CN117757807A - Tomato SlMIS1 gene and application thereof in improving high temperature resistance of tomatoes - Google Patents
Tomato SlMIS1 gene and application thereof in improving high temperature resistance of tomatoes Download PDFInfo
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Abstract
The invention belongs to the technical field of crop genetic engineering and molecular biology, and relates to tomatoesSlMIS1Gene and application thereof in improving high temperature resistance of tomatoes, and geneSlMIS1The nucleotide sequence of (2) is shown as SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2; tomato obtained by CRISPR/Cas9 gene editing technologySlMIS1The gene knockout mutant strain was found to be higher in temperature than the wild type,SlMIS1mutant strain [ (]slmis1) Higher temperature resistance, explainingSlMIS1The gene negatively regulates the high temperature resistance of tomatoes; the invention relates to a cultureThe new variety of the tomato crop with high temperature resistance provides new gene resources, has better application value, and simultaneously provides experimental thought and theoretical basis for researching the molecular mechanism of plant response to high temperature.
Description
Technical Field
The invention belongs to the technical fields of crop genetic engineering and molecular biology, and relates to a tomato SlMIS1 gene and application thereof in improving high temperature resistance of tomatoes.
Background
The temperature can directly or indirectly influence the growth and development of crops, thereby influencing the yield and quality of the crops. The high temperature often affects the flowering time of crops, which leads to the problems of pollen abortion, fertilization failure, low fruit setting rate and the like, thereby directly affecting the crop yield. Secondly, the photosynthesis is weakened by influencing the synthesis of chlorophyll, so that the synthesis of organic matters is reduced, and the crop yield is indirectly influenced. The research of the key genes of crops for coping with high temperature and the improvement of the tolerance of the crops to high temperature by genetic engineering means are of great significance.
The mitogen-activated protein kinase (Mitogen Activated Protein Kinase, MAPK) cascade pathway is one of the major pathways of plant cells to convert extracellular stimulus signals into intracellular responses, an important central node for signal transduction, known as the "neural" of plant cells, thought to be widely involved in the growth and development of plants and in the adaptation process to the environment. The MAPK cascade is a highly conserved important signaling module in eukaryotes that acts downstream of sensors or plasma membrane receptors to regulate plant growth, development and adaptation to changing environments by coordinating various cellular responses.
The biological function of the MAPK cascade is largely determined by the downstream target proteins phosphorylated by MAPKs, most of which are Transcription Factors (TFs), but proteins involved in hormone biosynthesis, hormone transport, RNA processing and microtubule regulation may also be substrates for MAPKs. The MAPK cascade is an important signaling module involved in the whole growth and development process of plants, and the diversity of downstream substrates enables the MAPK cascade to regulate different biological processes. Extensive molecular, cellular and biochemical studies have also shown that MAPK substrates play an important role in plant growth and development processes, including cytokinesis, proliferation, and leaf senescence. MAPK substrates also play an important role in abiotic stress signaling.
Tomato (Solanum lycopersicum) is a perennial herb plant of the subgenera of Solanaceae, is rich in multiple vitamins, has higher nutritive value and medicinal value, and plays an important role in fruits and vegetables. In addition, the optimal growth temperature of tomatoes is about 28 ℃, and the growth and development of tomatoes are extremely easy to be influenced by high temperature. And tomato genome is smaller, easy to transform, and is a common mode plant. The research on tomatoes not only can directly promote the development of the tomato industry, but also can provide experimental thought and theoretical basis for researching the response mechanism of other plants to high temperature.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides a tomato SlMIS1 gene and application thereof in improving the high temperature resistance of tomatoes.
Relates to tomato SlMIS1 gene and how to utilize site-directed mutant SlMIS1 S44A (dephosphorylation mutant), slMIS1 S44D (sustained phosphorylation mutants) functional and physiological biochemical responses of SlMIS1 Ser-44 phosphorylation under high temperature stress were explored. The invention particularly relates to an application of how to knock out the gene by using CRISPR/Cas9 means so as to improve the high temperature resistance of tomatoes.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the application of the SlMIS1 gene as a negative regulatory factor in improving the high temperature resistance of tomatoes is characterized in that the nucleotide sequence of the SlMIS1 gene is shown as SEQ ID NO. 1.
The amino acid sequence of the SlMIS1 code is shown as SEQ ID NO. 2.
The application method specifically comprises the following steps:
step 1, designing a target sequence sgRNA of a SlMIS1 gene;
step 2, constructing an expression plasmid: using a kit, firstly synthesizing Oligo, then preparing Oligo dimer, and connecting Oligo dimer to a vector BGK015 to obtain an expression plasmid; the expression plasmid is introduced into tomato plants through genetic transformation, and the SlMIS1 gene is knocked out by using CRISPR/Cas9 gene editing technology, so that the heat resistance of tomatoes is improved.
Further, the target sequence sgrnas of the designed SlMIS1 gene is: 5'-AGGTAGATCGAGCAACCGAT-3'.
Further, the preparation of Oligo dimer is specifically: buffer interconnect 18 mul，UP Oligo 1μl，Low Oligo 1μl，H 2 O was supplemented to 20. Mu.l; heating at 95 ℃ for 3 minutes after mixing, and then slowly reducing to 20 ℃ at 0.2 ℃/sec; wherein the primer sequences are as follows:
UP Oligo：gtttAGGTAGATCGAGCAACCGAT
Low Oligo：aaacATCGGTTGCTCGATCTACCT。
further, the construction of the expression plasmid is specifically: CRISPR/Cas Vector 2. Mu.l, oligo dimer 1. Mu.l, enzyme Mix 1. Mu.l, H 2 O was supplemented to 10. Mu.l; the components were mixed on ice and reacted at room temperature of 20℃for 1 hour after mixing.
Further, the expression plasmid is introduced into tomato plants by genetic transformation, specifically: the expression plasmid was transformed into Agrobacterium competent cells GV3101, and positive colonies were selected for Agrobacterium-mediated tomato genetic transformation by leaf disc method.
The invention firstly constructs the SlMIS1 and the SlMIS1 based on the earlier study S44A And SlMIS1 S44D Is used, genetic transformation is carried out through agrobacterium-mediated infection by using a leaf disc method, tomato high Wen Biaoxing and physiological analysis are studied under high temperature stress, and the analysis of tomato phenotype, DAB and NBT staining and antioxidant guard enzyme activity assay are analyzed under high temperature stress, so that under high temperature stress, compared with wild type, the SlMIS1-OE and the SlMIS1 are discovered S44A Reduced high temperature tolerance in OE overexpressing plants, whereas SlMIS1 S44D OE plants are not distinguishable from wild-type plants.
The SlMIS1 knockout strain is obtained by CRISPR/Cas9 gene editing technology, and after high-temperature treatment, the SlMIS1 knockout mutant is found to be more resistant to high temperature compared with a wild type.
The invention has the following technical effects:
(1) According to the invention, through constructing the overexpression material of the SlMIS1 gene and carrying out heat resistance analysis, the result preliminarily clarifies the functional mechanism of the SlMIS1 gene in the process of responding to high temperature stress of tomatoes, and can provide new gene resources for cultivating new varieties of high temperature resistant tomato crops.
(2) According to the invention, a mutant plant of the SlMIS1 gene is obtained through a CRISPR/Cas9 gene editing technology for the first time, and functional research is carried out. The result of heat resistance analysis shows that the mutant plant is more resistant to high temperature than the wild type, and the result is further verified. Therefore, the invention provides a new breeding idea for breeding workers.
Drawings
FIG. 1 shows the results of SlMIS1-OE, slMIS1 in example 1 S44A -OE and SlMIS1 S44D Positive identification result of transgenic tomato with over-expressed OE (A: identification of SlMIS1-OE positive plants; B: slMIS 1) S44A -OE positive plant identification; c: slMIS1 S44D -OE positive plant identification).
FIG. 2 shows the results of the examples 1 of SlMIS1-OE and SlMIS1 S44A -OE and SlMIS1 S44D Determination of the relative expression level of OE tomato (A: relative expression level of SlMIS1 in overexpressed transgenic tomato; B: slMIS1 in overexpressed transgenic tomato) S44A Is a relative expression level of (2); c: overexpression of SlMIS1 in transgenic tomato S44D Is a relative expression level of (2); differential analysis was performed, n=3, p being represented by<0.05 represents q<0.01 represents q<0.001 represents q<0.0001)。
FIG. 3 is a schematic representation of the overexpression of SlMIS1 plants and overexpression of SlMIS1 under prolonged high temperature stress in example 1 S44A Plants, over-expressed SlMIS1 S44D Plants (SlMIS 1-OE, slMIS 1) S44A -OE、SlMIS1 S44D -OE) growth phenotype (a: high temperature phenotype of SlMIS1-OE plants; b: slMIS1 S44A -an OE plant high temperature phenotype; c: slMIS1 S44D -high temperature phenotype of OE plants).
FIG. 4 shows the overexpression of SlMIS1 and the overexpression of SlMIS1 under high-temperature stress in example 1 S44A And overexpressing SlMIS1 S44D Tomato leaves DAB (A) and NBT (B) stained phenotypes.
FIG. 5 is a graph showing overexpression of SlMIS1 and overexpression of SlMIS1 under high temperature stress in example 1 S44A And overexpressing SlMIS1 S44D And wild tomato leaf antioxidant enzyme activity changes (OE-S1, OE-S9, OE-S26 are over-expressed SlMIS1 plant phenotype, OE-A38, OE-A69, OE-A72 are over-expressed SlMIS 1) S44A Plant phenotype, OE-D1, OE-D4, OE-D5 is overexpressed SlMIS1 S44D Plant phenotype. Control: pairs not subjected to high temperature treatmentA group is irradiated; HT:45 ℃ for 6 hours; * Represents p<0.05 represents q<0.001)。
FIG. 6 shows the sequencing results of the sgRNA sequence of the SlMIS1 knockout tomato line in example 2.
FIG. 7 is a phenotype control of wild type tomato and SlMIS1 knockout mutant tomato in example 3 (A: phenotype of wild type tomato and SlMIS1 knockout mutant tomato before high temperature treatment, B: phenotype of wild type tomato and SlMIS1 knockout mutant tomato under conditions of incubator simulated high temperature (42.+ -. 2 ℃ C./36.+ -. 2 ℃ C. (16 h/8 h), continuous treatment for about 8 days).
Detailed Description
The following detailed description and drawings of the present invention will be presented in terms of detailed embodiments and procedures based on the technical scheme of the present invention, but the scope of the present invention is not limited to the following specific examples, and the terms used in the present invention are only for the purpose of describing the specific examples and are not intended to limit the present invention.
Example 1: overexpression of SlMIS1 tomato plants
The specific operation method is as follows:
(1) Construction of an over-expression vector: specific upstream and downstream primers (c-SlMIS 1-pBI121-F (SEQ ID NO. 3): 5'-gatgacgatgacaagggatccATGTCAATTAACCACCGGACTTCC-3' were designed with BamH I and Sac I as cleavage sites;
c-SlMIS1-pBI121-R (SEQ ID NO. 4): 5'-atcggggaaatcatagagctcTCACCGTCTATCGGCGGTT-3') and amplifying the DNA fragment of the SlMIS1 by using tomato cDNA as a template under a high-fidelity enzyme system, wherein the high-fidelity amplification system is shown in table 1, the PCR amplification program is shown in table 2, and the carrier construction primer sequence is shown in table 3. Connection to pCE2
TA/Blunt-Zero (Northey, C601), transforming competent E.coli cells DH 5. Alpha., plating, selecting single colony for bacterial liquid identification, transferring positive colony into LB liquid medium containing 50mg/L kanamycin, culturing overnight at 37 ℃ with shaking table 220rpm, extracting plasmid and plant expression vector pBI121 (which is an over-expression vector), performing double enzyme digestion at BamHI and SacI enzyme digestion sites, performing T4 connection, transforming competent E.coli cells, sequencing after bacterial liquid identification, and extracting plasmid after confirming no errors.
TABLE 1 high fidelity amplification system
TABLE 2PCR amplification procedure
TABLE 3 construction of PBI121-SlMIS1 vector
Note that: the primer sequences are lower-case letters as carrier homology arms.
(2) Agrobacterium-mediated tomato genetic transformation: pBI121-SlMIS1, pBI121-SlMIS1 S44A 、pBI121-SlMIS1 S44D The recombinant plasmid was transformed into Agrobacterium competent cell GV3101 (well known as century Co.), and positive colonies were selected for Agrobacterium-mediated tomato genetic transformation by leaf disc method. Specific primers (35S-5' -F (SEQ ID NO. 5): AGATTAGCCTTTTCAATTTCAGAAA, c-SlMIS1-pBI 121-R) were designed to identify tissue culture seedlings by PCR, and positive plants were selected.
(3) Analysis of SlMIS1 expression level: leaf samples of transgenic tomatoes were collected, wrapped with tinfoil, marked, rapidly cooled in liquid nitrogen and stored at-70 ℃. Extraction of total RNA from samples (total RNA from tomato samples was extracted according to the procedure of RNA isolater Total RNA Extraction Reagent (Nuo-uzan Co.) protocol), synthesis of cDNA (employedIII RT SuperMix for qPCR (+gDNA wind) (Norwezan Co.) after reverse transcription of the extracted RNA to cDNA, its concentration was detected), qRT-PCR was performed to detect SlMIS1/SlMIS1 in transgenic tomato according to the qRT-PCR primers of Table 4, the qRT-PCR amplification system of Table 5 and the qRT-PCR amplification program of Table 6 S44A /SlMIS1 S44D Is a relative expression level of (a).
TABLE 4 tomato qRT-PCR primers
TABLE 5qRT-PCR amplification System
TABLE 6qRT-PCR amplification procedure
FIG. 1 shows the results of SlMIS1-OE, slMIS1 in example 1 S44A -OE and SlMIS1 S44D -positive identification of OE over-expressed transgenic tomato. Collecting tissue culture seedling leaf extraction DNA as a template, and taking 35S-5' -F and c-SlMIS1-pBI121-R as upstream and downstream primers for PCR identification, wherein an over-expression plasmid is used as a positive control, and wild tomato DNA is used as a negative control. Co-identification of 15 strains of SlMIS1-OE transgenic positive seedlings and 15 strains of SlMIS1 S44A -OE transgenic positive seedlings and 19 strains SlMIS1 S44D -OE transgenic positive seedlings.
FIG. 2 shows the results of the examples 1 of SlMIS1-OE and SlMIS1 S44A -OE and SlMIS1 S44D -relative expression amount determination of OE tomato. Collecting leaf samples of the over-expressed positive transgenic tomatoes, extracting total RNA, synthesizing cDNA, performing qRT-PCR, and detecting the SlMIS1/SlMIS1 in the over-expressed transgenic tomatoes by taking wild tomato cDNA as a control S44A /SlMIS1 S44D Is a relative expression level of (a). The results showed that SlMIS1/SlMIS1 in overexpressing positive plants S44A /SlMIS1 S44D The expression quantity of the expression is obviously higher than that of a wild plant, which indicates that the SlMIS1-OE and the SlMIS1 are successfully constructed S44A -OE and SlMIS1 S44D -OE transgenic tomato material.
FIG. 3 is a schematic representation of the overexpression of SlMIS1 plants and overexpression of SlMIS1 under prolonged high temperature stress in example 1 S44A Plants, over-expressed SlMIS1 S44D Plants (SlMIS 1-OE, slMIS 1) S44A -OE、SlMIS1 S44D -OE). Wherein OE-S1, OE-S9, OE-S26 are over-expressed SlMIS1 plant phenotypes, OE-A38, OE-A69, OE-A72 are over-expressed SlMIS1 S44A Plant phenotype, OE-D1, OE-D4, OE-D5 is overexpressed SlMIS1 S44D Plant phenotype. The wild type and the over-expression transgenic tomato with 5 leaf periods and consistent growth vigor are selected to recover growth after 6d treatment at 42+/-2 ℃/36+/-2 ℃ (16 h/8 h), and the phenotype is observed. The results show that after high temperature treatment, slMIS1-OE and SlMIS1 S44D The OE plants grow worse than the WT plants, the leaves wilt and the growing points droop; while SlMIS1 S44A OE plants were not distinguished from WT plants (FIG. 3-B). This suggests that SlMIS1 negatively regulates tomato high temperature tolerance and Ser-44 phosphorylation plays a key role.
FIG. 4 shows the overexpression of SlMIS1 and the overexpression of SlMIS1 under high-temperature stress in example 1 S44A And overexpressing SlMIS1 S44D Tomato leaves DAB (A) and NBT (B) stained phenotypes. After high temperature treatment (45 ℃ C., 6 h), the mixture was subjected to WT and SlMIS1-OE, slMIS1 S44A -OE、SlMIS1 S44D DAB staining of leaves of OE plants to detect peroxidase active sites. The results show that SlMIS1-OE and SlMIS1 S44D The OE plant leaves are more stained parts and darker than the WT plant leaves, indicating that after high temperature treatment the SlMIS1-OE and SlMIS1 S44D More hydrogen peroxide accumulates in the leaves of OE plants and oxidative damage is more severe; slMIS1 S44A OE plant leaves were not significantly different from WT leaves (FIG. 4-A). NBT staining results were similar to DAB staining results, and SlMIS1-OE and SlMIS1 compared to WT plant leaves S44D -darker leaf staining of OE plants, slMIS1 S44A The amount of blue deposition of OE leaves tended to coincide with WT (FIG. 4-B); indicating that under high temperature stress, slMIS1-OE and SlMIS1 S44D Super-oxyanions in leaves of OE plants compared to WT and SlMIS1 S44A OE plant leaves accumulate more.
FIG. 5 is a graph showing overexpression of SlMIS1 and overexpression of SlMIS1 under high temperature stress in example 1 S44A And overexpressing SlMIS1 S44D And wild-type tomato leaf antioxidant enzyme activity. After high temperature treatment, there was no significant difference in POD, APX and GR enzyme activity levels in WT and OE plants, whereas SlMIS1-OE and SlMIS1 S44D SOD Activity of OE plantsBelow WT, but the difference was not statistically significant. In addition, it is notable that under high temperature stress, slMIS1-OE and SlMIS1 S44D CAT enzyme activity of OE plants was significantly lower than that of WT plants, whereas SlMIS1 S44A CAT enzyme activity in OE plants was not different from that of WT.
Example 2: slMIS1 CRISPR/Cas9 knockout mutant material acquisition
In order to clearly knock out the influence of the SlMIS1 gene on the high temperature resistance of tomato plants, we designed the target sequence sgRNA of the SlMIS1 gene and constructed an expression plasmid through a CRISPR/Cas9 vector BGK 015. The expression plasmid is introduced into tomato plants through genetic transformation, and the function of the expression plasmid is studied by knocking out the SlMIS1 through CRISPR/Cas9 technology.
The specific operation method is as follows:
(1) SlMIS1 sgRNA design and knockout vector construction: CRISPR-P2 website (http:// CRISPR) was utilized.
Design of target sequence sgRNA of SlMIS1 Gene by hzau.edu.cn/cgi-bin/CRISPR2/CRISPR (SEQ ID NO. 10): 5'-AGGTAGATCGAGCAACCGAT-3'. Using a kit, firstly synthesizing Oligo, and then preparing Oligo dimer, wherein the preparation of Oligo dimer specifically comprises: buffer Anneal 18. Mu.l, UP Oligo 1. Mu.l, low Oligo 1. Mu.l, H 2 O was supplemented to 20. Mu.l; heating at 95 ℃ for 3 minutes after mixing, then slowly decreasing to 20 ℃ at about 0.2 ℃/sec; wherein:
UP Oligo (SEQ ID NO. 11): gtttAGGTAGATCGAGCAACCGAT Low Oligo (SEQ ID NO. 12): aaacATCGGTTGCTCGATCTACCT and ligating the Oligo dimer to the vector BGK 015: CRISPR/Cas Vector 2. Mu.l, oligo dimer 1. Mu.l, enzyme Mix 1. Mu.l, H 2 O was supplemented to 10. Mu.l; mixing the components uniformly on ice, and reacting for 1 hour at room temperature of 20 ℃; and transforming the connection product into escherichia coli competent cells DH5 alpha, selecting single colony for bacterial liquid identification, screening positive colony for shaking, extracting plasmid, sending to Nanjing qing department biological limited company for sequencing, and carrying out tomato tissue culture after confirming.
(2) The genetic transformation procedure was consistent with the transformation in example 1, and the sequencing results of the sgRNA sequence of the SlMIS1 knockout tomato line are shown in fig. 6.
Example 3: heat resistance analysis of SlMIS1 overexpressing and knockout tomato plants
SlMIS1 is a negative regulatory factor in the process of tomato coping with high temperature stress. Under high temperature stress, the wild type shows better heat resistance than the overexpressing strain, and the knockdown strain shows high temperature tolerance.
The specific operation method is as follows:
(1) Over-expression and knocking out plants and wild plants of the T1 generation of the SlMIS1 tomato, which are identified as positive, are grown to a 5-leaf stage in a constant-temperature illumination incubator at the temperature of 28 ℃/16h and 25 ℃/8h, the tomato plants with the same growth condition are taken to be soaked overnight until soil is completely wetted, high-temperature stress treatment is carried out for 6d at the temperature of 42+/-2 ℃/36+/-2 ℃ (16 h/8 h), then the growth is recovered under the conditions of 25 ℃/20 ℃ (14 h/10 h) and 70% relative humidity, and the growth phenotype of the plants is observed. Each set was provided with 3 biological replicates.
(2) DAB, NBT staining: wild type and overexpressing SlMIS1 positive plants of 5 leaf stage with consistent growth vigor were selected and stained after 6h treatment at 45 ℃. The experiments were set up in two groups: control and high temperature treatment groups, 3 biological replicates per group.
(3) Well-grown overexpressed SlMIS1 positive and wild-type pentaleaf tomato plants were sampled (about 3 leaves) after 6h of high temperature treatment at 0h and 45℃respectively, with 3 biological replicates. Adding 1.6ml of enzyme liquid extract, grinding in liquid nitrogen, centrifuging at 4deg.C and 12000 Xg for 30min, and collecting supernatant as crude enzyme liquid for tomato extraction. And the activities of the five antioxidant guard enzymes APX, CAT, GR, SOD and POD were measured at the corresponding wavelengths (for specific methods, reference Mo Shuangrong, 2021) and repeated 3 times.
In conclusion, the invention discovers that the SlMIS1 is taken as a negative regulatory factor to participate in the high temperature stress process of tomatoes, and mainly shows that the growth is obviously inhibited, more oxidative damage is accumulated and the activity of antioxidant protective enzymes is obviously reduced.
Analysis of heat resistance of SlMIS1 CRISPR/Cas9 knockout material: wild type and SlMIS1 mutant plants with consistent growth are placed in a greenhouse for high-temperature treatment, the daytime temperature is about 55 ℃, the night temperature is about 30 ℃, and the treatment is continued for about 10 days. The results show that the SlMIS1 mutant plants are more refractory than the wild type plants.
FIG. 7 is a phenotypic control of wild type tomato versus SlMIS1 knockout mutant tomato in example 3. Wild tomatoes and SlMIS1 knockout mutant tomatoes (SlMIS 1-1 and SlMIS 1-2) with consistent growth are selected in the experiment, high temperature treatment (42+/-2 ℃/36+/-2 ℃ (16 h/8 h) is carried out under the condition that an incubator simulates high temperature, and the treatment is carried out continuously for about 8 days), and phenotypic results are recorded. The results show that the SlMIS1 knockout mutant tomato is in good condition, while wild type tomato plants are more affected by high temperature stress.
By combining the researches, the invention discovers that the SlMIS1 gene negatively regulates the high temperature resistance of tomatoes, and the knockout mutant can promote the growth of tomatoes and obviously improve the tolerance of tomatoes to high temperature.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Meanwhile, the above embodiments are only for illustrating the technical idea of the present invention, and the protection scope of the present invention is not limited thereto, and any modification made on the basis of the technical scheme according to the technical idea of the present invention falls within the protection scope of the present invention.
SEQ ID NO.1：
ATGGCAGACA ACAAACCACT CATTTCTTCT TCCGGCGACC GGCGCAAACC AAGCCGGTTA CAGCGCAGAG CGCCGGCGTC GATACAGGTA GATCGAGCAA CCGATTGGAA TGTTGCGATA CCGCTTCTCT CGCCGCTGAT AACTTCTCCG ACGTCTCCTG AATCGGACAA CTTGAAGGCG GCGATAAATG CTTTCTCCAG CTCGGTTCAA AAGGAAGAGG TGAAGAAAGA GCATACGGAG AAGCCGGTAA TGGTGTTTAA GAAGTGGCAG CATCCGGCGT CGCCGTTCTG TTACGAGCCG GCTCCGTTGG TGCCGTTTGT ATGTGCAGGA AGCTCCGATA GACGATGA SEQ ID NO.2：
MADNKPLISS SGDRRKPSRL QRRAPASIQV DRATDWNVAIPLLSPLITSP TSPESDNLKA AINAFSSSVQ KEEVKKEHTE KPVMVFKKWQ HPASPFCYEP APLVPFVCAG SSDRR*
Note that: * Representing the stop codon TAA.
Claims (7)
- The application of the SlMIS1 gene as a negative regulatory factor in improving the high temperature resistance of tomatoes is characterized in that the nucleotide sequence of the SlMIS1 gene is shown as SEQ ID NO. 1.
- 2. The use according to claim 1, wherein the amino acid sequence encoded by SlMIS1 is shown in SEQ ID No. 2.
- 3. The use according to claim 1 or 2, characterized in that the method of use is in particular:step 1, designing a target sequence sgRNA of a SlMIS1 gene;step 2, constructing an expression plasmid: using a kit, firstly synthesizing Oligo, then preparing Oligo dimer, and connecting Oligo dimer to a vector BGK015 to obtain an expression plasmid; the expression plasmid is introduced into tomato plants through genetic transformation, and the SlMIS1 gene is knocked out by using CRISPR/Cas9 gene editing technology, so that the heat resistance of tomatoes is improved.
- 4. The use according to claim 3, wherein the target sequence sgRNA of the designed SlMIS1 gene is: 5'-AGGTAGATCGAGCAACCGAT-3'.
- 5. Use according to claim 3, characterized in that the preparation of the Oligo dimer is in particular: buffer Anneal 18. Mu.l, UP Oligo 1. Mu.l, low Oligo 1. Mu.l, H 2 O was supplemented to 20. Mu.l; heating at 95 ℃ for 3 minutes after mixing, and then slowly reducing to 20 ℃ at 0.2 ℃/sec; wherein the primer sequence is as follows:UP Oligo：gtttAGGTAGATCGAGCAACCGATLow Oligo：aaacATCGGTTGCTCGATCTACCT。
- 6. use according to claim 3, characterized in that the construction of the expression plasmid is in particular: CRISPR/Cas Vector 2. Mu.l, oligo dimer 1. Mu.l, enzyme Mix 1. Mu.l, H 2 O was supplemented to 10. Mu.l; the components were mixed on ice and reacted at room temperature of 20℃for 1 hour after mixing.
- 7. Use according to claim 3, characterized in that the expression plasmid is introduced into tomato plants by genetic transformation, in particular: the expression plasmid was transformed into Agrobacterium competent cells GV3101, and positive colonies were selected for Agrobacterium-mediated tomato genetic transformation by leaf disc method.
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