DsRNA of medicago sativa MsGI gene and application of dsRNA in salt stress resistance of medicago sativa

Abstract

The invention discloses dsRNA of a medicago sativa MsGI gene and application of the dsRNA in salt stress resistance of medicago sativa. The nucleotide sequence of the dsRNA of the alfalfa MsGI gene in the invention is as shown in SEQ ID No. 1. The dsRNA of the medicago sativa MsGI gene can be applied to improvement of environmental salt stress resistance of medicago sativa. The dsRNA of the medicago sativa MsGI gene can target the medicago sativa MsGI gene, the mRNA level of the medicago sativa MsGI gene is reduced in an RNA interference mode, active oxygen generated by the medicago sativa under salt stress is eliminated, accumulation of salt ions in the medicago sativa root system is reduced, and therefore the resistance of the medicago sativa to environmental salt stress is remarkably enhanced, and the medicago sativa MsGI gene has a good application prospect. And the biological stimulant for external use is prepared, so that the biological stimulant for external use is convenient to use, quick in effect, green and safe, and has a good market application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biostimulants, and particularly relates to a dsRNA of Medicago sativa MsGI gene and its application in salt stress resistance of Medicago sativa. Background Art

[0002] With the increase of global climate change and extreme climate, the stress resistance of crops has gradually attracted attention. Among them, salt stress in the environment is one of the main environmental challenges threatening agriculture worldwide. Excessive salt will damage plants by causing osmotic stress, ionic toxicity and oxidative stress, seriously affecting the growth and development of plants. Therefore, it is crucial to improve the resistance of crops to high-salt environments to cope with the increasingly serious threat of environmental salt stress.

[0003] Alfalfa (Medicago sativa L.) is an important leguminous forage grass in China and even in the world. It is known as the "king of forage grasses" because of its high forage yield, rich nutrition and good palatability, and plays an important role in the development of animal husbandry production in China. In addition, alfalfa has strong adaptability and developed roots, and can grow well in a mild salt stress environment. However, at present, salt stress is still one of the main environmental factors affecting the yield of alfalfa in Northeast China, seriously restricting the development of agriculture and animal husbandry in this region.

[0004] The main means to improve the resistance of alfalfa to high-salt environments in the prior art are: exploring salt-tolerant genes in alfalfa and using transgenic means to cultivate new alfalfa varieties resistant to salt stress, so as to increase the yield of alfalfa. For example: Chinese Patent CN105925589A disclosed a salt-tolerant and alkali-resistant gene MsFLS of alfalfa, its encoded protein and application on September 7, 2016. The full length of this salt-tolerant and alkali-resistant gene MsFLS is 1068bp, encoding 355 amino acids. A plant expression vector containing this salt-tolerant and alkali-resistant gene MsFLS of alfalfa was constructed, and the constructed plant expression vector was transformed into tobacco tissues by Agrobacterium-mediated method, and plants with enhanced salt and alkali resistance were screened. This MsFLS gene provides a theoretical basis for studying the molecular mechanism of alfalfa stress resistance and breeding. However, the breeding cycle of this method is long, the investment is large, the price of transgenic seeds is high, and there are doubts about the long-term safety, which limits the large-scale planting of alfalfa. Summary of the Invention

[0005] The purpose of the present invention is to provide a dsRNA of Medicago sativa MsGI gene and its application in salt stress resistance of Medicago sativa, aiming to solve the problem that the method of using the salt-tolerant gene of alfalfa to cultivate new alfalfa varieties resistant to salt stress in the prior art limits the large-scale planting of alfalfa due to the long breeding cycle, large investment, high price of transgenic seeds and doubts about long-term safety.

[0006] In order to solve the above technical problems, the present invention is mainly implemented through the following technical solutions:

[0007] In one aspect, the present invention provides a dsRNA of an alfalfa MsGI gene, wherein the nucleotide sequence of the dsRNA of an alfalfa MsGI gene is shown as SEQ ID No.1.

[0008] RNA interference (RNAi) refers to the phenomenon of highly conserved, highly efficient and specific degradation of homologous mRNA (messenger ribonucleic acid) induced by double-stranded RNA (dsRNA) in the evolutionary process. RNAi technology is a biological mechanism that induces gene silencing by targeting complementary mRNA for degradation. In the present invention, the dsRNA (double-stranded RNA) of the MsGI gene (MsGigantea gene) of alfalfa (Medicago sativa L.) can target the MsGI gene of alfalfa, reduce the mRNA level of the MsGI gene of alfalfa by RNA interference, knock down the MsGI gene of alfalfa, achieve the purpose of silencing the key negative regulatory gene of alfalfa stress resistance, remove the active oxygen produced by alfalfa under salt stress, reduce the accumulation of salt ions in the root system of alfalfa, thereby significantly enhancing the resistance of alfalfa to environmental salt stress and promoting the growth of alfalfa.

[0009] As a preferred embodiment, the nucleotide sequence of the alfalfa MsGI gene is shown in SEQ ID No. 2. The alfalfa MsGI gene is a key negative regulatory gene in alfalfa salt stress. After the alfalfa MsGI gene function is lost, it will promote the removal of active oxygen in leaves and roots, alleviating the adverse effects of alfalfa under salt stress.

[0010] As a preferred embodiment, the amino acid sequence of the alfalfa MsGI gene is shown in SEQ ID No. 3. The alfalfa MsGI gene of the present invention is used as a target gene for RNAi, and knocking down the alfalfa MsGI gene will enhance the removal of active oxygen under salt stress in alfalfa, thereby enhancing the salt resistance of alfalfa.

[0011] In another aspect, the present invention provides a use of a dsRNA of the alfalfa MsGI gene in improving the resistance of alfalfa to environmental salt stress.

[0012] In the present invention, the dsRNA of the Medicago sativa MsGI gene can target the Medicago sativa MsGI gene, reduce the mRNA level of the Medicago sativa MsGI gene by RNA interference, scavenge the reactive oxygen species generated by Medicago sativa under salt stress, reduce the accumulation of salt ions in the roots of Medicago sativa, thereby significantly enhancing the resistance of Medicago sativa to environmental salt stress and promoting the growth of Medicago sativa. The present invention first proposes to improve the stress resistance of Medicago sativa by RNA interference technology, and silence the target gene of Medicago sativa - the Medicago sativa MsGI gene by external application of double-stranded RNA, thereby improving the ability of Medicago sativa to resist abiotic stress - salt stress resistance.

[0013] As a preferred embodiment, the dsRNA expression vector of the Medicago sativa MsGI gene is an aqueous solution. In the present invention, the dsRNA of the Medicago sativa MsGI gene is prepared into an aqueous solution. As an external biological stimulant, it is convenient to use, green and safe, has a quick effect, promotes the growth of Medicago sativa, has good market application prospects, and is worthy of large-scale popularization and application. The roots of Medicago sativa can be directly immersed in the aqueous solution for cultivation. Of course, the dsRNA of the Medicago sativa MsGI gene can also be prepared into various forms such as foliar fertilizer and root fertilizer, which is a new type of biological stimulant for Medicago sativa cultivation.

[0014] As a preferred embodiment, the concentration of the dsRNA of the Medicago sativa MsGI gene in the aqueous solution is 150 - 200 mg / L. The aqueous solution containing the dsRNA of the Medicago sativa MsGI gene in the present invention has good stability, and the active ingredient is not easily decomposed or precipitated. Both water and the active ingredient can be effectively absorbed by Medicago sativa, reducing waste; in addition, the concentration of dsRNA in the aqueous solution containing the dsRNA of the Medicago sativa MsGI gene in the present invention can be adjusted according to actual conditions to meet the needs of different growth stages of Medicago sativa.

[0015] As a preferred embodiment, the method for preparing the aqueous solution comprises the following steps: 1) Download the mRNA sequence of Medicago truncatula, a related species of alfalfa, from the NCBI database, save it, select alfalfa in the alfalfa database, input the mRNA sequence of Medicago truncatula, perform a homologous alignment, analyze the protein sequence using an mRNA translation software, analyze the domain using the SMART website, and perform a phylogenetic tree analysis for identification to obtain the nucleotide sequence of the alfalfa MsGI gene; 2) Submit the nucleotide sequence of the alfalfa MsGI gene to the siDirect version 2.1 server, predict small interfering RNAs, select the region within a sequence length of 200 - 500 bp that generates the most small interfering RNAs as the dsRNA template. The nucleotide sequence of the dsRNA template is as shown in SEQ ID No.4. Design primers, amplify. The nucleotide sequence of the upstream primer is as shown in SEQ ID No.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.6; 3) Take the seedlings of alfalfa, extract RNA, use the reverse-transcribed cDNA as a template, perform PCR amplification, subject the product to agarose gel electrophoresis and phenol-chloroform extraction to obtain DNA, and clone it into an empty plasmid to obtain a recombinant plasmid; 4) After the recombinant plasmid is sequenced, it is transferred into an engineering bacterium, inoculated at a ratio of 1:100, induced to ferment to produce dsRNA, collect the bacterial cells, purify them, and dissolve them in enzyme-free ddH 2 O to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

[0016] The aqueous solution containing the dsRNA of the alfalfa MsGI gene in the present invention uses the target gene MsGigantea gene as a template, designs upstream and downstream primers, adds a vector sequence to the 5′ segment of the primers, and finally is amplified by PCR (Polymerase Chain Reaction) and ligated with the vector and transferred into an engineering bacterium for fermentation. This preparation method has a short technological process, is easy to operate, convenient to control, and suitable for large-scale production. When this aqueous solution containing the dsRNA of the alfalfa MsGI gene encounters salt stress during the cultivation of alfalfa, the roots of alfalfa are thicker, the leaves are less poisoned by salt, the wilting degree is lighter, the reactive oxygen species generated by alfalfa under salt stress are removed, and the accumulation of salt ions in the roots of alfalfa is reduced, thereby significantly enhancing the resistance of alfalfa to environmental salt stress.

[0017] In the present invention, RNA, i.e., Ribonucleic Acid, ribonucleic acid; NCBI database, i.e., National Center for Biotechnology Information Database, the full name is the National Center for Biotechnology Information Database of the United States; the SMART website is used to analyze the domains of the obtained protein sequences, and its website is http: / / smart.embl - heidelberg.de; the siDirect version 2.1 server is a server for online designing of highly efficient and target - specific siRNA; DNA, i.e., Deoxyribonucleic Acid, deoxyribonucleic acid; cDNA, i.e., Complementary DNA, complementary DNA; enzyme - free ddH 2 O, Double Distilled Water free of RNase and DNase, double - distilled water free of ribonuclease and deoxyribonuclease, is a specially treated double - distilled water. By various methods, ribonuclease (RNase) and deoxyribonuclease (DNase) in it are removed or inactivated. The purpose is to prevent these enzymes from degrading nucleic acids (such as DNA, RNA) in the experiment and ensure the accuracy and reliability of the experimental results.

[0018] As a preferred embodiment, in step 4), the induced fermentation is carried out by adding IPTG for induced fermentation. The conditions for induced fermentation are: 0.5 mM IPTG, 37 °C, 200 r / min, 8 h. The present invention utilizes the stress - resistant dsRNA interference technology of plants to design and synthesize dsRNA sequence fragments. The dsRNA sequence fragments have a stress - resistant effect on plants and are a dsRNA application technology for improving plant stress resistance. The present invention uses IPTG (isopropyl - β - D - thiogalactoside) for induced fermentation. The induced fermentation conditions are mild, the dosage of IPTG is small, the cost is low, the fermentation speed is fast, and a large amount of dsRNA can be rapidly produced.

[0019] As a preferred embodiment, the empty plasmid in step 3) is the L4440 plasmid, and the engineered bacterium in step 4) is HT115. HT115 in the present invention is a commonly used engineered bacterium belonging to Escherichia coli; HT115 has a wide source, is convenient to obtain materials, is inexpensive and easy to get, and is convenient to use. The L4440 plasmid is an interfering and silencing expression plasmid with a size of 2790 bp, having a T7 promoter and a PUC replicon, and is mainly used for interference and silencing.

[0020] As a preferred embodiment, the method for preparing the aqueous solution comprises the following steps: 1) Download the mRNA sequence of Medicago truncatula, a related species of Medicago sativa, from the NCBI database, save it, select Medicago sativa in the Medicago sativa database, input the mRNA sequence of Medicago truncatula, perform a homologous alignment, analyze the protein sequence with an mRNA translation software, analyze the domain on the SMART website, and perform a phylogenetic tree analysis for identification to obtain the nucleotide sequence of the Medicago sativa MsGI gene; 2) Submit the nucleotide sequence of the Medicago sativa MsGI gene to the siDirect version 2.1 server, predict small interfering RNAs, select the region within a sequence of 200 - 500 bp that generates the most small interfering RNAs as the dsRNA template. The nucleotide sequence of the dsRNA template is as shown in SEQ ID No.4. Design primers for in vitro amplification. The nucleotide sequence of the upstream in vitro amplification primer is as shown in SEQ ID No.7, and the nucleotide sequence of the downstream in vitro amplification primer is as shown in SEQ ID No.8. Perform PCR amplification and purify with phenol-chloroform to obtain the dsRNA in vitro synthesis template; 3) Using the T7 high-yield transcription kit, in the following synthesis system: 8 μL of NTP, 4 μL of 5×TranscrptAid Reaction Buffer, 1 μg of the dsRNA in vitro synthesis template, 2 μL of TranscrptAid Enzyme Mix, and supplement with enzyme-free ddH 2 O to 20 μL, and synthesize overnight at 37°C; 4) Digest the remaining dsRNA in vitro synthesis template with DNAase I, extract with phenol-chloroform, and precipitate with absolute ethanol to obtain an aqueous solution containing the dsRNA target sequence of the Medicago sativa MsGI gene.

[0021] The aqueous solution containing dsRNA of Medicago sativa MsGI gene of the present invention can also be synthesized in vitro. Using the target gene MsGigantea gene as a template, the upstream and downstream primers for in vitro amplification are designed, and the T7 promoter sequence (TAATACGACTCACTATAGGG) is added to the 5′ end of the upstream and downstream primers respectively. Through PCR amplification, it is synthesized overnight at 37°C in a synthesis system composed of a T7 high-yield transcription kit (TranscriptAid T7 HighYield Transcription Kit (Thermoscientific, K0441)); this synthesis method can obtain dsRNA with higher purity. The obtained aqueous solution containing dsRNA of Medicago sativa MsGI gene is labeled with UTP-cy3 and can be detected by fluorescence. The whole process requires light-shielding operation. Among them, NTP, that is, Nucleoside TriPhosphate, nucleoside triphosphate; TranscrptAid Reaction Buffer, reaction buffer; the dsRNA in vitro synthesis template is the dsRNA in vitro synthesis template obtained in step 2); TranscrptAid EnzymeMix, enzyme mixture; DNAase I, that is, DNA enzyme I, also called deoxyribonuclease I.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows: The dsRNA of Medicago sativa MsGI gene in the present invention can target the Medicago sativa MsGI gene, reduce the mRNA level of the Medicago sativa MsGI gene by means of RNA interference, scavenge the reactive oxygen species generated by Medicago sativa under salt stress, and reduce the accumulation of salt ions in the roots of Medicago sativa; when this aqueous solution containing dsRNA of Medicago sativa MsGI gene encounters salt stress during the cultivation of Medicago sativa, the roots of Medicago sativa are thicker, the leaves are less poisoned by salt, and the wilting degree is lighter, which promotes the growth of Medicago sativa and significantly enhances the resistance of Medicago sativa to environmental salt stress; this aqueous solution containing dsRNA of Medicago sativa MsGI gene is convenient to use, has a quick effect, is green and safe, has a good market application prospect, and is worthy of large-scale popularization and application. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0024] Figure 1 It is the electrophoresis detection result diagram of the aqueous solution obtained in Example 1 of the present invention;

[0025] Figure 2 Electrophoresis detection result diagram after the aqueous solution obtained in the first embodiment of the present invention is digested by RNase III;

[0026] Figure 3 In the present invention, alfalfa phenotypic trait photo diagram cultured with enzyme-free ddH 2 O as a control sample;

[0027] Figure 4 Alfalfa phenotypic trait photo diagram cultured with the aqueous solution obtained in the first embodiment of the present invention;

[0028] Figure 5 In the present invention, alfalfa after sodium chloride stress with enzyme-free ddH 2 O as a control sample, NBT staining photo diagram;

[0029] Figure 6 Alfalfa NBT staining photo diagram after sodium chloride stress cultured with the aqueous solution obtained in the first embodiment of the present invention;

[0030] Figure 7 In the present invention, alfalfa after sodium chloride stress with enzyme-free ddH 2 O as a control sample, DAB staining photo diagram;

[0031] Figure 8 Alfalfa DAB staining photo diagram after sodium chloride stress cultured with the aqueous solution obtained in the first embodiment of the present invention;

[0032] Figure 9 Root fluorescence imaging diagram of alfalfa cultured with enzyme-free ddH 2 O as a control sample in the present invention;

[0033] Figure 10 Root fluorescence imaging diagram of alfalfa cultured with the aqueous solution obtained in the second embodiment of the present invention;

[0034] Figure 11 Leaf fluorescence imaging diagram of alfalfa cultured with enzyme-free ddH 2 O as a control sample in the present invention;

[0035] Figure 12 Leaf fluorescence imaging diagram of alfalfa cultured with the aqueous solution obtained in the second embodiment of the present invention. Detailed implementation manners

[0036] The technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts fall within the scope of protection of the present invention.

[0037] A dsRNA of Medicago sativa MsGI gene of the present invention, wherein the nucleotide sequence of the dsRNA of Medicago sativa MsGI gene is shown in SEQ ID No.1.

[0038] Preferably, the nucleotide sequence of the Medicago sativa MsGI gene is shown in SEQ ID No.2.

[0039] Furthermore, the amino acid sequence of the Medicago sativa MsGI gene is shown in SEQ ID No.3.

[0040] An application of a dsRNA of Medicago sativa MsGI gene of the present invention in improving the resistance of Medicago sativa to environmental salt stress.

[0041] Preferably, the dsRNA expression vector of the Medicago sativa MsGI gene is an aqueous solution.

[0042] Furthermore, the concentration of the dsRNA of the Medicago sativa MsGI gene in the aqueous solution is 150 - 200 mg / L.

[0043] Specifically, the preparation method of the aqueous solution includes the following steps:

[0044] 1) Download the mRNA sequence of the Medicago sativa relative species Medicago truncatula in the NCBI database, save it, select Medicago sativa in the Medicago sativa database, input the mRNA sequence of Medicago truncatula, perform homology alignment, analyze the protein sequence with mRNA translation software, analyze the domain on the SMART website, and perform phylogenetic tree analysis and identification to obtain the nucleotide sequence of the Medicago sativa MsGI gene;

[0045] 2) Submit the nucleotide sequence of the Medicago sativa MsGI gene to the siDirect version 2.1 server, predict small interfering RNA, select the region with the most small interfering RNA generated within the sequence of 200 - 500 bp as the dsRNA template, the nucleotide sequence of the dsRNA template is shown in SEQ ID No.4, design primers, amplify, the nucleotide sequence of the upstream primer is shown in SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.6;

[0046] 3) Take the seedlings of Medicago sativa, extract RNA, use the obtained cDNA by reverse transcription as a template, perform PCR amplification, electrophorese the product on an agarose gel and extract it with phenol-chloroform to obtain DNA, and clone it into an empty plasmid to obtain a recombinant plasmid;

[0047] 4) After the recombinant plasmid is sequenced, it is transferred into an engineering bacterium, inoculated at a ratio of 1:100, induced to ferment to produce dsRNA, collect the bacterial cells, purify them, and dissolve them in enzyme-free ddH 2 O to obtain an aqueous solution containing the dsRNA target sequence of the Medicago sativa MsGI gene.

[0048] Preferably, in step 4), the induced fermentation is induced by adding IPTG, and the induced fermentation conditions are: 0.5 mM IPTG, 37 °C, 200 r / min, 8 h.

[0049] Preferably, the empty plasmid in step 3) is the L4440 plasmid, and the engineering bacterium in step 4) is HT115.

[0050] Specifically, the preparation method of the aqueous solution includes the following steps:

[0051] 1) Download the mRNA sequence of the Medicago sativa related species Medicago truncatula in the NCBI database, save it, select Medicago sativa in the Medicago sativa database, input the mRNA sequence of Medicago truncatula, perform a homologous alignment, analyze the protein sequence with an mRNA translation software, analyze the domain on the SMART website, and perform a phylogenetic tree analysis for identification to obtain the nucleotide sequence of the Medicago sativa MsGI gene;

[0052] 2) Submit the nucleotide sequence of the Medicago sativa MsGI gene to the siDirect version 2.1 server, predict small interfering RNAs, select the region that produces the most small interfering RNAs within a sequence length of 200 - 500 bp as the dsRNA template. The nucleotide sequence of the dsRNA template is as shown in SEQ ID No. 4. Design primers for in vitro amplification. The nucleotide sequence of the upstream in vitro amplification primer is as shown in SEQ ID No. 7, and the nucleotide sequence of the downstream in vitro amplification primer is as shown in SEQ ID No. 8. Perform PCR amplification and purify it with phenol-chloroform to obtain the dsRNA in vitro synthesis template;

[0053] 3) Use the T7 high-yield transcription kit. In the following synthesis system: 8 μL of NTP, 4 μL of 5×TranscrptAid Reaction Buffer, 1 μg of the dsRNA in vitro synthesis template, 2 μL of TranscrptAid Enzyme Mix, and supplement it to 20 μL with enzyme-free ddH 2 O and synthesize overnight at 37 °C;

[0054] 4) Digest the remaining dsRNA in vitro synthesis template with DNAase I, extract with phenol-chloroform, and precipitate with absolute ethanol to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

[0055] Example 1

[0056] A method for preparing an aqueous solution of dsRNA containing the alfalfa MsGI gene of the present invention includes the following steps:

[0057] 1) In the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), search for and download the mRNA sequence of the alfalfa related species Medicago truncatula, and then save it; in the alfalfa database (https: / modms.lzu.edu.cn / alfalfa / blast / blastPage), select alfalfa (M. sativa cultivar XinJiangDaYe) and input the above mRNA sequence of Medicago truncatula for homologous alignment. Then, parse the protein sequence through the mRNA translation software (https: / / web.expasy.org / translate / ), analyze the domain using the SMART website (http: / / smart.embl-heidelberg.de / ), and perform phylogenetic tree analysis and identification to obtain the nucleotide sequence of the alfalfa MsGI gene (as shown in SEQ ID No. 2), and the amino acid sequence of the alfalfa MsGI gene is as shown in SEQ ID No. 3;

[0058] SEQ ID No. 2 is:

[0059] ATGACTTCTTCTATGGCTGCTACAAGTGAAAGGTGGATTGACCGTCTTCAATTTTCTTCAT

[0060] TGTTCTGGCCTCCCCCACAAGATGTTCAGCAAAAAAAGGATCAAATTGCTGCATATGTTG

[0061] AGTACTTGATTCAGTTTACATCAGAACAATTTGCTGACGATATTGCCGAGTTGATCCGCA

[0062] ACCGTTACCCGTCGAAGGAAATTATTCTCTTTGATGACGTTTTGGCAACATTTGTCCTTCA

[0063] TCATCCAGAGCATGGGCATGCAGTTGTACTTCCAATTATTTCGTGTATTATCGATGGTACA

[0064] CTAGTCTATGACAAGACTAGTCCTCCATTTGCTTCTTTGATATCTTTAGTCTGCCCAAAAG

[0065] ACGAGAATGAATATTCAGAACAATGGGCTCTTGCATGCGGTGAAATTTTGCGTATTTTAA

[0066] CTCATTACAATCGGCCCATTTACAAAACGGAAAGGCAATCCAGTGAAACAGAAAGAAG

[0067] CAGTAGTGGCAGCCATGCGACAACTAGTGAGCCACTTAATGGGAAAGCTGTCAATAATG

[0068] CTTTGGCACAACAAGAGAAAAAACCTATCAGGCCCTTGTCCCCCTGGATTACTGATATAT

[0069] TGTTAGTTGCGCCCGTAGGCATTAGAAGTGACTATTTCCGGTGGTGCAGTGGTGTTATGG

[0070] GCAAATATGCAGCAGGAGAACTCAAGCCTCCATCAATTGCTACTTCCCGTGGTTCTGGG

[0071] AAGCATCCTCAACTCGTGCCTTCTACTCCCAGATGGGCTGTTGCCAATGGTGCTGGTGTT

[0072] ATATTAAGTGTTTGTGATGATGAAGTTGCTCGCAATGAAACTGCTATTTTAACAGCAGCA

[0073] GCTGTTCCTGCACTTCTGCTTCCTCCTCCAACAACAGCTCTGGATGAACATCTTGTTGCT

[0074] GGACTACCAGCTCTAGAGCCATATGCCCGTTTATTTCACAGATACTATGCAATTGCTACTC

[0075] CAAGTGCTACACAAAGACTTCTTCTTGGACTTCTAGAAGCACCCCCGTCGTGGGCTCCG

[0076] GATGCCCTTGATGCTGCTGTGCAGCTTGTGGAGCTTCTTCGAGCTGCTGAAGAGTATGCT

[0077] TCTGGCATAAGGCTTCCTAGAAATTGGATGCATTTGCATTTCTTGCGTGCAATAGGGACT

[0078] GCAATGTCCATGAGAGCTGGCATAGCTGCTGATGCTGCAGCTGCTTTGCTTTTCCGTATA

[0079] CTTTCACAGCCAGCATTGCTTTTTCCTCCTCTTAGACAAGTTGATGGAGTTGAAGTTCAG

[0080] CATGAACCTTTGGGTGAAGTTCCTGCTGCTGAAGCCTCTATTGACGCTACTGCCCAAGG

[0081] CATTGCGTCAATGCTTTGCGCCCATGGTCCAGAAGTTGAATGGAGAATTTGCACCATTTG

[0082] GGAAGCTGCTTATGGCCTGATTCCTGCAAGTTCCTCAGCCGTAGATCTTCCAGAGATCAT

[0083] AGTTGCAGCCCCACTACAACCTCCCATACTATCATGGAATTTGTACATACCTCTACTTAAG

[0084] GTCCTGGAATATCTTCCTCGTGGAAGCCCGTCAGAGGCATGCCTTATGAAAATATTTGCT

[0085] GCTACAGTTGAAGCTATTCTTCAGAGGACATTTCCACCCGAGTCCAGCAGAGAACAAAA

[0086] CAGAAAAGCAAATTACCTCTTTGGTCTTGGTTCAGCCTCTAAAAACCTTGCTGTGGCAG

[0087] AGCTTCGTACTATGGTTCATTCACTTTTTTTAGAATCATGTGCATCTGTAGAGCTTTCTTC

[0088] GCGCCTACTTTTTGTTGTCTTAACTGTCTGTGTCAGTCATGAAGCCCAATTCAGTGGAAG

[0089] CAAGAAGCCTAGAGGTGAAGATAATTATTCAGTTGAGGAAATCATTGAGGACTTGCACG

[0090] CAATATCAGAAATCCGGAAAGAAAGAAAAAATAGGAAAGTGAAGAAGCAAGGTCCTGT

[0091] AGCAGCATTTGATTCTTATGTTATGGCTGCTGTTTGTGCTCTTGCCTGTGAGCTTCAGTTG

[0092] TTTCCTTTGATGTCACGGGGAAATAATCATTCAGTTTCCAATAATGTGCAAGATATAGCCA

[0093] AGCCCGTTACACTACACGGATCATCCCAAGATTTGCAGAATGGCCTAGATTCGGCAGTCC

[0094] GCCATACTCACAGAATTTTAGCAATTTTAGAGGCACTATTTTCATTAAAGCCGTCTTCTGT

[0095] TGGCACTCCTTGGAGTTACAGCTCAAATGAGATAGTGGCAGCAGCTATGGTTGCTGCAC

[0096] ATGTTTCTGAACTATTTAGACGGTCAAAAGCTTGCATGCATGCTCTGTCTGTTCTGATACG

[0097] TTGCAAATGGAACAAAGAAATTCACTCCAGGGCATCATCATTGTATAATCTCATAGATATC

[0098] CACAGTAAAGTTGTTGCATCTATAGTGAATAAGGCAGAGCCATTAGAAGCAACCTTGATT

[0099] CACGCACCTATTTACAAGGACGCACTTGTTTGTCATGACGGTAAAAGAAAGAATCGGAG

[0100] TGAAAATGGTAGCTGCTCTGACCCTGGGCAGACGTCGATTGTACCTTCGGCTGATTCCA

[0101] CACCATCAAAACATATCCATAAATCTGGAAGAACTCCATGTTCAGATGAAGAAGCATCA

[0102] GGGTATAACTTGGGTAAAAGTGTCACCAGTTTCTCGTTGGAGGCATCTGATCTCGCCAAT

[0103] TTCCTCACAATGGACAGGCATATAGGCTTGAATTGCAATACACAGATCTTTCTGATATCCA

[0104] TGCTGTCTGAGAAACAGGAGTTATGTTTCTCTGTAGTTTCTCTACTATGGCACAAATTGA

[0105] TTGCATCTCCTGAAACTCAACCATGTTCTGAAAGCACTTCTGCCCAGCAGGGCTGGAGA

[0106] CAGGTTGTCGATGCATTATGCAATGTTGTATCAGCCTCGCCAGCAAAAGCAGCTACAGCT

[0107] GTTGTTCTTCAGGCGGAGAAGGAATTGCAGCCCTGGATAGCCAAGGATGATGATCTAGG

[0108] TCAGAAGATGTGGAGAATCAATCAGCGGATCGTTAAATTGATAGTGGAACTTATGAGGA

[0109] ATCATGATAGCGCGGAGTCATTGGTAATTCTGGCAAGTGCATCAGATTTACTTTTACGAG

[0110] CCACAGATGGGATGCTTGTTGATGGAGAAGCTTGTACTTTACCACAGCTGGAGCTATTGG

[0111] AAGCGACAGCTAGAGCAATTCAGCCAGTGCTTGAGTTTGGAGAACCAGGATTGGCTGT

[0112] AGCAGATGGCCTTTCAAACCTTTTGAAGTGTCGTCTAGCAGCTACCATTAGGTGCCTTTG

[0113] TCATCCAAGTGCACATGTCCGTGCTCTGAGTGTCTCAGTTCTCCGTGACATTTTGCATAC

[0114] CGGTTCAATAAGGTGTAGTCCTAAACCACTGCGAATAAACGGCAGCCACAATCCCTCTTA

[0115] TCCATACTTCAAGTTGGACGTCGTTGATTGGCAAGCTGACATAGAAAAATGCTTGGCATG

[0116] GGAAGCTCACAGCAGAATTTCTGCTGGATTGCCTATAAAGTTTCTTGACACTGCTGCTAA

[0117] AGAACTAGGCTGCGCTATTTCCTCTTTGGTGGCGAACGAAGAATTTCAGCACATTCTGC

[0118] GTGTGTTGAACACAAATGTTGATGGAAAGCAGAAGATCCAGTTCGCAATGACCTCAATC

[0119] AAGGGTATTGGTAGGAGATTCGCTAACATCTGCTGTAAGAAGGCCGATGTTGACATGAA

[0120] CAAGAGGGCTGGTGAATTGAGTGCCGCAGAGTTGGATAATATTATGACAGTGGTTGCGA

[0121] ATCCTCGTCAATTCAAGGTTCCAGATTGGTTTTTGAACAGAAAGAAGGATTACAAGGAT

[0122] GGCAAATTCTCTCAGGTTGTCTCCAATCAACTCGACATGAAGTTGAGAGATGATTTGGA

[0123] GAGACTCAAGAAGATCCGTAATCACCGGGGTTTGAGGCACTACTGGGGCCTTCGTGTTC

[0124] GTGGTCAGCATACCAAGACTACAGGCCGCAGGGGAAAGACTGTTGGTGTCTCAAAGAA

[0125] GCGT

[0126] SEQ ID No.3 is

[0127] MTSSMAATSERWIDRLQFSSLFWPPPQDVQQKKDQIAAYVEYLIQFTSEQFADDIAELIRNR

[0128] YPSKEIILFDDVLATFVLHHPEHGHAVVLPIISCIIDGTLVYDKTSPPFASLISLVCPKDENEYS

[0129] EQWALACGEILRILTHYNRPIYKTERQSSETERSSSGSHATTSEPLNGKAVNNALAQQEKKPI

[0130] RPLSPWITDILLVAPVGIRSDYFRWCSGVMGKYAAGELKPPSIATSRGSGKHPQLVPSTPRWA

[0131] VANGAGVILSVCDDEVARNETAILTAAAVPALLLPPPTTALDEHLVAGLPALEPYARLFHRYY

[0132] AIATPSATQRLLLGLLEAPPSWAPDALDAAVQLVELLRAAEEYASGIRLPRNWMHLHFLRAI

[0133] GTAMSMRAGIAADAAAALLFRILSQPALLFPPLRQVDGVEVQHEPLGEVPAAEASIDATAQ

[0134] GIASMLCAHGPEVEWRICTIWEAAYGLIPASSSAVDLPEIIVAAPLQPPILSWNLYIPLLKVLE

[0135] YLPRGSPSEACLMKIFAATVEAILQRTFPPESSREQNRKANYLFGLGSASKNLAVAELRTMV

[0136] HSLFLESCASVELSSRLLFVVLTVCVSHEAQFSGSKKPRGEDNYSVEEIIEDLHAISEIRKERK

[0137] NRKVKKQGPVAAFDSYVMAAVCALACELQLFPLMSRGNNHSVSNNVQDIAKPVTLHGSS

[0138] QDLQNGLDSAVRHTHRILAILEALFSLKPSSVGTPWSYSSNEIVAAAMVAAHVSELFRRSKA

[0139] CMHALSVLIRCKWNKEIHSRASSLYNLIDIHSKVVASIVNKAEPLEATLIHAPIYKDALVCHD

[0140] GKRKNRSENGSCSDPGQTSIVPSADSTPSKHIHKSGRTPCSDEEASGYNLGKSVTSFSLEASD

[0141] LANFLTMDRHIGLNCNTQIFLISMLSEKQELCFSVVSLLWHKLIASPETQPCSESTSAQQGW

[0142] RQVVDALCNVVSASPAKAATAVVLQAEKELQPWIAKDDDLGQKMWRINQRIVKLIVELMR

[0143] NHDSAESLVILASASDLLLRATDGMLVDGEACTLPQLELLEATARAIQPVLEFGEPGLAVAD

[0144] GLSNLLKCRLAATIRCLCHPSAHVRALSVSVLRDILHTGSIRCSPKPLRINGSHNPSYPYFKL

[0145] DVVDWQADIEKCLAWEAHSRISAGLPIKFLDTAAKELGCAISSLVANEEFQHILRVLNTNVD

[0146] GKQKIQFAMTSIKGIGRRFANICCKKADVDMNKRAGELSAAELDNIMTVVANPRQFKVPD

[0147] WFLNRKKDYKDGKFSQVVSNQLDMKLRDDLERLKKIRNHRGLRHYWGLRVRGQHTKTT

[0148] GRRGKTVGVSKKR

[0149] 2) Submit the nucleotide sequence of the alfalfa MsGI gene (SEQ ID No.2) obtained above to the siDirect version 2.1 server (http: / / sidirect2.rnai.jp / ) for prediction of small interfering RNA (siRNA). Select the region that produces the most siRNAs within the sequence with a length of 200 - 500 bp as the dsRNA template. The nucleotide sequence of the dsRNA template is as shown in SEQ ID No.4. Design upstream and downstream primers, and add homologous arm sequences to the 5′ ends of the upstream and downstream primers for subsequent recombinant plasmid ligation. The nucleotide sequence of the upstream primer is as shown in SEQ ID No.5, and the nucleotide sequence of the downstream primer is as shown in SEQ ID No.6, and then perform amplification;

[0150] SEQ ID No.4 is:

[0151] TGACTTCTTCTATGGCTGCTACAAGTGAAAGGTGGATTGACCGTCTTCAATTTTCTTCATT

[0152] GTTCTGGCCTCCCCCACAAGATGTTCAGCAAAAAAAGGATCAAATTGCTGCATATGTTG

[0153] AGTACTTGATTCAGTTTACATCAGAACAATTTGCTGACGATATTGCCGAGTTGATCCGCA

[0154] ACCGTTACCCGTCGAAGGAAATTATTCTCTTTGATGACGTTTTGGCAACATTTGTCCTTCA

[0155] TCATCCAGAGCATGGGCATGCAGTTGTACTTCCAATTATTTCGTGTATTATCGATGGTACA

[0156] CTAGTCTATGACAAGACTAGTCCTCCATTTGCTTCTTTGATATCTTTAGTCTGCCCAAAAG

[0157] ACGAGAATGAATATTCAGAACAATGGGCTCTTGCATGCGGTGAAATTTTGCGTATTTTAA

[0158] CTCATTACAATCGGCCCATTTACAAAACGGAAAGGCAATCCAGTGAAACAGAAAGAAG

[0159] CAGTAGTGGCAGCCATGCGACAACTAGTGAGCCACTTAATGGGAAAGCTGTCAATAATG

[0160] CTTTGGC

[0161] SEQ ID No.5 is: TACTCAGAGCTCTGACTTCTTCTATGGCTGCTACA

[0162] SEQ ID No.6 is: TACTCAGTCGACGCCAAAGCATTATTGACAGCTT

[0163] 3) Take the seedlings of alfalfa that have grown for 2 weeks, extract RNA, reverse transcribe it into cDNA, use the obtained cDNA as a template for PCR amplification, electrophorese the product on agarose gel and extract it with phenol-chloroform to obtain DNA, and clone it into the L4440 empty plasmid to obtain a recombinant plasmid;

[0164] 4) After the recombinant plasmid was sequenced, it was transferred into the engineering bacterium HT115, inoculated at a ratio of 1:100, and induced to ferment by IPTG. The induction fermentation conditions were: 0.5 mM IPTG, 37 °C, 200 r / min, 8 h to produce dsRNA. The bacterial cells were collected, dissolved in ultrapure water containing lysozyme, sonicated, extracted and purified with phenol-chloroform, and dissolved in double-distilled water without enzymes (ddH 2 O) to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

[0165] The dsRNA target sequence containing the alfalfa MsGI gene is shown in SEQ ID No. 1. SEQ ID No. 1 is: UGACUUCUUCUAUGGCUGCUACAAGUGAAAGGUGGAUUGACCGUCUUCAAUUUUCUUCAUUGUUCUGGCCUCCCCCACAAGAUGUUCAGCAAAAAAAGGAUCAAAUUGCUGCAUAUGUUGAGUACUUGAUUCAGUUUACAUCAGAACAAUUUGCUGACGAUAUUGCCGAGUUGAUCCGCAACCGUUACCCGUCGAAGGAAAUUAUUCUCUUUGAUGACGUUUUGGCAACAUUUGUCCUUCAUCAUCCAGAGCAUGGGCAUGCAGUUGUACUUCCAAUUAUUUCGUGUAUUAUCGAUGGUACACUAGUCUAUGACAAGACUAGUCCUCCAUUUGCUUCUUUGAUAUCUUUAGUCUGCCCAAAAGACGAGAAUGAAUAUUCAGAACAAUGGGCUCUUGCAUGCGGUGAAAUUUUGCGUAUUUUAACUCAUUACAAUCGGCCCAUUUACAAAACGGAAAGGCAAUCCAGUGAAACAGAAAGAAGCAGUAGUGGCAGCCAUGCGACAACUAGUGAGCCACUUAAUGGGAAAGCUGUCAAUAAUGCUUUGGC

[0166] Experiment 1

[0167] In the aqueous solution of dsRNA containing the alfalfa MsGI gene obtained in Example 1, agarose gel was added for agarose gel electrophoresis test; as shown in the appendix Figure 1 It can be seen that there is an obvious band in the agarose gel electrophoresis result.

[0168] To verify that the obtained band is the dsRNA of Medicago sativa MsGI gene, RNase III (i.e., Ribonuclease III) was added to the above aqueous solution of dsRNA containing Medicago sativa MsGI gene for digestion, and then, agarose gel electrophoresis test was carried out again. Attached Figure 2 It can be seen that the band with a size of about 500 bp was degraded after digestion by RNase III, which proves that the obtained band is the dsRNA of Medicago sativa MsGI gene.

[0169] Using Image J image processing software, the gray value was calculated to obtain the concentration of dsRNA; then, enzyme-free double-distilled water was continuously added for dilution to make the concentration of the aqueous solution of dsRNA containing Medicago sativa MsGI gene be 200 mg / L.

[0170] Experiment 2

[0171] Twenty 2-week-old seedlings of Medicago sativa Xinjiang Daye with good and consistent growth were selected and randomly divided into two groups, with 10 plants in each group. One group was the experimental group and the other group was the control group; the seedlings of Medicago sativa in the experimental group were cultured in the aqueous solution of dsRNA containing Medicago sativa MsGI gene with a concentration of 200 mg / L obtained in Experiment 1, and the seedlings of Medicago sativa in the control group were cultured in enzyme-free double-distilled water with the same dosage as the experimental group. The seedlings of both groups of Medicago sativa were cultured for 48 h.

[0172] Leaf tissues and root tissues of Medicago sativa were taken, total RNA was extracted, and cDNA was obtained by reverse transcription. Upstream and downstream specific primers were designed. The sequence of the upstream specific primer was as shown in SEQ No.9, SEQ No.9 was ATGACTTCTTCTATGGCTGC, and the sequence of the downstream specific primer was as shown in SEQ No.10, SEQ No.10 was TGCCAAAACGTCATCAAAGAG; using MsActin-2 as the internal reference gene, upstream and downstream primers of the internal reference gene were designed. The sequence of the upstream primer was as shown in SEQ No.11, SEQNo.11 was CCAGAAGTCCTTTTCCAACCATC, and the sequence of the downstream primer was as shown in SEQ No.12, SEQ No.12 was CAGACTCATCATATTCACCCTT. The mRNA expression level of the target gene MsGigantea was detected, and each tissue was sampled 3 times. The experimental results are listed in Table 1.

[0173] Table 1 mRNA expression levels of MsGigantea gene in Medicago sativa tissues after cultivation under different conditions

[0174]

[0175] The data in Table 1 were subjected to paired comparison by Student's t - test, and statistical analysis was performed using a two - tailed paired test. Compared with the control group formed by alfalfa cultivated with enzyme - free double - distilled water, in the experimental group formed by alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention, the mRNA level of the target gene MsGigantea in the leaf tissue decreased, and the p - value was less than 0.005, showing extremely significant; the mRNA level of the target gene MsGigantea in the root tissue also decreased, and the p - value was less than 0.05, showing significant. Therefore, the mRNA level of the target gene MsGigantea in the leaf tissue of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention decreased extremely significantly, and the mRNA level of the target gene MsGigantea in the root tissue of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention decreased significantly. This indicates that the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention can be absorbed by the root system of alfalfa and significantly reduces the expression of the alfalfa MsGigantea gene.

[0176] Experiment 3

[0177] Again, 100 two - week - old seedlings of Xinjiang big - leaf alfalfa with good and consistent growth were selected and randomly divided into two groups, with 50 plants in each group. One group was the experimental group, and the other group was the control group. The alfalfa seedlings in the experimental group were cultivated in the dsRNA aqueous solution containing the alfalfa MsGI gene with a concentration of 200 mg / L obtained in Experiment 1, and the alfalfa seedlings in the control group were cultivated in enzyme - free double - distilled water with the same dosage as the experimental group. The seedlings of both groups of alfalfa were cultivated for 48 h. Then, sodium chloride was added to the culture solutions of both groups of alfalfa seedlings to make the concentration of sodium chloride in the culture solution 150 mM to simulate salt stress conditions for 3 d. Observations were made every 12 h to observe the growth of alfalfa seedlings and record by taking pictures.

[0178] As can be seen from the appendix Figure 3 It can be seen that when the control group formed by alfalfa cultivated with enzyme - free double - distilled water was stressed with sodium chloride addition for 3 d, the degree of leaf wilting of alfalfa seedlings was large, and some leaves were even severely wilted. At the same time, the roots of alfalfa seedlings in the control group were thin and short, and the development was retarded. However, as can be seen from the appendix Figure 4 It can be seen that when the experimental group formed by alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention was stressed with sodium chloride addition for 3 d, the degree of leaf wilting of alfalfa seedlings was small, and the growth was uniform. At the same time, the roots of alfalfa seedlings in the experimental group were thick and strong, and the development was good.

[0179] Experiment 4

[0180] Take the alfalfa seedlings treated for 3 days in Experiment 3, with 3 plants in each group. Separate the roots and measure the sodium ion concentration in the roots. Repeat the experiment 2 times to obtain the results of 3 parallel experiments, which are listed in Table 2.

[0181] Table 2 Sodium ion content in the roots of alfalfa after cultivation under different conditions

[0182]

[0183] Analysis of the data in Table 2 by the method of Experiment 2 shows that, compared with the control group of alfalfa cultivated with enzyme-free double-distilled water, in the experimental group of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention, the sodium ion content in the roots is reduced, and the p-value is less than 0.05, showing significance. Therefore, alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention can significantly reduce the sodium ion content in its roots when encountering sodium chloride stress.

[0184] Experiment 5

[0185] Take another group of 3 alfalfa seedlings treated for 3 days in Experiment 3. Collect the root tissues and detect the enzyme activities of catalase (CAT) and peroxidase (POD) through a kit. Repeat the experiment 2 times to obtain the results of 3 parallel experiments, which are listed in Table 3.

[0186] Analysis of the data in Table 3 by the method of Experiment 2 also shows that, compared with the control group of alfalfa cultivated with enzyme-free double-distilled water, in the experimental group of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention, the enzyme activity of catalase in the root tissues increases, and the p-value is less than 0.05, showing significance; the enzyme activity of superoxide dismutase in the root tissues also increases, and the p-value is also less than 0.05, showing significance. Therefore, alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention can significantly enhance the activities of two oxidases, catalase and superoxide dismutase, in its roots when encountering sodium chloride stress.

[0187] Table 3 Enzyme activities in the roots of alfalfa after cultivation under different conditions

[0188]

[0189] Experiment 6

[0190] Take another group of 3 alfalfa seedlings treated for 3 days in Experiment 3. Separate the roots and detect the content of malondialdehyde (MDA) in the roots by the thiobarbituric acid colorimetric method. Repeat the experiment 2 times to obtain the results of 3 parallel experiments, which are listed in Table 4.

[0191] Table 4 Content of malondialdehyde in the roots of alfalfa cultivated under different conditions

[0192]

[0193] Analysis of the data in Table 4 using the method of Experiment 2 shows that, compared with the control group of alfalfa cultivated with enzyme-free double-distilled water, in the experimental group of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention, the content of MDA in the roots decreased, and the p-value was less than 0.05, showing significance. Therefore, alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention can significantly increase the content of malondialdehyde, an index of membrane lipid peroxidation in its roots, when encountering sodium chloride stress.

[0194] Experiment 7

[0195] Take the alfalfa seedlings treated for 3 days in Experiment 3 again, 3 plants in each group. Separate the roots and leaves, and place them in NBT staining solution and DAB staining solution respectively. Immerse them in the dark for 24 hours for staining, rinse with distilled water, soak in 95% anhydrous ethanol, and heat at 80 °C until the original color completely fades. Take pictures and repeat 2 times. Observe the tissue reactive oxygen species situation by taking clear pictures.

[0196] From Figure 5 It can be seen that when the control group of alfalfa cultivated with enzyme-free double-distilled water was added with sodium chloride stress for 3 days, whether it was the roots or the leaves, after staining in the NBT staining solution, the coloring degree was very deep and the pictures were cloudy; however, from the attached Figure 6 It can be seen that when the experimental group of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention was added with sodium chloride stress for 3 days, whether it was the roots or the leaves, after staining in the NBT staining solution, the coloring degree was relatively light and the pictures were clear and transparent. This shows that under salt stress conditions, compared with the control group of alfalfa cultivated with enzyme-free double-distilled water, the experimental group of alfalfa cultivated with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention alleviated the accumulation of superoxide anions in the roots and leaves of alfalfa.

[0197] From Figure 7 It can be seen that when the control group of alfalfa cultivated with enzyme-free double-distilled water was added with sodium chloride stress for 3 days, whether it was the roots or the leaves, after staining in the DAB staining solution, the coloring degree was very deep and the pictures were dull; however, from the attached Figure 8It can be seen that when the alfalfa formed by the experimental group cultured with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention was subjected to sodium chloride stress for 3 days, whether it was the root or the leaf, after staining with the DAB staining solution, the coloring degree was relatively light, and the photo was clear and transparent. This indicates that under salt stress conditions, compared with the control group formed by alfalfa cultured with enzyme-free double-distilled water, the experimental group formed by alfalfa cultured with the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention alleviated the accumulation of hydrogen peroxide in the roots and leaves of alfalfa.

[0198] In summary, the dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention can scavenge the reactive oxygen species generated by alfalfa under salt stress, reduce the accumulation of sodium ions in the roots of alfalfa, and thus significantly enhance the salt tolerance of alfalfa seedlings.

[0199] Example 2

[0200] A method for preparing a dsRNA aqueous solution containing the alfalfa MsGI gene of the present invention comprises the following steps:

[0201] 1) In the NCBI database (https: / / www.ncbi.nlm.nih.gov / ), search for and download the mRNA sequence of Medicago truncatula, a closely related species of alfalfa, and then save it; in the alfalfa database (https: / modms.lzu.edu.cn / alfalfa / blast / blastPage), select alfalfa (M. sativa cultivar XinJiangDaYe) and input the above mRNA sequence of Medicago truncatula for homologous alignment, then parse the protein sequence through an mRNA translation software (https: / / web.expasy.org / translate / ), analyze the domain using the SMART website (http: / / smart.embl-heidelberg.de / ), and perform phylogenetic tree analysis and identification to obtain the nucleotide sequence of the alfalfa MsGI gene, as shown in SEQ ID No. 2;

[0202] 2) Submit the nucleotide sequence of the alfalfa MsGI gene obtained above (SEQ ID No. 2) to the siDirect version 2.1 server (http: / / sidirect2.rnai.jp / ) for small interfering RNA (siRNA) prediction. Select the region that generates the most siRNAs within the sequence with a length of 200 - 500 bp as the dsRNA template. The nucleotide sequence of the dsRNA template is as shown in SEQ ID No. 4. Then design upstream and downstream in vitro amplification primers, and add the T7 promoter sequence - TAATACGACTCACTATAGGG to the 5′ ends of the upstream in vitro amplification primer and the upstream and downstream in vitro amplification primers respectively. The nucleotide sequence of the upstream in vitro amplification primer is as shown in SEQ ID No. 7, and the nucleotide sequence of the downstream in vitro amplification primer is as shown in SEQ ID No. 8, where SEQ ID No. 7 is GTAATACGACTCACTATAGGGATGACTTCTTCTATGGCTGC and SEQ ID No. 8 is GTAATACGACTCACTATAGGGTGCCAAAACGTCATCAAAGAG. Perform PCR amplification and phenol-chloroform purification to obtain the dsRNA in vitro synthesis template;

[0203] 3) Using the TranscriptAid T7 High Yield Transcription Kit, in the following synthesis system: 8 μL of NTP (the UTP part is labeled with cy3), 4 μL of 5× TranscrptAid Reaction Buffer, 1 μg of the dsRNA in vitro synthesis template obtained in step 2), 2 μL of TranscrptAid Enzyme Mix, supplement with enzyme-free ddH 2 O to 20 μL and synthesize overnight at 37°C;

[0204] 4) Use DNAase I to digest the remaining dsRNA in vitro synthesis template, perform phenol-chloroform extraction, and precipitate with absolute ethanol to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

[0205] The dsRNA target sequence containing the alfalfa MsGI gene is as shown in SEQ ID No. 1.

[0206] Experiment 8

[0207] The dsRNA aqueous solution containing the Medicago sativa MsGI gene obtained in Example 2 was measured for the concentration of dsRNA using a ultra-micro spectrophotometer, and the concentration of the dsRNA aqueous solution containing the Medicago sativa MsGI gene was found to be 200 mg / L. The dsRNA synthesized in Example 2 was labeled with (UTP-cy3), and the whole process required light-shielding operation.

[0208] Experiment 9

[0209] Twenty 2-week-old seedlings of Medicago sativa cv. Xinjiang Daye with consistent growth vigor were selected and randomly divided into two groups, with 10 plants in each group. One group was the experimental group, and the other group was the control group. The Medicago sativa seedlings in the experimental group were cultured in the dsRNA aqueous solution containing the Medicago sativa MsGI gene obtained in Example 2, and the Medicago sativa seedlings in the control group were cultured in the same amount of enzyme-free double-distilled water as the experimental group. After culturing in the dark for 24 h, the root and leaf tissues of the Medicago sativa seedlings were taken, mounted with a water-soluble mounting medium, and observed and imaged under a fluorescence microscope.

[0210] As can be seen from Figure 9 It can be seen that no red fluorescence was observed in the root tissue of Medicago sativa in the control group. However, as can be seen from Figure 10 It can be seen that obvious red fluorescence could be observed in the root tissue of Medicago sativa in the experimental group. As can be seen from the attached figure Figure 11 It can be seen that no red fluorescence was observed in the leaf tissue of Medicago sativa in the control group either. However, as can be seen from the attached figure Figure 12 It can be seen that obvious red fluorescence could be observed in the root tissue of Medicago sativa in the experimental group. This indicates that Medicago sativa can absorb dsRNA in the dsRNA aqueous solution containing the Medicago sativa MsGI gene through its roots and conduct it upward to the leaf tissue.

[0211] Therefore, compared with the prior art, the beneficial effects of the present invention are as follows: The dsRNA of the Medicago sativa MsGI gene in the present invention can target the Medicago sativa MsGI gene, reduce the mRNA level of the Medicago sativa MsGI gene by RNA interference, scavenge the reactive oxygen species generated by Medicago sativa under salt stress, and reduce the accumulation of salt ions in the roots of Medicago sativa; when this aqueous solution containing dsRNA of the Medicago sativa MsGI gene encounters salt stress during the cultivation of Medicago sativa, the roots of Medicago sativa are thicker, the leaves are less damaged by salt toxicity, and the wilting degree is lighter, which promotes the growth of Medicago sativa and significantly enhances the resistance of Medicago sativa to environmental salt stress; this aqueous solution containing dsRNA of the Medicago sativa MsGI gene is convenient to use, has a quick effect, is green and safe, has good market application prospects, and is worthy of large-scale popularization and application.

[0212] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A dsRNA of the MsGI gene of alfalfa, characterized in that: The nucleotide sequence of the dsRNA of the alfalfa MsGI gene is shown in SEQ ID No.

1.

2. The dsRNA of the alfalfa MsGI gene according to claim 1, characterized in that: The nucleotide sequence of the alfalfa MsGI gene is shown in SEQ ID No.

2.

3. The dsRNA of the alfalfa MsGI gene according to claim 2, characterized in that: The amino acid sequence of the alfalfa MsGI gene is shown in SEQ ID No.

3.

4. Use of the dsRNA of the alfalfa MsGI gene according to any one of claims 1 to 3 in improving the resistance of alfalfa to environmental salt stress.

5. The use of the dsRNA of the alfalfa MsGI gene according to claim 4 in improving the resistance of alfalfa to environmental salt stress, characterized in that: The dsRNA expression vector of the alfalfa MsGI gene is an aqueous solution.

6. The use of the dsRNA of the alfalfa MsGI gene according to claim 5 in improving the resistance of alfalfa to environmental salt stress, characterized in that: The concentration of the dsRNA of the alfalfa MsGI gene in the aqueous solution is 150-200 mg / L.

7. The use of the dsRNA of the alfalfa MsGI gene according to claim 5 in improving the resistance of alfalfa to environmental salt stress, characterized in that: The method for preparing the aqueous solution comprises the following steps: 1) Downloading the mRNA sequence of Medicago truncatula, a close species of alfalfa, from the NCBI database, saving it, selecting alfalfa from the alfalfa database and inputting the mRNA sequence of Medicago truncatula, performing homology comparison, parsing the protein sequence using mRNA translation software, analyzing the structural domain using the SMART website, and performing phylogenetic tree analysis and identification to obtain the nucleotide sequence of the MsGI gene of alfalfa; 2) Submit the nucleotide sequence of the MsGI gene of alfalfa to the siDirect version 2.1 server for small interfering RNA prediction, select the region with the most small interfering RNA in the sequence of 200-500 bp in length as the dsRNA template, the nucleotide sequence of the dsRNA template is shown in SEQ ID No.4, design primers, amplify, the nucleotide sequence of the upstream primer is shown in SEQ ID No.5, and the nucleotide sequence of the downstream primer is shown in SEQ ID No.6; 3) Take alfalfa seedlings, extract RNA, use the cDNA obtained by reverse transcription as a template, amplify by PCR, and obtain DNA by agarose gel electrophoresis and phenol chloroform extraction. Then clone it into an empty plasmid to obtain a recombinant plasmid; 4) After sequencing, the recombinant plasmid is transferred into the engineered bacteria, inoculated at a ratio of 1:100, induced to ferment and produce dsRNA, the bacteria are collected, purified, and dissolved in enzyme-free ddH2O to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

8. The use of the dsRNA of the alfalfa MsGI gene according to claim 7 in improving the resistance of alfalfa to environmental salt stress, characterized in that: In step 4), the induction fermentation is to add IPTG to induce fermentation, and the induction fermentation conditions are: 0.5 mM IPTG, 37° C., 200 r / min, 8 h.

9. The use of the dsRNA of the alfalfa MsGI gene according to claim 7 in improving the resistance of alfalfa to environmental salt stress, characterized in that: The empty vector plasmid in step 3) is L4440 plasmid, and the engineered bacteria in step 4) is HT115.

10. The use of the dsRNA of the alfalfa MsGI gene according to claim 5 in improving the resistance of alfalfa to environmental salt stress, characterized in that: The method for preparing the aqueous solution comprises the following steps: 1) Downloading the mRNA sequence of Medicago truncatula, a close species of alfalfa, from the NCBI database, saving it, selecting alfalfa from the alfalfa database and inputting the mRNA sequence of Medicago truncatula, performing homology comparison, parsing the protein sequence using mRNA translation software, analyzing the structural domain using the SMART website, and performing phylogenetic tree analysis and identification to obtain the nucleotide sequence of the MsGI gene of alfalfa; 2) Submitting the nucleotide sequence of the alfalfa MsGI gene to the siDirect version 2.1 server for small interfering RNA prediction, selecting the region with the largest amount of small interfering RNA produced within the sequence of 200-500 bp in length as a dsRNA template, the nucleotide sequence of the dsRNA template is shown in SEQ ID No.4, designing in vitro amplification primers, the nucleotide sequence of the upstream in vitro amplification primer is shown in SEQ ID No.7, the nucleotide sequence of the downstream in vitro amplification primer is shown in SEQ ID No.8, PCR amplification, and phenol-chloroform purification to obtain a dsRNA in vitro synthesis template; 3) Using the T7 high-yield transcription kit, in the following synthesis system, NTP 8 μL, 5× TranscrptAid Reaction Buffer 4 μL, dsRNA in vitro synthesis template 1 μg, TranscrptAid Enzyme Mix 2 μL, supplemented to 20 μL with enzyme-free ddH2O, and synthesized at 37°C overnight; 4) using DNAase I to digest the remaining dsRNA in vitro synthesis template, extracting with phenol chloroform, and precipitating with anhydrous ethanol to obtain an aqueous solution containing the dsRNA target sequence of the alfalfa MsGI gene.

Images