Application of SlMOT1 gene in regulating salt resistance of tomato

Abstract

The present invention relates to the field of biotechnology, and specifically to SlMOT1 Application of genes in regulating salt resistance of tomatoes. The present invention discloses a protein as shown in (a) or (b); (a) a protein composed of the amino acids shown in SEQ ID NO. 1; (b) a derivative protein with the same function after one or more amino acid residues are replaced and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO. 1. The present invention proposes and verifies for the first time that SlMOT1 Genes and their encoding SlMOT1 The salt tolerance function of protein in plants can be applied to tomato salt resistance breeding and variety improvement, providing more options for tomato resistance breeding.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and specifically to SlMOT1 Application of genes in regulating salt tolerance in tomato. Background Art

[0002] Salt stress caused by soil salinization can inhibit plant growth, cause crop yield reduction or even crop failure, and seriously threaten agricultural development and food security. Ways to resolve this contradiction include: controlling the expansion of saline-alkali land, improving saline-alkali land, and improving the salt tolerance of crops to utilize saline-alkali land. Among them, improving crop salt tolerance and cultivating salt-tolerant varieties is a green and effective way, which requires exploring the key genes that regulate crop salt tolerance and studying the molecular genetic mechanism of salt tolerance. Therefore, analyzing the molecular mechanism of plant salt tolerance has become one of the important research directions for researchers.

[0003] Tomato (Solanum lycopersicum) is rich in nutrients and is an important source of trace elements necessary for humans. It is an important global fruit and vegetable dual-purpose economic crop. China's tomato planting area exceeds 1 million hectares, with a total output of nearly 60 million tons, making it the world's largest tomato producer and consumer. Wild tomatoes originated in the coastal areas of South America and have excellent characteristics of salt resistance, barrenness tolerance, and wide adaptability. However, artificial breeding of tomatoes focuses on yield and ignores the excellent traits of wild tomatoes such as salt resistance and barrenness tolerance, which has led to the gradual loss of salt resistance in modern cultivated tomatoes. With the excessive consumption of freshwater resources, the area of ​​arable land has gradually decreased, and the salt stress caused by the intensification of soil salinization has inhibited the growth and development of crops, causing a significant reduction in crop yields, becoming one of the main abiotic stresses threatening agricultural production and food security. With the continuous growth of the global population, the demand for vegetables such as tomatoes has continued to increase, forming an increasingly acute contradiction with the salinization of arable land and the lack of freshwater resources. Therefore, one of the effective ways to resolve this contradiction is to restore the salt-resistant traits of cultivated tomatoes and make full use of saline-alkali land to expand the tomato planting area to increase the total tomato yield.

[0004] Molybdenum ion transporter (MOT1) is the main transporter for molybdate absorption. It has been isolated and identified in species such as Arabidopsis, rice, and soybean, but there are no related reports in tomatoes.

[0005] The present invention uses wild salt-resistant tomato materials as research objects and clones SlMOT1 Gene, based on Agrobacterium genetic transformation system, and CRISPR / Cas9-mediated gene editing technology, was successfully applied to tomato regeneration system, providing an effective technical means for tomato genetic transformation to knock out target genes, and obtained SlMOT1 Targeted knockout strains were identified as tomato salt resistance genes, and their absence reduced the salt resistance of tomatoes, laying the foundation for genetic breeding of tomato salt resistance. Summary of the invention

[0006] The invention provides a tomato derived from wild species SlMOT1 The coding sequence and the protein it encodes were identified, and it was proved that their deletion would reduce the salt resistance of tomatoes. They can be applied to tomato salt resistance breeding and variety improvement, providing more options for tomato resistance breeding.

[0007] To achieve the above object, the present invention provides a protein, wherein the protein is (a) or (b);

[0008] (a) a protein consisting of the amino acids shown in SEQ ID NO. 1;

[0009] (b) a derivative protein having the same function as the amino acid sequence shown in SEQ ID NO.1, wherein one or more amino acid residues are substituted and / or deleted and / or added;

[0010] The protein of one of the above (a) or (b), which improves the survival rate of tomatoes under salt stress compared with wild-type plants.

[0011] In some specific embodiments, the present invention provides a protein having an amino acid sequence with 80% identity to the sequence shown in SEQ ID NO.1 and having enhanced salt tolerance; preferably, 85% identity, more preferably, 90% identity, more preferably, 95% identity, and most preferably, 99% identity.

[0012] The present invention also provides a gene encoding the above-mentioned protein, wherein the nucleotide sequence of the gene is (a), (b) or (c);

[0013] (a) the nucleotide sequence shown in SEQ ID NO. 2;

[0014] (b) a nucleotide sequence that hybridizes with and encodes the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions;

[0015] (c) a nucleotide sequence encoding a gene having 80% or more homology to the nucleotide sequence shown in SEQ ID NO. 2.

[0016] Those skilled in the art fully understand that, since the same amino acid may be determined by a variety of different codons, the nucleotide sequence encoding the above-mentioned protein is not limited to just one type. It can be a nucleotide sequence that can also encode the mutant amino acid sequence of the present invention by mutating one or more nucleotides of the RNase inhibator mutant nucleotide sequence shown in SEQ ID NO. 1 to form a synonymous mutation, or a nucleotide sequence that can encode the mutant amino acid sequence of the present invention can be designed based on codon optimization.

[0017] In some specific embodiments, the present invention provides a protein whose gene nucleotide sequence has 80% identity with the sequence shown in SEQ ID NO. 2; preferably, it has 85% identity, more preferably, it has 90% identity, more preferably, it has 95% identity, and most preferably, it has 99% identity.

[0018] The recombinant vector, expression cassette, transgenic cell line or recombinant bacteria containing the above gene also fall within the protection scope of the present invention.

[0019] The use of any of the above proteins, genes, recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria in tomato cultivars for resisting salt stress also falls within the protection scope of the present invention.

[0020] The present invention also provides SlMOT1 Use of genes as selection markers using high salt and / or ABA as selection pressure.

[0021] Further, providing SlMOT1 The application of the gene in breeding transgenic plants tolerant to high salt and / or ABA.

[0022] Further, providing SlMOT1 Application of genes in genetic engineering to adjust salt tolerance of tomatoes.

[0023] Furthermore, by knocking out SlMOT1 Gene, can significantly reduce the salt tolerance of tomatoes.

[0024] Furthermore, the knockout is achieved by targeted knockout of the gene using the Crispr / Cas9 method to obtain a knockout mutant plant.

[0025] Furthermore, the target site sequences for targeted knockout are shown in the sequence listings SEQ ID NO.3 and SEQ ID NO.4.

[0026] Furthermore, the plant is tomato.

[0027] Beneficial effect: The present invention provides a gene with the function of regulating tomato salt tolerance SlMOT1 , the present invention SlMOT1 The gene encodes a regulatory subunit of the molybdenum ion channel, which can regulate the balance of molybdenum ions in cells; targeted knockout in the wild type TS-21 SlMOT1 Gene can significantly reduce the salt resistance of tomatoes, so SlMOT1 It can be used in tomato salt-resistant breeding and variety improvement, providing more options for tomato resistance breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Picture 1 for SlMOT1 Gene-protein subcellular localization;

[0029] Picture 2 for SlMOT1 Targeted knockout obtained the gene sequences of two independent deletion mutants;

[0030] Picture 3 for SlMOT1 Phenotypic analysis of deletion mutants under high salt stress;

[0031] Among them, 3a is the phenotypic difference diagram of plants treated with salt stress, and 3b is the fresh weight measurement diagram of plants under salt stress;

[0032] Picture 4 for SlMOT1 Changes in ABA content in leaves of deletion mutants under high salt stress. DETAILED DESCRIPTION

[0033] In order to enable those skilled in the art to better understand the technical solution of the present invention, the following is a detailed description of the present invention.

[0034] The present invention is described in detail. The experimental methods in the following examples without specifying specific conditions are generally carried out according to conventional

[0035] Conditions or conditions recommended by the manufacturer. The test materials used in the following examples, unless otherwise specified, are purchased from conventional biochemical reagent stores. Unless otherwise specified, percentages and parts are calculated by weight. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to the recorded contents can be applied to the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0036] Example 1 SlMOT1 Gene and subcellular localization studies

[0037] (1) The plant materials used in this study were salt-tolerant wild tomato material TS-21 and cultivated material TS-670. They were germinated and grown for 7 days on 1 / 4MS medium supplemented with 1% agar at 23 ± 1°C and a 16 h light / 8 h dark photoperiod. The 7-day-old plants were transplanted to 1 / 4MS medium containing 150 mM NaCl and 1% agar and incubated in a plant growth climate chamber (23 ± 2°C, 16 h light / 8 h dark photoperiod) for 6 h, 24 h, and 72 h. Leaves of the treated plants were taken and RNA was extracted using the Vazyme (RC401-01) Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. The obtained RNA was converted into cDNA using HiScriptIII RT SuperMix (Vazyme, R323) for qRT-PCR. The PCR products were SlMOT1 Hap1 and SlMOT1 Hap2

[0038] Amplification primers are as follows:

[0039]

[0040] The reaction system is as follows:

[0041]

[0042] The reaction procedure is as follows:

[0043]

[0044] (2) SlMOT1 Hap1 and SlMOT1 Hap2 The pCAMBIA1300-GFP vector was constructed and fused with the green fluorescent protein encoding gene to express under the promoter of the tobacco mosaic virus 35S promoter, and Agrobacterium transformation was performed using the product instructions of Weidi Biotechnology GV3101 (Weidi Biotechnology, AC1001). The genetic transformation operation was performed using the Agrobacterium infection method, and the recipient materials were TS-21 and TS-670, respectively, including the following processes: pre-culture, co-culture, callus induction, and bud regeneration stage.

[0045] (3) Screening of positive plants: The vector contains the hygromycin resistance gene. When an appropriate amount of hygromycin purple is added to the tissue culture medium, positive plants can grow normally, while negative plants turn yellow and cannot grow. The leaves of positive seedlings are labeled with FM4-64 fluorescent dye and SlMOT1 is observed using a laser confocal microscope. Hap1 -GFP and SlMOT1 Hap2 -GFP fluorescence ( Picture 1 ).

[0046] like Picture 1 As shown, SlMOT1 Hap1 -GFP and SlMOT1 Hap2 -GFP green fluorescence can be used with FM4-64

[0047] The red fluorescence overlapped with that of SlMOT1 Hap1 and SlMOT1 Hap2 In both wild and cultivated tomatoes,

[0048] The membrane is involved in the transport of molybdenum ions. SlMOT1 Genes are the object. SlMOT1The protein encoded by the protein was studied for its effect on tomato's high salt resistance, as the reduction of molybdenum content leads to a decrease in the molybdenum cofactor coenzyme activity of the key enzyme in ABA synthesis, thereby reducing the activity.

[0049] Example 2 SlMOT1 Construction of gene-targeted knockout mutants

[0050] Creation of the tomato variety TS-21 background using the Crispr-Cas9 system SlMOT1 Mutant.

[0051] According to target gene SlMOT1 The target site for knockout was designed by using the cDNA of SEQ ID NO. 2. The website used was: http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR; the target site sequence is as follows:

[0052] SlMOT1-F (SEQ ID NO.3): 5'-ATTGTGTGATTGTTAATGGTGC-3'

[0053] SlMOT1-R (SEQ ID NO.4): 5'-AAACGCACCATTAACAATCACAA-3'

[0054] Add adapters, synthesized by a primer synthesis company, and then amplify the fragments. Then, gradient annealing is performed to form a double strand of two oligodeoxyribonucleotides.

[0055] The reaction system is as follows:

[0056]

[0057] The reaction procedure is as follows:

[0058]

[0059] Will Bsa I digested the binary vector plasmid containing the Cas9 expression frame, and the reaction system was as follows:

[0060]

[0061] The reaction procedure is:

[0062]

[0063] PCR products a and the vector plasmid after restriction digestion b For purification, use T4 ligase ( NEB , M0202V) operation instructions to connect, the specific operations are as follows:

[0064] The following connection system is configured on ice:

[0065]

[0066] The reaction procedure is:

[0067]

[0068] Use the instructions of the Weidi Biotech DH5α Chemically Competent Cell product to transform E. coli. The specific steps are as follows:

[0069] a. Take out the DH5α competent cells from −80°C and quickly put them in ice. After 5 minutes, wait for the bacterial block to melt, add 5 μL of the above ligation product and gently mix it by hand at the bottom of the EP tube, and let it stand in ice for 25 minutes;

[0070] b. Heat shock in a 42°C water bath for 45 seconds, quickly return to ice and let stand for 2 minutes (shaking will reduce transformation efficiency);

[0071] c. Add 700 μL of LB without antibiotics, mix well and recover at 37°C, 200 rpm for 60 minutes;

[0072] d. Centrifuge at 5000 rpm for 1 min to collect the cells, take about 100 μL of the supernatant, gently blow to resuspend the cells and spread on the LB plate containing Kan;

[0073] e. Place the plate upside down in a 37°C incubator overnight.

[0074] Identification of positive clones: Use the specific primers M13F / sgRNA on the vector for amplification and sequence the positive clones. Extract plasmids from the positive clones with correct sequencing and set aside;

[0075] M13F: 5'-GTAAAACGACGGCCAG-3'

[0076] sgRNA: 5'-AAACGCACCATTAACAATCACAA-3'

[0077] Example 3 Agrobacterium-mediated genetic transformation of tomato

[0078] 1. Agrobacterium Transformation

[0079] The positive plasmid was transformed into Agrobacterium using the product instructions of Weidi Biotech GV3101 (Weidi Biotech, AC1001). The specific operation is as follows:

[0080] (1) Thaw the GV3101 Agrobacterium competent cells stored at −80°C on ice;

[0081] (2) Add 1 μL of the plasmid DNA to be transformed into every 50 μL of competent cells, mix gently, and place on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min;

[0082] (3) Add 700 μL of LB liquid medium without antibiotics and culture at 28°C with shaking for 2 hours;

[0083] (4) Pipette about 100 μL of bacterial solution and spread it on an LB plate containing Kan. Invert the plate and incubate at 28°C for 2-3 days.

[0084] (5) Colony PCR identification.

[0085] 2. Agrobacterium-mediated genetic transformation of tomato

[0086] The recipient material for genetic transformation was TS-21, and the following process was involved:

[0087] (1) Pre-culture: Select 28-day-old sterile tomato seedlings, cut stem segments with a length of 0.5 to 1 cm without axillary buds, and place them on a plate for pre-culture for 2 days;

[0088] (2) Co-cultivation: Shake 20 mL of successfully transformed Agrobacterium (LB+Kan+Rif) to OD600 = 0.6-0.8 at 8000 rpm for 10 min, resuspend the bacteria in 20 ml of liquid MS20, transfer the above stem segments to the bacterial solution, infect for 10-15 min, transfer to sterile filter paper to absorb the bacterial solution, place on a plate, and seal with tin foil for co-cultivation for 2 days;

[0089] (3) Regeneration culture: Transfer the co-cultivated tomato stem segments to the plate of regeneration culture medium and place them in a light incubator at 22°C with alternating conditions of 16 h light (light intensity 2000 lx) / 8 h dark. Replace the culture medium every two weeks until regenerated seedlings grow out.

[0090] (4) Screening of positive plants: The vector contains a separate promoter driving the eYGFPuv tag protein. Use a 365nm ultraviolet flashlight to irradiate and preliminarily identify positive plants through fluorescence detection. Then transfer the positive seedlings to the rooting medium.

[0091] 3. NptII Resistance gene detection

[0092] Using the Kan resistance gene NptII Screening of positive plants, the specific operation is as follows:

[0093] (1) Cut the leaves of transgenic plants and extract DNA using the CTAB method;

[0094] (2) Use the following primers to amplify the Kan resistance gene:

[0095] seqFP:5'-TAGGCGTAACCGGATTGATGC-3'

[0096] seqRP:5'-TGTATTGCCCTGCTAACCCAC-3'

[0097] (3) The PCR reaction system is as follows:

[0098]

[0099] (4) PCR amplification conditions are as follows:

[0100]

[0101] (5) Detect the PCR product using 1% agarose gel electrophoresis.

[0102] To determine the reliability of eYGFPuv label fluorescence screening of positive seedlings, the Kana resistance gene NptII Conduct PCR testing to amplify positive plants SlMOT1 The fragment of the gene containing the sgRNA target site was sequenced to determine the type of targeted gene editing knockout mutation (see Picture 2 ), of which two independent allele-positive plants were named slmot1-1 and slmot1-2.

[0103] Example 4 Salt tolerance test of transgenic plants

[0104] The control group TS-21 , slmot1-1 and slmot1-2 Planted in substrate blocks, 18 days later, 100 mM NaCl and 150 mM NaCl were used for salt stress treatment. Plant height was counted one week after treatment ( Picture 3 ). Picture 3 As can be seen in a, after salt stress treatment, the growth of TS-21 strain was significantly better than slmot1-1 and slmot1-2 strain, Picture 3 As can be seen in b, SlMOT1-1 and SlMOT1-2 The deletion mutant is more sensitive to high salt stress than TS-21; SlMOT1 The loss of gene function leads to a significant decrease in salt resistance.

[0105] Example 5 SlMOT1 Effects of genes on abscisic acid (ABA) content

[0106] The control group TS-21 , slmot1-1 and slmot1-2 Planted in 1 / 4MS liquid medium, 10 days later, 100 mM NaCl was used for salt stress treatment, and leaves were taken at 0, 7 and 21 days to detect ABA content ( Picture 4 ). Picture 4 As can be seen in Figure 2, after salt stress treatment, slmot1-1 and slmot1-2 Compared with TS-21, the ABA content was lower; SlMOT1 The loss of gene function leads to a decrease in salt-induced ABA content in the leaves and a significant reduction in salt resistance, laying the foundation for the breeding of salt-resistant tomatoes.

[0107] Finally, it should be noted that the above description is only a preferred embodiment of the present invention. Under the guidance of the present invention, those skilled in the art can make a variety of similar expressions without violating the purpose and claims of the present invention, and such changes all fall within the protection scope of the present invention.

Claims

1. An application of the SlMOT1 gene as a selection marker for high salt selection pressure in salt-tolerant wild tomato TS-21, characterized in that: The nucleotide sequence of the S1MOT1 gene is shown in SEQ ID NO.2.

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