electrophoresis figure of JcSnRK2 gene transgenic Arabidopsis thaliana in embodiment 3, in the figure, figure (A) is the PCR identification result of JcSnRK2 gene transgenic Arabidopsis genome, and figure (B) is the RT-PCR identification result of RNA; 1 ~ Lane 6: PCR result of transgenic Arabidopsis; Lane 7: PCR result of non-transgenic Arabidopsis.

Fig. 22 is the root growth analysis figure of snf2.8 and JK2 in embodiment 3, wherein, figure (A) is the root length condition figure of seedlings of snf2.8 and JK2 after MS culture medium grows for 1 week, figure (B) is Statistical graph of the root length of snf2.8 and JK2 seedlings grown in MS medium for 1 week. Figure (C) is a graph of root growth of snf2.8 and JK2 seedlings cultured in soil for 45 days.

Figure 23 is a statistical diagram of the germination rates of snf2.8 and JK2 in Example 3.

Figure 24 is a graph showing the growth status of snf2.8 and JK2 under salt stress in Example 3, wherein the upper row and the lower row are respectively snf2.8 and JK2 grown for 1 month on MS medium containing different concentrations of NaCl status map.

Fig. 25 is the salt resistance analysis figure of snf2.8 and JK2 plant among the embodiment 3, and wherein, figure A is the growth state figure after the seedling of 30 days of growth is stressed by 450Mm NaCl solution for 2 weeks and 1 month, and figure B is growth 45-day-old seedlings were subjected to 450Mm NaCl solution stress for 2 weeks and 1 month after the growth status chart.

Figure 26 is a graph of the growth status of snf2.8 and JK2 under drought stress in Example 3, wherein, Figure A is the general situation of snf2.8 and JK2 grown on MS medium containing different concentrations of mannitol for 1 month, Panel B shows the specific phenotypes of snf2.8 and JK2 grown on MS medium containing different concentrations of mannitol for 1 month.

Fig. 27 is the drought resistance analysis diagram of snf2.8 and JK2 plants in Example 3, wherein, the upper row and the lower row are the growth status graphs of snf2.8 and JK2 seedlings growing for 30 days and 45 days respectively after stopping water supply for 1 month .

Fig. 28 is a graph showing the detection results of water loss rate of detached leaves in Example 3.

Fig. 29 is a graph showing the detection results of the expression levels of JcSnRK2 and its downstream genes AREB1, LEA, RD29A, and RD29B under drought stress in Example 3.

Detailed ways

The present invention will be further described below in conjunction with embodiment. In the following examples, where no specific experimental conditions are indicated, all are conventional conditions well known to those skilled in the art, such as Molecular Cloning by Sambrook et al.: Laboratory Manual (NewYork: Cold Spring Harbor Laboratory Press, 1989), The conditions and experimental procedures described in the General Manual of Laboratory Animals (National Center for Laboratory Animal Regulations, November 2004): Basic Technical Guidelines, Fifth Edition (John Wiley & Sons, Inc, 2005), or the conditions and experimental procedures recommended by the manufacturer step.

In the following examples, Jatropha curcas mature seeds and Jatropha curcas flowers are collected from Shuijiao Village, Fenghuang Town, Sanya City, Hainan Province, and Jatropha curcas roots, stems, and leaves are taken from Jatropha curcas seedlings cultivated with Jatropha curcas mature seeds , Cultivation operation: sow mature Jatropha curcas seeds in flowerpots, and cultivate them in a greenhouse (30-35°C) under 24h full light conditions for one to two months.

Embodiment 1: Molecular cloning of Jatropha curcas JcSnRK2 gene

1. Extraction of Jatropha curcas leaves total RNA

The total RNA of Jatropha curcas leaves was extracted using Plant RNeasy Kit (Qiagen), and the specific steps were as follows:

1) Take Jatropha curcas leaves ≤0.1g, fully grind with liquid nitrogen, add 450 μl RLC (beta-mercaptoethanol was added in advance), and shake vigorously;

2) Transfer the liquid into a purple filter column, put it in a 2ml collection tube, centrifuge at 14,000rpm for 2min at room temperature, put the filtrate into a 1.5ml EP tube, be careful not to inhale the precipitate;

3) Add anhydrous ethanol equivalent to 1/2 volume of the filtrate, beat and mix well;

4) Transfer the sample, including the precipitate, into a pink filter column, centrifuge at 10,000rpm for 15s at room temperature, and discard the filtrate;

5) Add 700μl RW1buffer, centrifuge at 10,000rpm for 15s at room temperature, and wash the column;

6) Add 500μl RPE, centrifuge at 10,000rpm for 15s at room temperature, wash the column, and repeat this operation once;

7) Centrifuge at 14,000rpm for 1min at room temperature, discard the filtrate;

8) Put the filter column into a 1.5ml collection tube, add 30-50μl RNase-free water, and centrifuge at 10,000rpm for 1min at room temperature to obtain the total RNA of Jatropha curcas leaves.

Its agarose gel electrophoresis picture is shown in Figure 1.

2. Obtaining the middle fragment of JcSnRK2 gene

2.1Reverse Transcriptase M-MLV synthesizes the first strand of cDNA

According to the operating instructions of reverse transcriptase M-MLV (TaKaRa), the total RNA of Jatropha curcas leaves obtained in step 1 was used as a template, and Oligo(dT) ₁₈ was used as a primer.

Add to RNase-free 0.2ml PCR tube

Jatropha curcas leaves total RNA 3μl (about 500ng)

Oligo(dT) ₁₈ (50μM) 1μl

Make up the volume to 6 μl with RNase-free ddH ₂ O.

After mixing, keep warm at 70°C for 10 minutes, then quickly cool on ice for more than 2 minutes, and then centrifuge briefly to collect the solution in the tube at the bottom of the EP tube. Add the following reagents sequentially on ice: 5×M-MLV Buffer 2 μl; dNTP (10 μM) 0.5 μl; RNase Inhibitor 0.3 μl; RTase M-MLV 0.5 μl; RNase-free ddH ₂ O 0.7 μl. After mixing evenly, put it into the PCR instrument again to react at 42°C for 1h, and at 70°C for 10min, and store it at -20°C for later use.

The sequence of primer Oligo(dT) ₁₈ is: 5′-GCTGTCAACGATACGCTACGTAACGGCATGACAGTG(T) ₁₈ -3′

2.2PCR

Add the following components to a 0.2ml EP tube on ice:

Cycling conditions: 95°C for 3min (pre-denaturation); 94°C for 30s (denaturation), 55°C for 30s (refolding), 72°C for 1min (extension), the denaturation-refolding-extension cycle is 35 cycles; 72°C for 10min (final extend).

The primer sequences are as follows:

P1:5′-GAGATNATNAAYCATAGRTCATTGA-3′

P2: 5′-GATCYTCAAAWGGATAWGCDCCAA-3′

Degenerate base codes: R(A,G),Y(C,T),M(A,C),K(G,T),S(G,C),W(A,T),H( A, T, C), B (G, T, C), V (G, A, C), D (G, A, T), N (A, T, G, C).

2.3 Electrophoresis separation, gel recovery target band

After the PCR product was subjected to 1% agarose gel electrophoresis, the target band was excised, and the fragment was recovered using the DNA Column Gel Recovery Kit (TIANGEN). The specific steps are as follows:

1) Add 500μl BL to the adsorption column CA2, centrifuge at 12,000rpm for 1min, and discard the filtrate;

2) Cut off the target band and put it into a clean EP tube, and weigh it;

3) Add about 500 μl PN sol, and sol in a water bath at 50°C for 10 minutes or sol at room temperature;

4) Pipette the solution (<700μl) into the CA2 column, place it at room temperature for 2min, centrifuge at 12,000rpm for 30-60s, and discard the filtrate;

5) Add 700μl PW to the CA2 column, centrifuge at 12,000rpm for 30-60s, and discard the filtrate;

6) Add 500μl PW to the CA2 column, centrifuge at 12,000rpm for 30-60s, and discard the filtrate;

7) Centrifuge at 12,000rpm for 2 minutes, and place at room temperature for several minutes to dry the CA2 column completely;

8) Put the CA2 column into a new centrifuge tube, add about 35 μl EB dropwise in the air, leave it at room temperature for 2 minutes, and centrifuge at 12,000 rpm for 2 minutes.

2.4 Ligation and transformation of vector

1) The fragment recovered from the gel is connected to the pMD19-T vector (TaKaRa), and the reaction system is as follows:

Gel Recovery Fragment 6μl

pMD19-T 1μl

Solution Ⅰ 3μl

After mixing, connect overnight at 16°C.

2) Preparation of competent E. coli

Escherichia coli (Escherichia coli) DH5α: purchased from TIANGEN company, adopting Liu Jinyuan's _CaCl method to prepare Escherichia coli competent cells, the steps are as follows:

Pick a single colony of Escherichia coli Top10 from the LB plate cultivated overnight at 37°C, inoculate it in 5ml LB liquid medium, and cultivate overnight at 37°C with shaking at 220rpm; transfer 100μl of the overnight culture to 50ml LB liquid medium, and incubate , 220rpm shaking culture for 2-4 hours, until the OD ₆₀₀ of the bacterial solution is 0.5. Transfer the culture medium to two pre-cooled 50ml sterile centrifuge tubes, put them in an ice bath for 10 minutes, then centrifuge at 5,000 rpm at 4°C for 5 minutes, discard the supernatant, resuspend the pellet with 0.1mol/l sterile CaCl ₂ , and place in an ice bath 30min. Centrifuge at 4°C and 5,000 rpm for 5 min, discard the supernatant, and resuspend the pellet with 0.1 mol/l sterile CaCl ₂ . Then add 500 μl of sterilized glycerin, mix well, aliquot into 50 μl tubes, freeze in liquid nitrogen and store in -70°C refrigerator for later use.

3) Transformation and identification of Escherichia coli

Take out the competent cells from the ultra-low temperature freezer, freeze and thaw in an ice bath. Add 10 μl of the ligation product above to the competent state, mix with a gun, place on ice for 30 minutes, then quickly place in a water bath at 42°C for heat shock for 45-90 seconds, and place on ice for 1-3 minutes. Add 1ml of liquid LB medium to the ultra-clean bench, and shake the bacteria at 37°C and 220rpm for 1h. Centrifuge at 4,000rpm for 2min at room temperature and discard the supernatant. The bacterial pellet was resuspended in 50 μl of liquid LB medium, and then evenly spread on LB plates (adding ampicillin at a final concentration of 50 μg/L as a screening), and cultured upside down at 37° C. overnight. Pick out a single colony for colony PCR and put it into 700 μl liquid LB (Amp+) medium to shake the bacteria for 2 to 3 hours. Select the bacterial liquid with successful colony PCR and send it to Beijing Huada Gene Technology Company for sequencing. The length of the obtained sequence is 466bp (Figure 2 ).

3. Obtaining the downstream fragment of JcSnRK2 gene

Two forward primers 3P1 and 3P2 were designed according to the middle fragment sequence, and two Oligo(dT) ₁₈ complementary primers AP1 and AP2 were used as reverse primers. Using the reverse transcription product as a template (synthesis of the first strand of cDNA is as described in 2.1 in this example), the first round of PCR amplification was performed with primers 3P1 and AP1. Then, using the first-round PCR product as a template, the second-round PCR amplification was performed with primers 3P2 and AP2. The reaction system is as in 2.2 of Example 1, cycle conditions: pre-denaturation at 95°C for 3 minutes, then 35 cycles (94°C for 30s, 58°C for 30s, 72°C for 1min), and extension at 72°C for 10min.

The primer sequences are as follows:

3P1: 5′-CTACAGTGGGAACACCAGCCTAT-3′

3P2: 5′-AGATGTTTGGTCTTGTGGGGTTA-3′

AP1: 5′-GTCAACGATACGCTACGTAACG-3′

AP2: 5′-TACGTAACGGCATGACAGTG-3′

The subsequent steps were as in 2.3 and 2.4 of Example 1, and the downstream fragment of the gene with a length of 625 bp was obtained by sequencing (see Figure 3).

4. Obtaining the upstream fragment of JcSnRK2 gene

4.1RT

With Jatropha curcas leaf total RNA as template, 5PR is used as reverse transcription primer, experimental procedure is as 2.1 in the present embodiment, and primer sequence is as follows:

5PR: 5′-GTGTTCCCACTGTAGACTTAGGTTG-3′

4.2 Removal of excess RNA

The reverse transcription product was treated with 2 μl RNase in a water bath at 37°C for 30 minutes.

4.3 Purification of cDNA

The cDNA purification kit used is DNA Fragment Purification Kit (TaKaRa), and the specific experimental steps are as follows:

1) Add 3 times the amount of DB Buffer to the reaction solution (if the amount of DB Buffer to be added is less than 100 μl, add 100 μl), then mix well;

2) Place the Spin Column on the Collection Tube;

3) Transfer the solution of the above step 1) to the Spin Column, centrifuge at 12,000rpm for 1min, and discard the filtrate; if the filtrate is added to the Spin Column and centrifuged once, the recovery rate of DNA can be improved;

4) Add 500μl Rinse A to the Spin Column, centrifuge at 12,000rpm for 30s, and discard the filtrate;

5) Add 700μl Rinse B (confirm that ethanol has been added) into the Spin Column, centrifuge at 12,000rpm for 30s, discard the filtrate; repeat this operation once;

6) Put the Spin Column on a new 1.5ml EP tube, add 25-30μl of sterilized water or Elution Buffer preheated to 60°C to the center of the Spin Column membrane, and let stand at room temperature for 1min;

7) Centrifuge at 12,000rpm for 1min to elute DNA.

4.3 cDNA tailing

Add the following components to a 0.2ml EP tube:

After denaturation at 94°C for 2 minutes, ice bath for 3 minutes, after brief centrifugation, add 1 μl TdT terminal transferase, mix gently and put into PCR machine, 37°C for 30 minutes, 65°C for 10 minutes.

4.4 Nested PCR: The first round of PCR uses the tailed cDNA as a template and AP1 and 5P1 as primers. The second round of PCR used the product of the first round of PCR as a template and AP2 and 5P2 as primers. The reaction system is as in 2.2 of Example 1, cycle conditions: pre-denaturation at 95°C for 3 minutes, followed by 35 cycles (94°C for 30s, 57°C for 30s, 72°C for 40s), and extension at 72°C for 7 minutes. The primer sequences are as follows:

5P1: 5′-TAGTTGTTGAAAGAAAAATCGTGCC-3′

5P2: 5′-ACTATGGCTAAATGAGTTGGTGTGA-3′

Subsequent steps were as in 2.3 and 2.4 of Example 1, and the upstream fragment of the gene with a length of 295 bp was obtained by sequencing (Figure 4).

5. Acquisition of the open reading frame (ORF) of the JcSnRK2 gene

According to the obtained middle fragment, 3' fragment and 5' fragment, the full-length cDNA of JcSnRK2 gene was spliced, and the specific primers SP1 and SP2 for amplifying the ORF were designed accordingly. Add the following components to a 0.2ml EP tube:

Amplify with 2 μl cDNA as template, cycle conditions are: 95°C for 3 minutes, 35 cycles (94°C for 30s, 57°C for 30s, 72°C for 1min), 72°C for 10min.

The full-length ORF of 1017bp was obtained by sequencing (Figure 5 and Figure 6). The primer sequences are as follows:

SP1: 5′-ATGGAGCGTTATGAGATATTGAGAG-3′

SP2: 5′-TCACAATGCACAAAACAAAATCACCAC-3′

6. Obtaining JcSnRK2 Genomic DNA

6.1 Extraction of DNA from leaves of Jatropha curcas

Use the Plant Genomic DNA Kit (TIANGEN) to extract the DNA of Jatropha curcas leaves, the specific method is as follows:

1) Take 0.1g of Jatropha curcas leaves and grind them fully with liquid nitrogen;

2) Quickly transfer the ground Jatropha curcas leaf powder to a centrifuge tube pre-filled with 700 μl 65°C preheating buffer GP1 (add mercaptoethanol to the preheated GP1 before the experiment, with a final concentration of 0.1%), and quickly invert After mixing, place the centrifuge tube in a 65°C water bath for 20 minutes, during which time the centrifuge tube was inverted several times to mix the sample;

3) Add 700μl chloroform, mix thoroughly, and centrifuge at 12,000rpm for 5min;

4) Transfer the upper aqueous phase into a new centrifuge tube, add 700 μl buffer GP2, and mix well;

5) Transfer the mixed liquid into the adsorption column CB3, centrifuge at 12,000rpm for 30s, and discard the waste liquid;

6) Add 500μl buffer GD to the adsorption column CB3, centrifuge at 12,000rpm for 30s, and discard the waste liquid;

7) Add 600μl rinse solution PW to the adsorption column CB3, centrifuge at 12,000rpm for 30s, and discard the waste solution;

8) Repeat step 7) once;

9) Put the adsorption column CB3 back into the collection tube, centrifuge at 12,000rpm for 2 minutes, and discard the waste liquid; place the adsorption column CB3 at room temperature for several minutes to completely dry the residual rinse solution in the adsorption material;

10) Transfer the adsorption column CB3 into a clean centrifuge tube, add 50-200 μl of elution buffer TE dropwise to the middle of the adsorption membrane, leave it at room temperature for 2-5 minutes, centrifuge at 12,000 rpm for 2 minutes, and collect the solution into the centrifuge tube middle.

6.2PCR

Using Jatropha curcas leaf DNA as a template and SP1 and SP2 as primers, the genomic DNA of JcSnRK2 was amplified using long Taq enzyme (Adelaide). The specific system is as follows:

Cycle conditions: 94°C for 2min, 10 cycles (94°C for 15s, 57°C for 30s, 68°C for 5min), 30 cycles (94°C for 15s, 57°C for 30s, 68°C for 5min (automatic extension 15s/cycle)), 68°C ℃ 10min.

6.3 Sequence Analysis

As in 2.3 and 2.4 in this example, after gel recovery, pMD19-T ligation, transformation of Escherichia coli, colony PCR, and sequencing, a JcSnRK2 genomic DNA fragment with a length of 2578bp was obtained (Figure 7). After sequence analysis, the genomic DNA contained The exon region of 1017bp and the intron region of 1561bp, as shown in Figure 8, the lengths of the 10 exons are 120, 76, 102, 52, 92, 94, 104, 101, 18 and 218bp respectively, The lengths of the nine introns are 227, 526, 128, 291, 89, 109, 97, 94 and 40 bp, and the genomic DNA contains multiple introns similar to other SnRK2 family members.

Embodiment 2: Analysis of Jatropha curcas JcSnRK2 gene

1. Bioinformatics analysis

Use the software DNAMAN, Primer Premier5.0 and the Blast program on NCBI to perform homology on the JcSnRK2 sequence (SEQ ID No: 1 in the sequence listing) and the amino acid sequence of the polypeptide encoded by the JcSnRK2 gene (SEQ ID No: 3 in the sequence listing) analysis and conserved region analysis.

2. Real-time quantitative PCR detection of JcSnRK2 gene expression pattern

2.1 Fluorescence quantitative PCR primer design

According to the cloned full-length sequence of JcSnRK2 cDNA, QP1 and QP2 were designed as fluorescent quantitative PCR primers to detect the expression level of JcSnRK2 gene, actin was selected as the internal reference gene, and the primers were ActP1 and ActP2. The primer sequences are as follows:

QP1: 5′-TGAAGATGAGGCACGATT-3′

QP2: 5′-CCACTGTAGACTTAGGTTGA-3′

ActP1: 5′-TGGAGCAGAGAGATTCCGATG-3′

ActP2: 5′-CACCACTGAGCACAATGTTACC-3′

2.2 Preparation of materials for analysis of gene expression patterns under different stress conditions

ABA (abscisic acid) treatment: After washing the soil of Jatropha curcas seedlings, put them into 1mM ABA solution for cultivation, and take leaves at 0h, 1h, 3h, 6h, 12h and 24h respectively.

Salt stress: Jatropha curcas seedlings were washed clean soil, cultured in 250mM NaCl solution, and leaves were taken at 0h, 1h, 3h, 6h, 12h, 24h and 48h.

Drought stress: After washing the soil of Jatropha curcas seedlings, they were cultured in a solution with a mass fraction of 20% PEG6000, and the leaves were collected at 0h, 1h, 3h, 6h, 12h, 24h and 48h.

Cold stress: the potted Jatropha curcas seedlings were cultured at 4°C, and the leaves were taken at 0h, 1h, 3h, 6h, 12h, 24h and 48h.

2.3RT

According to the extraction method of the Jatropha curcas leaf total RNA of embodiment 1, extract their total RNA respectively from the root, stem, leaf of Jatropha curcas seedling, flower and Jatropha curcas seedling and 2.2 different stress conditions For RNA, the obtained total RNA was reverse-transcribed using iScript ^TM cDNA Synthesis Kit from BIO-RAD Company. Add the following components to a 0.2ml RNase-free siliconized EP tube with a siliconized pipette tip:

The reaction was performed on the PCR instrument according to the following program: 5 min at 25°C, 30 min at 42°C, and 5 min at 85°C. The resulting cDNA was diluted 10 times with Nuclease-free water and stored at -20°C for future use.

2.4 Fluorescence quantitative PCR

The kit used for fluorescent quantitative PCR is SsoFast ^TM EvaGreen from BIO-RAD Superemix, PCR reaction was carried out on the IQ5 PCR instrument (BIO-RAD company), and the following components were added to a 0.2ml siliconized EP tube with a siliconized pipette tip:

The reaction program was denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 10 s and 60°C for 1 min, and finally melting curve analysis from 65°C to 95°C to remove primer dimers and other non-specific amplification. Each sample was repeated three times to avoid sampling errors. The experimental data was analyzed using iQ5-Cycler software, and the analysis method was the ΔΔCt method.

3. Results

3.1 Homology analysis

Taking the amino acid sequence of the polypeptide encoded by the JcSnRK2 gene sequence described in SEQ ID No: 1 in the sequence listing (SEQ ID No: 3 in the sequence listing) as the query sequence, Blastp was performed on NCBI, and it was found that it was similar to Medicago truncatula (Medicago truncatula, XM_003615109 ) and Arabidopsis thaliana (Arabidopsis thaliana, NM_120165) have the highest homology of SnRK2 gene, which are 88% and 85%, respectively. Zea Mays, NM_001154177) were 78%, 78% and 77% homologous. According to the amino acid sequence homology of SnRK2 in these six plants, the phylogenetic tree was drawn with DNAMAN software, and the result was slightly different from that of NCBI Blastp. It can be seen from Figure 9 that tomato is the closest relative to JcSnRK2, followed by rice, corn and wheat.

At present, Arabidopsis thaliana is the most in-depth research on the function of SnRK2 protein. Among its 10 family members, except SnRK2.9, all of them can be induced by NaCl and drought stress. SnRK2.7 and SnRK2.8 are activated by ABA, and the overexpression of SnRK2.8 improves the cold resistance of plants under water shortage. In order to deduce the biological function of JcSnRK2 protein, amino acid sequence homology analysis was carried out between it and 10 members of SnRK2 protein family in Arabidopsis, and their registration numbers on NCBI were SnRK2. ), SnRK2.3(At5g66880), SnRK2.4(At1g10940), SnRK2.5(At5g63650), SnRK2.6(At4g33950), SnRK2.7(At4g40010), SnRK2.8(At1g78290), SnRK2.9(At0)2303 , SnRK2.10 (At1g60940), using DNAMAN software to draw a phylogenetic tree, as shown in Figure 10, as can be seen from Figure 10, the amino acid sequence of the polypeptide encoded by the JcSnRK2 gene of the present invention is the same as that of the ABA-induced type in Arabidopsis The family members SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7 and SnRK2.8 have a close homology relationship, so it is preliminarily speculated that the JcSnRK2 gene may be activated by ABA and participate in the biological response activities of Jatropha curcas against salt and drought.

3.2 Conserved area analysis

By comparing with SnRK2.2, SnRK2.3, SnRK2.6, SnRK2.7 and SnRK2.8, as shown in Figure 11, the amino acid sequence encoded by the JcSnRK2 gene of the present invention contains three conserved regions shared by the SnRK2 family, That is, ATP bindingdomain (GXGXXGX), activation loop (DFGYSKSSVLHSQPKSTVGTPAYIAPE) and SnRK Box, and the ABA Box unique to ABA-stimulating members, where the activation loop is the target site of protein kinase phosphorylation, and the Ser and Thr residues marked with * in the figure Plays a crucial role in the activity of SnRK2. In addition, the SnRK2 protein family has an acidic patch (D/E) rich in glutamic acid or aspartic acid at the C-terminus. Based on this, SnRK2 is divided into two groups, SnRK2a and SnRK2b. The acidic patch of SnRK2a JcSnRK2 is rich in aspartic acid D, and SnRK2b is rich in glutamic acid E. The JcSnRK2 cloned in Example 1 has acidic patches and is mainly composed of aspartic acid, with a ratio of about 53%, so it is classified into the SnRK2a subfamily .

3.3 Analysis of tissue expression specificity

As shown in Figure 12, the JcSnRK2 gene of the present invention is expressed in the detected tissues, and the expression level is the highest in roots, followed by leaves and stems, and less flowers and seeds, which is similar to that of Arabidopsis SnRK2.8 The tissue expression patterns of members such as ZmSAPK8 in maize and TaSnRK2.4 in wheat are the same.

3.4 Analysis of gene expression patterns under different stress conditions

1) ABA treatment: as shown in Figure 13, the expression of the JcSnRK2 gene of the present invention is relatively slightly induced after 1 hour of ABA treatment, down-regulated to a level similar to that at 0 hour at 3 hours, and gradually increased at 6 to 24 hours High reaches a maximum value and then decreases again. It shows that JcSnRK2 can be induced by ABA.

2) Salt stress: As shown in Figure 14, the JcSnRK2 gene of the present invention was stimulated at 1 hour of salt stress, and the expression level increased significantly, then decreased at 3 hours, then maintained a relatively stable level, and increased to Then down again. It shows that the JcSnRK2 gene can be induced by salt stress and is rapidly activated at the initial stage of induction.

3) Drought stress: As shown in Figure 15, the expression of the JcSnRK2 gene of the present invention is similar to that of salt stress at 1 hour of drought stress, that is, the expression level of the gene increases significantly, and then decreases to a lower level at the stage of 3 hours to 6 hours , gradually decreased after being up-regulated at 12h. It shows that JcSnRK2 gene responds rapidly to drought stress, and there is a periodic regulation process.

4) Cold stress: As shown in Figure 16, although the gene level also changed during the whole cold stress process, the magnitude was relatively small and irregular, indicating that JcSnRK2 was not significantly affected by cold stress.

Based on various analyzes of the above-mentioned JcSnRK2 gene, it was shown that this gene is the closest to Arabidopsis SnRK2.8, so a eukaryotic recombinant plasmid was constructed to transform Arabidopsis SnRK2.8-deleted plants, and its function was further tested.

Example 3: Construction and functional verification of eukaryotic recombinant plasmids

1. Materials

1.1 Plant material

Arabidopsis thaliana SnRK2.8 deletion mutant snf2.8 seeds (Arabidopsis thaliana seeds that use T-DNA insertion method to mutate SnRK2.8) were purchased from the Arabidopsis Information Resource website located in Stanford, USA (The Arabidopsis InformationResource , TAIR), which is the most authoritative Arabidopsis genome database and Arabidopsis genome annotation system in the world, with rich data resources and the latest annotation information (http://www.arabidopsis.org/index.jsp).

The Arabidopsis thaliana SnRK2.8 deletion mutant snf2.8 seeds were cultured in a light incubator with 16 hours of light at 24°C and 8 hours of darkness at 18°C, nutrient soil: vermiculite = 1:1.

1.2 Strains

Escherichia coli (Escherichia coli) DH5α: purchased from TIANGEN company;

Agrobacterium tumefaciens LBA4404: preserved by the Key Laboratory of Biological Resources and Ecological Environment, Ministry of Education, School of Life Sciences, Sichuan University, and can be purchased from China Plasmid Vector Strain Cell Line Gene Collection Center.

1.3 Plasmids

pMD19-T: Reconstructed from the pUC19 vector, the EcoRV recognition site is inserted between the XbaI and SalI recognition sites at the multiple cloning site of the pUC19 vector, and Ampr is a high-efficiency cloning PCR product (TA cloning) Special carrier, purchased from TaKaRa;

Plant binary expression vector pBI121: Contains kanamycin resistance gene nptⅡ, CaMV35S promoter, GUS (β-glucuronidase, GUS) reporter gene, and is preserved by the Key Laboratory of Biological Resources and Ecological Environment, Ministry of Education, School of Life Sciences, Sichuan University, It can be purchased from China Plasmid Vector Strain Cell Line Gene Collection Center.

1.4 Media and solutions

LB liquid medium: tryptone 10g/L, yeast extract 5g/L, NaCl 5g/L. LB solid medium: add 12.5g/L agar powder on the basis of liquid medium.

YEB liquid medium: beef extract 5g/L, yeast extract 5g/L, peptone 5g/L, sucrose 1g/L, MgSO ₄ ·7H ₂ O 0.5g/L. YEB solid medium: add 15g/L agar powder on the basis of liquid YEB medium.

Infection solution: used for Agrobacterium to infect Arabidopsis inflorescences, the formula is MS liquid medium plus 5% sucrose.

The above-mentioned culture medium and solutions were all sterilized under high pressure at 121°C for 20 minutes for later use, and antibiotics were added when the culture medium was cooled to about 50°C.

2. Experimental method

2.1 Obtaining the full-length ORF of JcSnRK2 gene

According to the sequencing analysis, the ORF sequence of the JcSnRK2 gene has no restriction sites of Xba I and BamH I, therefore, we designed the primers for constructing eukaryotic recombinant plasmids as follows (bold letters are protected bases, italics are enzymes cutting point):

Xba-F1: 5′-GCGTCTAGAATGGAGCGTTATGAGATATTGAGAG-3′

Bam-R1: 5′-CGCGGATCCTCACAATGCACAAACAAAATCACCAC-3)

RT-PCR was carried out according to the experimental method in Example 1, and the cycle conditions were: 95°C for 3min, 35 cycles (94°C for 30s, 57°C for 30s, 72°C for 1min), and 72°C for 10min. Then the gel was recovered, connected to the pMD19-T vector, transformed into Escherichia coli and then sequenced.

2.2 pET-JcSnRK2 plasmid extraction

The bacterial liquid with successful sequencing was selected for expansion culture, and the plasmid pET-JcSnRK2 was obtained by using the plasmid extraction kit Plasmid Mini Kit I (Omega). All operations were carried out at room temperature, and the specific steps were as follows:

1) Take 1.5-5ml of bacterial solution, centrifuge at 10,000rpm for 1min, and discard the supernatant;

2) Add 250μl Solution I/RNase A to the pellet, and shake to suspend the bacteria;

3) Add 250μl Solution II, shake lightly several times, and wait for 2 minutes after the bacterial solution is clarified;

4) Add 350μl Solution III, turn it up and down several times until white flocs appear;

5) Centrifuge at 13,000rpm for 10min, take the supernatant or take the supernatant when standing for use;

6) Put the Hibind DNA binding column in the collection tube, add 200μl Buffer GPS, and place it at room temperature for 3-5 minutes;

7) Centrifuge at 12,000rpm for 2min, discard the filtrate;

8) Take the supernatant from step 5) and put it in a Hibind DNA binding column, centrifuge at 13,000rpm for 1min, and discard the filtrate;

9) Add 500μl Buffer HB, centrifuge at 10,000rpm for 1min, and discard the filtrate;

10) Add 700μl DNA Wash Buffer (containing absolute ethanol), centrifuge at 10,000rpm for 1min, and discard the filtrate;

11) Repeat step 10) once;

12) Empty column, centrifuge at 13,000rpm for 2min, discard the filtrate, place at room temperature for 3-5min, evaporate ethanol to dryness;

13) Put the Hibind DNA binding column on a new centrifuge tube, take 30-50 μl of sterile water or Elution Buffer and drop it into the binding column;

14) Place at room temperature for 1-2 minutes, centrifuge at 13,000 rpm for 2 minutes, and store at -20°C for later use.

2.3 Construction of eukaryotic recombinant plasmid pBI121-JcSnRK2

The construction method is shown in Figure 17, and the operation is as follows:

pET-JcSnRK2 and pBI121 were digested with Xba I and BamH I (purchased from TaKaRa) respectively, and the reaction system was as follows:

Mix gently, and after 12-16 hours in a 37°C water bath, run gel electrophoresis to recover the JcSnRK2 gene fragment with restriction sites and the pBI121 vector restriction fragment (Figure 18). For specific steps, see 2.3 of Example 1. T4 ligase (purchased from TaKaRa) was used to ligate the above two double-digested gel recovery products, and the reaction system was as follows:

After mixing gently, incubate at 16°C for 12h. For the transformation of the ligated product, see 2.4 of Example 1. Colonies with correct sequencing were selected for expansion and culture, and plasmid pBI121-JcSnRK2 was extracted.

2.4 Preparation of Competent Agrobacterium

1) Pick a single colony of Agrobacterium LBA4404 from the YEB solid medium, inoculate it into 10ml YEB liquid medium (containing 100μg/ml rifampicin), shake the bacteria overnight at 28°C and 200rpm;

2) Take 1ml of the bacterial liquid, add it to 50ml of YEB liquid medium, shake the bacteria at 28°C and 200rpm until the OD ₆₀₀ is about 0.5.

3) Centrifuge at 5,000rpm at 4°C for 5min, discard the supernatant;

4) The bacteria were resuspended in 10ml of pre-cooled 20mM CaCl ₂ and kept in ice bath for 30min;

5) 4°C, 5,000rpm, centrifuge for 5min, discard the supernatant;

6) The cells were resuspended with about 1ml of pre-cooled 20mM CaCl ₂ and stored at -70°C after aliquoting.

2.5 Transformation of Agrobacterium

1) Take out the competent Agrobacterium from the ultra-low temperature refrigerator, freeze and thaw in an ice bath;

2) Add 10 μl of the eukaryotic recombinant plasmid pBI121-JcSnRK2 constructed in Example 2.3 into the competent Agrobacterium, mix gently, and ice-bath for 30 minutes;

3) Place in a water bath at 37°C for 5 minutes, then quickly place on ice for 3-5 minutes;

4) Add 800 μl liquid YEB medium under sterile conditions, and shake the bacteria at 28°C and 175rpm for 3-6 hours;

5) Centrifuge at 4,000rpm for 2min at room temperature, discard the supernatant;

6) The bacteria were resuspended in about 80 μl liquid YEB medium, spread on the plate, and cultured at 28°C for two days;

7) Pick a single colony, perform colony PCR with JcSnRK2 full-length specific primers SP1 and SP2 (Figure 19), and select cultures with successful PCR for subsequent experiments.

2.6 Agrobacterium infection of Arabidopsis

1) Add the Agrobacterium with successful PCR obtained in step 2.5 into YEB liquid medium (containing 100 μg/ml rifampicin and 80 μg/ml kanamycin), and shake the bacteria at 28°C until the OD ₆₀₀ is about 0.6;

2) Centrifuge at 4,000 rpm for 2 minutes at room temperature, and resuspend the bacterial pellet with an appropriate amount of infection solution;

3) Select the inflorescence of Arabidopsis thaliana and soak it in the above solution for about 1 minute;

4) Plants were cultured overnight in the dark and then returned to the light incubator, and continued to culture under the conditions of 16 hours of light at 24°C and 8 hours of darkness at 18°C.

2.7 Identification of transgenic Arabidopsis

2.7.1 GUS staining method

Take the sample to be tested and place it in X-Gluc staining reaction solution (50mmol/L Na ₃ PO ₄ , 0.1% Trition-100, 20% methanol, 0.5mg/ml X-Gluc, pH 7.0), and keep it in an oven at 37°C Observe overnight after destaining with 95% ethanol.

2.7.2 PCR detection of gene integration

According to the operation of 6.1 in Example 1, the DNA of transgenic Arabidopsis leaves was extracted, and PCR was performed using the DNA as a template and the full-length specific primers SP1 and SP2 of JcSnRK2. The cycle conditions were: 95°C for 3min, 35 cycles (94°C 30s, 57°C 30s, 72°C 1min), 72°C 10min. PCR products were analyzed by 1% agarose gel electrophoresis.

2.7.3 RT-PCR detection of gene transcription and expression

The total RNA of the transgenic Arabidopsis was extracted according to the method in Example 1, and the first strand of cDNA was synthesized by reverse transcription with M-MLV reverse transcriptase. Using the reverse transcription product as a template, PCR was carried out with the full-length specific primers SP1 and SP2 of JcSnRK2. The reaction uses Arabidopsis 18S rRNA as a reference gene. The primer sequences are as follows:

At18sF: 5′-GAGTCATCAGCTCGCGTTGAC-3′

At18sR: 5′-CTTCACCGGATCATTCAATCG-3′

The cycle conditions are: 95°C for 3min, 35 cycles (94°C for 30s, 57°C for 30s, 72°C for 1min), 72°C for 10min. PCR products were analyzed by 1% agarose gel electrophoresis.

2.8 Screening of transgenic Arabidopsis seeds

1) Thoroughly soak the filter paper with 80 μg/ml kan (kanamycin) aqueous solution. As for the petri dish, sprinkle the harvested T0 generation seeds evenly on the filter paper, cultivate in the dark at 4°C for two days, and then culture at room temperature, every two days Make up kan water once;

2) One week later, the germinated kan-resistant Arabidopsis seedlings were transplanted into pots with nutrient soil: vermiculite = 1:1, and grown into _T1 generation plants in the light incubator, which were successfully verified by GUS and PCR Harvest Arabidopsis thaliana T ₂ generation seeds from each plant and make a record;

3) Arabidopsis thaliana T ₂ generation seeds were germinated on MS solid medium containing 80 μg/ml kan, and the differentiation of tolerance:sensitivity = 3:1 appeared, and the kan-resistant Arabidopsis seedlings were selected for cultivation Arabidopsis T ₃ generation seeds (i.e., plant transformants), showing 100% kan resistance, were named JK2 for subsequent experiments.

2.9 Salt and drought tolerance verification of transgenic Arabidopsis

2.9.1 Root growth

The seeds of Arabidopsis mutant snf2.8 and JK2 were soaked in 70% ethanol for 1 min, then in 5% NaClO aqueous solution for 5-7 min, and washed several times with sterilized ddH ₂ O. Evenly placed on MS solid medium, cultured in the dark at 4°C for 2 days, then placed in a light incubator for culture, and the root length was counted after 1 week.

Snf2.8 seeds and JK2 seeds were cultured in soil (nutrient soil: vermiculite = 1:1) for about 45 days, and the development of the root system of the seedlings was observed.

2.9.2 Seed germination rate

The seeds of Arabidopsis mutant snf2.8 and JK2 were soaked in 70% ethanol for 1 min, then treated with 5% NaClO aqueous solution for 5-7 min, and washed several times with sterilized ddH ₂ O. Put Arabidopsis snf2.8 seeds and JK2 seeds evenly on MS, MS+150mM NaCl, MS+200mM mannitol solid medium with about 90 seeds/petri dish, culture in dark at 4°C for 2 days, then put them in light Cultivate in an incubator, and after 2 weeks, take two green leaves as the standard to count the germination rate.

2.9.3 Salt resistance verification

The aseptic seedlings of Arabidopsis mutants snf2.8 and JK2 were cultured on MS medium for 2 weeks and had basically the same growth status, and were transferred to petri dishes with NaCl content of 150mM, 200mM, and 250mM, and cultured in a light incubator for 30 days. Observe the green seedling rate.

Seeds of Arabidopsis mutant snf2.8 and JK2 were cultured in soil for 30 days and 45 days, respectively, and the seedlings with similar growth conditions were selected, and water was supplied from the bottom of the pot with 450mM NaCl solution for 1 month. Observe the growth of seedlings.

2.9.4 Verification of drought tolerance

1) Select the sterile seedlings of snf2.8 and JK2 that were cultured on MS medium for 2 weeks and have basically the same growth status, and move them to MS estimation medium with mannitol content of 200mM, 250mM, and 300mM, and cultivate them in a light incubator for 30 days. Observe for leaf shrinkage.

2) After the Arabidopsis mutant snf2.8 seeds and JK2 seeds were cultured in soil for 30 days and 45 days, respectively, the seedlings with similar growth conditions were selected, and the water supply was stopped for 1 month to observe the growth conditions.

3) The fresh leaves of Arabidopsis thaliana mutants snf2.8 and JK2 cultured in soil for about 30 days were taken and weighed, and recorded as FW (freshweight, fresh weight). They were then placed in a 24°C incubator for dark culture, and the detached leaves of the two were taken out at different time points (0h, 0.5h, 1h, 2h, 3h, 4h, 5h, 6h, 7h, 8h) and weighed again. It is DTW (designated time weight, designated time weight). Then it was baked in an oven at 85°C for 24 hours and then weighed, which was recorded as DW (dought weight, dry weight). Repeat 3 times, and calculate the water loss rate (WLR) of the two isolated leaves respectively:

WLR=(DTW-DW)/(FW-DW)×100%

The smaller the WLR of the detached leaves, the stronger the early resistance.

2.9.5 Verification of the regulation of osmotic stress response genes

Four downstream genes AREB1, LEA, RD29A, and RD29B in Arabidopsis thaliana in response to osmotic stress were selected for real-time quantitative PCR to detect changes in their expression levels, thereby inferring the function of JcSnRK2.

Total RNA was extracted from leaves of Arabidopsis thaliana under drought stress for 0 h and 4 h, and detected according to the fluorescent quantitative PCR method described in Example 2. QP1 and QP2 were used as primers for detecting the expression of JcSnRK2 gene, and the primer sequences are shown in 2.1 of Example 2. Select Arabidopsis thaliana 18S rRNA as the internal reference gene, and the primers are At18sF and At18sR, and the primer sequences are shown in 2.7.3 of this embodiment.

The reaction program was denaturation at 95°C for 2min, followed by 45 cycles of 95°C for 10s and 60°C for 1min, and finally melting curve analysis from 65°C to 95°C.

The primer sequences are as follows:

AREB1-F: 5′-CATACCAGCAATCGCAACAG-3′

AREB1-R: 5′-CTCCAAGTCCCCACAAGACCA-3′

LEA-F: 5′-TCCAACCCACTCACACCACCA-3′

LEA-R: 5′-CAGAAGCCAAACCCTCCTCA-3′

RD29A-F: 5′-GCAATGAGCATGAGCAAGATC-3′

RD29A-R: 5′-TCGGAAGACACGACAGGAAAC-3′

RD29B-F: 5′-TCTGACCACACCAAACCCATT-3′

RD29B-R: 5′-CCTCTCTTTTTCGCTTCCCAG-3′

3. Results

3.1 Identification of transgenic plants

3.1.1 GUS staining identification

The recombinant plasmid of the present invention contains the open reading frames of JcSnRK2 and GUS at the same time, and the 35S promoter successively activates the expression of these two genes, and whether the transformation is successful can be identified by staining. As shown in Fig. 20, as identified by GUS staining, the Arabidopsis seedlings, siliques and flowers transfected with JcSnRK2 all had blue color after the successful expression of the GUS gene, which proved that the transformation was successful and the JcSnRK2 gene was expressed in multiple tissues.

3.1.2 PCR detection

The DNA and total RNA of the leaves of the Arabidopsis mutants snf2.8 and JK2 were extracted respectively, and PCR and RT-PCR were performed respectively. As shown in Figure 21, the DNA and total RNA of the leaves of the snf2.8 plants were not amplified A band was obtained, and a specific fragment of 1017 bp was amplified from the DNA and total RNA of the leaves of the JK2 plant, indicating that the Jatropha curcas JcSnRK2 gene had been integrated into the genomic DNA of Arabidopsis thaliana and expressed successfully.

3.2 Results and analysis of Arabidopsis transgenic plants

3.2.1 Plant root growth is promoted

The observation of root growth of Arabidopsis plants showed that the root growth of JK2 Arabidopsis plants was significantly faster than that of the Arabidopsis mutant snf2.8, as shown in Figure 22(A) and Figure 22(B), cultured on MS solid After one week of growth on the base, the average root length of JK2 Arabidopsis plants was 1.7 cm, while the root length of Arabidopsis mutant snf2.8 plants was 1.3 cm, and the former was more than 1.3 times the root growth rate of the latter. After 45 days of cultivation in soil, as shown in Figure 22(C), the root system of JK2 Arabidopsis plants is obviously more developed and has more lateral roots. The above two phenomena are beneficial to the performance of resistance to drought adversity.

3.2.2 Increased germination rate of plant seeds under osmotic stress

Observation on the germination rate of Arabidopsis plants showed (Figure 23): the germination rate of JK2 Arabidopsis seeds under osmotic stress was higher than that of Arabidopsis mutant snf2.8 seeds. The MS solid medium containing 150mM NaCl and 200mM mannitol was used to simulate salt stress and drought stress respectively. After 2 weeks of growth, both of them could grow two green leaves in normal environment, while under salt stress, Arabidopsis The germination rate of mutant snf2.8 seeds was 79.56%, while the germination rate of JK2 Arabidopsis seeds was maintained at 87.64%. Under drought stress, the germination rate of Arabidopsis mutant snf2. The germination rate of mustard seeds was 84.61%. Although both salt stress and drought stress can reduce the germination rate, the inhibitory effect on JK2 Arabidopsis seeds is relatively small, indicating that the expression of JcSnRK2 can improve the resistance of seeds to osmotic stress during germination.

3.2.3 Increased salt resistance of plants

In MS solid culture dishes with NaCl contents of 150mM, 200mM, and 250mM respectively, cultured in a light incubator for 30 days, the results are shown in Table 1 and Figure 24. The growth and development were severely inhibited, and the green seedling rate in the MS solid culture dish with NaCl content of 150mM was only 7.25%, but no green seedlings appeared in the following two concentrations, and the appearance rate of the 3rd and 4th leaves To count the growth rate of Arabidopsis thaliana seedlings, NaCl severely inhibited the growth of Arabidopsis mutant snf2.8 seedlings, and the four-leaf ratios were 27.54%, 17.14% and 6.25% respectively. The seedlings of JK2 Arabidopsis grew well in 150mM NaCl, with a green seedling rate of 57.39% and a four-leaf rate of 80%, which were nearly 8 times and 3 times that of the Arabidopsis mutant snf2.8, respectively. When the NaCl concentration increased, the growth of JK2 Arabidopsis seedlings was also inhibited, but the salt tolerance of the Arabidopsis mutant snf2.8 was significantly improved. When the NaCl concentration was 200mM, the green shoot rate and four-leaf rate remained It is about 2 times that of the Arabidopsis mutant snf2.8 in 150mM NaCl, and when the Arabidopsis mutant snf2.8 has no green shoots, JK2 Arabidopsis still maintains 15.63% green shoots. When the NaCl concentration reached 250mM, no green shoots appeared in both, but the growth rate of JK2 Arabidopsis seedlings was still twice that of the Arabidopsis mutant snf2.8.

Table 1 Statistics of green seedling rate and four leaf rate under salt stress

In addition, after 30 days and 45 days of cultivation in the soil, water was supplied from the bottom of the pot with 450 mM NaCl solution. After 2 weeks, as shown in Figure 25, the plants of the Arabidopsis mutant snf2.8 all experienced varying degrees of leaf dehydration. Although there was no obvious yellowing phenomenon, the plants all appeared soft and collapsed; The Arabidopsis thaliana plants all showed a better growth trend, especially the plants after 45 days still showed a normal growth state. After 1 month, more than half of the leaves of Arabidopsis mutant snf2.8 plants showed obvious and severe yellowing phenomenon no matter it was 30 days or 45 days old, while the leaves of JK2 Arabidopsis plants only showed slight yellowing Phenomenon. It indicated that the expression of JcSnRK2 enhanced the salt resistance of the plants.

3.2.3 Increased drought resistance of plants

Mannitol was used to simulate drought conditions, as shown in Figure 26. The Arabidopsis mutant snf2.8 grew well on the first and second leaves when the mannitol concentration was 200mM and 250mM, and the third and fourth leaves appeared to varying degrees However, when the concentration increased to 300mM, severe wilting occurred in all four leaves. However, JK2 Arabidopsis can maintain relatively normal growth under the first two concentrations of mannitol, and when grown on MS medium containing 300mM mannitol, the development of the third and fourth slices is also inhibited, which is similar to that of the Arabidopsis mutant snf2 .8 The traits were similar when the concentration of mannitol was 250mM, and there was no situation that all four leaves were inhibited.

In addition, after 30 days and 45 days of cultivation in the soil, the water supply was stopped for 1 month, and the growth conditions were observed. The results are shown in Figure 27. The 30-day-old Arabidopsis mutant snf2.8 plants were severely affected by drought stress, and some leaves were completely dry and yellow. Although the 45-day-old Arabidopsis mutant snf2.8 plants still maintained the whole plant Green, but most of the leaves are curled; while the 30-day and 45-day JK2 Arabidopsis plants grow better than the Arabidopsis mutant snf2.8, the 30-day JK2 Arabidopsis plants have no dead leaves, and the 45-day Only a few leaves of the JK2 Arabidopsis plants were slightly curled.

The water loss rate of the isolated leaves of JK2 Arabidopsis was significantly lower than that of the Arabidopsis mutant snf2.8 (Fig. 28).

It indicated that the expression of JcSnRK2 improved the drought resistance of the plants.

3.2.4 Mechanism of JcSnRK2 drought resistance

The downstream genes AREB1, LEA, RD29A, and RD29B that partially responded to osmotic stress were selected, and the changes in their expression levels were checked by real-time fluorescent quantitative PCR. , RD29A, and RD29B genes were all up-regulated, and the up-regulated levels in JK2 Arabidopsis were stronger than those in Arabidopsis mutant snf2.8. It shows that JcSnRK2 can be activated by hyperosmotic stress, which may be similar to Arabidopsis SnRK2.8, and it can initiate stress response by interacting with ABF/AREB and so on.

Claims (4)

1. A gene obtained from Jatropha curcas clone is characterized in that the nucleotide sequence of the gene is as shown in SEQ ID No: 1 in the sequence listing. 2. A eukaryotic recombinant plasmid, characterized in that the recombinant plasmid consists of the nucleotide sequence shown in SEQ ID No: 1 in the sequence listing and a eukaryotic expression vector. 3. The eukaryotic recombinant plasmid according to claim 2, characterized in that the eukaryotic expression vector is pBI121, pBI221 or pBin438. 4. The application of the eukaryotic recombinant plasmid described in claim 2 or 3 in improving plant salt resistance and drought tolerance.