Application of tomato SlMT5 gene in improving high temperature stress resistance of plants

Abstract

The invention provides application of a tomato SlMT5 gene in improving high temperature stress resistance of plants, and belongs to the technical field of plant genetic engineering.Experiments show that under high temperature stress, an overexpressed plant containing the SlMT5 gene shows remarkably enhanced heat-resistant phenotype compared with a wild type, specifically, the withering degree and the heat damage index of the plant are remarkably reduced, and the heat-resistant phenotype of the overexpressed plant containing the SlMT5 gene is remarkably improved. The relative water content of the leaves is obviously increased. Meanwhile, the overexpressed plant can better maintain the photosynthesis capacity, and the maximum photochemical efficiency (Fv / Fm) and chlorophyll content of the overexpressed plant are remarkably higher than those of a wild type. In the aspect of oxidation resistance, the activity of antioxidant enzymes (SOD, POD and CAT) of overexpressed plants is remarkably enhanced, accumulation of active oxygen (and) and membrane lipid peroxidation products (MDA) is remarkably reduced, and the content of osmotic regulation substances (proline, ascorbic acid and soluble sugar) is remarkably increased; the invention provides key gene resources and technical support for cultivating new varieties of high-temperature-resistant crops.

Description

TECHNICAL FIELD

[0001] The application relates to the field of plant genetic engineering technology, in particular to application of a tomato SlMT5 gene in improving plant resistance to high-temperature stress. BACKGROUND

[0002] Tomato (Solanum lycopersicum) is the largest vegetable crop in terms of global cultivation area and yield. It is loved by growers and consumers for its high nutritional and economic value. Based on huge market demand, the year-round production of tomato is a daunting task. However, the tolerance of tomato to high-temperature environment is poor, which directly limits its planting potential in some hot regions / hot seasons. More seriously, the continuous rise of global temperature makes it difficult for greenhouse and greenhouse vegetables to grow in summer, and also aggravates the occurrence of various diseases, causing reduced resistance and weakened growth of plants, which poses a serious threat to yield and quality. Therefore, the research on tomato heat tolerance should be highly valued by breeders. Identifying high-temperature stress response genes and cultivating high-quality tomato varieties with heat tolerance and taste is an important economic measure to adapt to climate change.

[0003] At present, the influence of high-temperature stress on plant growth and development has gradually become a global research topic. Tomato is a warm crop, and its suitable growth temperature is 15-30℃, and it is easily damaged by high temperature (Khavari-Nejad, R. (1981). Growth of tomato plants in different oxygen concentrations.). High temperature above 32℃ will seriously affect the growth and development, fruit quality and yield of tomato (Felix Amuji, C. et al. (2020). The Effect of Co-occurring Heat and Water Stress on Reproductive Traits and Yield of Tomato (Solanum lycopersicum). The Horticulture Journal 89, 530-536; Shaheen, M.R. et al. (2016). Morpho-physiological evaluation of tomato genotypes under high temperature stress conditions. Journal of the Science of Food and Agriculture 96, 2698-2704.).

[0004] The research on tomato heat tolerance mainly focuses on the changes of economic traits and the response of physiological and biochemical metabolism, but the mechanism of how to respond to high temperature stress at the molecular level is relatively less. The biological functions of most heat response genes are still unknown. Therefore, dissecting the molecular regulatory network of tomato heat tolerance, identifying and cloning heat tolerance related genes, and systematically studying the expression patterns and regulatory mechanisms of these genes under high temperature stress will lay a solid molecular foundation for breeding new tomato varieties with heat tolerance. The relevant results not only open up a new path for improving tomato heat tolerance through genetic engineering technology, but also provide valuable gene resources for heat tolerance molecular breeding and variety optimization of other crops.

[0005] Metallothioneins (MTs) are a class of low-molecular-weight (7-10 kDa), cysteine-rich metal-binding proteins. Studies have found that MTs can participate in plant responses to various stress conditions, including hormone treatment, low temperature stress, high temperature stress, salt stress, and drought, by scavenging reactive oxygen species (Ahn, Y.O. et al. (2011). Three Brassica rapa metallothionein genes are differentially regulated under various stress conditions. Molecular Biology Reports 39, 2059-2067; Mekawy, A.M.M. et al. (2020). Constitutive overexpression of rice metallothionein-like gene OsMT-3a enhances growth and tolerance of Arabidopsis plants to a combination of various abiotic stresses. Journal of Plant Research 133, 429-440; Patankar, H.V. et al. (2019). Overexpression of a Metallothionein 2A Gene from Date Palm Confers Abiotic Stress Tolerance to Yeast and Arabidopsis thaliana. International Journal of Molecular Sciences 20; Xue, T. et al. (2008). Cotton metallothionein GhMT3a, a reactive oxygen species scavenger, increased tolerance against abiotic stress in transgenic tobacco and yeast. Journal of Experimental Botany 60, 339-349; Yang, M. et al. (2015).Characterization of a Type 1 Metallothionein Gene from the Stresses-Tolerant Plant Ziziphus jujuba. International Journal of Molecular Sciences 16, 16750-16762.) It is worth noting that although MT is conserved in plants and other forms of life, the growth of Arabidopsis mutants containing multiple knockout MT genes differs little from wild type (R. Benatti, M. et al. (2014). Metallothionein deficiency impacts copper accumulation and redistribution in leaves and seeds of Arabidopsis. New Phytologist 202, 940-951.). This finding lays a solid theoretical foundation for the application of MT genes in breeding. SUMMARY

[0006] The present application aims to overcome the deficiencies of the prior art and provide the application of tomato SlMT5 gene in improving plant resistance to high temperature stress. The application has important production significance in improving plant resistance to high temperature, photosynthetic capacity and antioxidant level.

[0007] Technical solution: The application of tomato SlMT5 gene in improving plant resistance to high temperature stress, the amino acid sequence of the tomato SlMT5 gene is shown as SEQ ID NO. 3.

[0008] Further, the full-length DNA sequence of the tomato SlMT5 gene is shown as SEQ ID NO: 1.

[0009] Further, the base sequence of the tomato SlMT5 gene has more than 90% homology with the sequence shown as SEQ ID NO: 1, and the DNA molecule encoding the amino acid sequence shown as SEQ ID NO: 3.

[0010] Further, the full-length CDS sequence of the tomato SlMT5 gene is shown as SEQ ID NO: 2.

[0011] Further, the base sequence of the tomato SlMT5 gene has more than 90% homology with the sequence shown as SEQ ID NO: 2, and the DNA molecule encoding the amino acid sequence shown as SEQ ID NO: 3.

[0012] Further, the tomato SlMT5 gene is prepared into an overexpression vector to be applied to improve the high-temperature resistance of the target plant.

[0013] Further, the target plant is a tomato.

[0014] A method for cultivating a high-temperature-resistant plant, comprising overexpressing a tomato SlMT5 gene in a target plant gene to obtain a high-temperature-resistant target plant.

[0015] Further, the tomato SlMT5 gene is constructed into a recombinant expression vector, and then the recombinant expression vector is transformed into a bacterial strain to obtain a recombinant bacterial strain carrying the tomato SlMT5 gene, and then the recombinant bacterial strain is used to infect a plant leaf, so that the plant carries the tomato SlMT5 gene, and finally the high-temperature resistance, photosynthesis capacity and antioxidant level of the plant are regulated through the expression of the tomato SlMT5 gene.

[0016] Advantages: Compared with the prior art, the application has the advantages that the overexpression of the SlMT5 gene can significantly enhance the high-temperature resistance of the tomato, and the application provides a gene resource for cultivating a new high-temperature-resistant tomato variety, and has a certain application value in the field of tomato molecular breeding, and lays a theoretical foundation for studying the high-temperature stress signal response and molecular mechanism of the tomato plant. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Relative expression amount of the tomato SlMT5 gene under high-temperature stress, h: hour;

[0018] Figure 2 SlMT5 gene expression amount detection of the T1 generation overexpression plant Figure 2 A) and the plant Figure 2 B), Figure 2 A In the column chart, different letters on the column body represent that the difference between the treatments reaches a significant level (P < 0.05); WT is a wild type; OE is overexpression;

[0019] Figure 3 Plant phenotype diagram of the tomato SlMT5 gene overexpression plant and the wild type plant after two days of normal temperature (25℃) and high-temperature (42℃) treatment Figure 3 A) Relative water content Figure 3 B) and heat damage index Figure 3 C); wherein the higher the degree of leaf wilting, the more serious the damage caused by high-temperature stress; the lower the relative water content, the more serious the damage caused by high-temperature stress; the higher the heat damage index, the more serious the damage caused by high-temperature stress; Figure 3In the BC bar chart, different letters on the bars indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild type; OE represents overexpression; Control represents room temperature control; HT represents high temperature stress.

[0020] Figure 4 Chlorophyll fluorescence parameters of leaves from tomato SlMT5 gene overexpressing plants and wild-type plants after two days of treatment at room temperature (25℃) and high temperature (42℃). Figure 4 A) and Fv / Fm ( Figure 4 B); Figure 4 In the bar chart (B), different letters on the bars indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild type; OE represents overexpression; Fv / Fm represents maximum photochemical efficiency.

[0021] Figure 5 The total chlorophyll content of leaves from tomato SlMT5 gene overexpressing plants and wild-type plants after two days of treatment at room temperature (25℃) and high temperature (42℃); different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild-type; OE represents overexpression;

[0022] Figure 6 The table shows the SOD, POD, CAT, MDA, catalase, and superoxide anion contents in the leaves of tomato SlMT5 gene overexpressing plants and wild-type plants after two days of treatment at room temperature (25℃) and high temperature (42℃). Different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild-type; OE represents overexpression.

[0023] Figure 7 The levels of proline, ascorbic acid (AsA), and soluble sugar in the leaves of tomato SlMT5 gene overexpressing plants and wild-type plants after two days of treatment at room temperature (25℃) and high temperature (42℃); different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild-type; OE represents overexpression;

[0024] Figure 8 The relative expression levels of the heat response gene in tomato SlMT5 gene overexpressing plants and wild-type plants after treatment at room temperature (25℃) and high temperature (42℃); different letters on the bars in the bar chart indicate that the differences between treatments reached a significant level (P < 0.05); WT represents wild-type; OE represents overexpression. Detailed Implementation

[0025] The above and / or other aspects of the present application will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings in which:

[0026] The nucleotide sequence is from left to right, 5' end to 3' end.

[0027]

[0028] SEQ ID NO. 2: ATGTCGTGCTGTGGAGGAAACTGTGGCTGTGGATCTAGCTGCAAGTGCGGCAACGGCTGTGGAGGTTGCAAGATGTACCCAGACATGAGCTACACCGAAAGCAGCACAACCACTGAGACTTTGGTGCTTGGGGTGGGACCTGAGAAGACAAGCTTCGGCGCCATGGAGATGGGTGAATCCCCTGTTGCTGAGAATGGCTGCAAATGTGGATCTGACTGCAAGTGCAACCCTTGCACTTGTTCTAAGTGA

[0029] SEQ ID NO. 3: MSCCGGNCGCGSSCKCGNGCGGCKMYPDMSYTESSTTTETLVLGVGPEKTSFGAMEMGESPVAENGCKCGSDCKCNPCTCSK

[0030] Example 1, Analysis of relative expression of tomato SlMT5 gene under high temperature stress.

[0031] The normal growth to four-leaf wild type tomato variety Micro-Tom was placed in a 42℃ / 25℃ (day 16h / night 8h) artificial climate chamber (RDN-1000A, Ningbo) for high temperature stress treatment, and the same position of tomato leaves were sampled at 0h, 0.5h, 1h, 2h, respectively. The total RNA was extracted using Bi-ospin Plant Total RNA extraction Kit polysaccharide polyphenol plant total RNA extraction kit of Hangzhou Boer Science and Technology Co., Ltd. The RNA in each sample was reverse transcribed using total RNA as template, and the specific method was performed according to HiScript II One Step RT-PCR (Novozyme Biotech Co., Ltd., Nanjing) kit to obtain cDNA product. The reverse transcription cDNA was used as template, eif was used as internal reference gene, and RT-PCR analysis was performed. The overexpression plants were detected using primers with sequences as shown in SEQ ID NO: 4 to 7 (Table 1). According to the reagent instruction of Hieffff UNICON Universal Blue qPCR SYBR Green Master Mix (Yixing Biotechnology Co., Ltd., Shanghai), the reaction solution was configured and the related reaction program was set, and the expression of each sample was calculated. The results show that the SlMT5 gene is a high temperature stress response gene of tomato, and its expression is directly induced by high temperature. On the other hand, it shows that the expression of the gene is activated rapidly, which provides a theoretical basis for enhancing the resistance of tomato to high temperature by transgenic overexpression in the future.

[0032] Table 1 Detection primer and internal standard primer for detecting the expression amount of positive seedlings by fluorescence quantitative PCR

[0033] Name Sequence No. FQ-PCR primer-F 5'-CTGTGGAGGAAACTGTGGCT-3' SEQ ID NO: 4 FQ-PCR primer-R 5'-AGGGGATTCACCCATCTCCA-3' SEQ ID NO: 5 Internal primer-F 5'-ATCCTTCAGAGCGGTGTTCA-3' SEQ ID NO: 6 Internal primer-R 5'-ATCTCAAGAGCCTCTGGTGG-3' SEQ ID NO: 7

[0034] Example 2, Construction of SlMT5 gene overexpression vector.

[0035] The CDS sequence of SlMT5 was obtained through the plant comparative genomics database Phytozome (https: / / phytozome-next.jgi.doe.gov / ), and was imported into the P2300 SYC-35S empty map in SnapGene®4.3.6 software. The CDS sequence of SlMT5 gene was inserted into the empty multiple cloning site by using In-Fusion® Cloning function, and specific primers OE-SlMT5-F (SEQ ID NO: 8) and OE-SlMT5-R (SEQ ID NO: 9) were designed for corresponding enzyme digestion sites. The cDNA obtained by reverse transcription of wild type Micro-Tom RNA was used as a template, and the target amplification fragment was obtained by PCR amplification reaction. Then, the pCAMBIA1300-35S-eGFP vector was linearized by KpnI and BamHI double enzyme digestion, and the homologous recombination enzyme (Nanjing Novozyme ClonExpress Il One Step Cloning Kit) was used. The SlMT5 gene fragment was connected with the linearized vector to obtain the overexpression vector driven by 35S promoter. The recombinant plasmid was sent to Shanghai Genesee Company for sequencing, and the detection primer sequence is shown as SEQ ID NO: 10 to 11 in Table 2. It was found that the sequence was completely consistent with the published sequence of tomato genome. It is proved that the construction of SlMT5 gene overexpression vector is successful.

[0036] Table 2 Construction primer and detection primer of overexpression vector

[0037] Name Sequence No. OE-SlMT5-F 5'-TTTGGAGAGGACAGGGTACCATGTCGTGCTGTGGAGGAAAC-3' SEQ ID NO: 8 OE-SlMT5-R 5'-TTTGGAGAGGACAGGGTACCATGTCGTGCTGTGGAGGAAAC-3' SEQ ID NO: 9 Detection primer-F 5'-CAAGGACGACGGCAACTACA-3' SEQ ID NO: 10 Detection primer-R 5'-GACTGGGTGCTCAGGTAGTG-3' SEQ ID NO: 11

[0038] Example 3, Preparation and identification of SlMT5 gene overexpression plant.

[0039] The correctly sequenced overexpression vector was transformed into Agrobacterium GV3101. Tomato cotyledons were infected with 30 mL of Agrobacterium GV3101 bacterial suspension with an OD of 0.5-0.6. Callus induction was then performed using tomato selection medium, adventitious shoot differentiation was performed using subculture medium, and rooting culture was performed using rooting medium to finally obtain rooted tissue culture seedlings.

[0040] The expression level of overexpressing plants was detected using real-time quantitative PCR. Primer sequences are shown in SEQ ID NO: 4 to 7 in Listing 1. Results are as follows: Figure 2 As shown, real-time quantitative PCR analysis revealed four overexpression lines. Two independent overexpression lines, OE-3 and OE-4, were selected for further research.

[0041] Example 4: Identification of the high-temperature resistance of plants overexpressing the SlMT5 gene.

[0042] Wild-type tomato plants that had reached the four-leaf stage and were overexpressing the Micro-Tom and SlMT5 genes (OE-3 and OE-4) were treated together at 42℃ for 48 h in a light incubator. The high-temperature stress phenotypes of each line were observed. Relative water content, heat damage index, chlorophyll fluorescence parameters (Fv / Fm), total chlorophyll content, antioxidant enzyme activities (SOD, POD, CAT), and reactive oxygen species (ROS) were measured. and Accumulation, malondialdehyde (MDA) content, and osmotic protective substances (proline, ascorbic acid, soluble sugars) content.

[0043] Phenotypic observation results of each strain are as follows Figure 3 As shown in Figure A, before high-temperature treatment, both wild-type and SlMT5-overexpressing plants grew vigorously without wilting. However, after two days of high-temperature treatment, the SlMT5-overexpressing plants showed less wilting and overall better growth than the wild-type plants, which exhibited severe wilting with drooping and curled leaves. The relative water content results are as follows: Figure 3 As shown in Figure B, after high-temperature treatment, the relative water content of the leaves of SlMT5-overexpressing plants was significantly higher than that of wild-type plants. The statistical results of the heat damage index are as follows: Figure 3 As shown in Figure C, the heat damage index of SlMT5 overexpressing plants was significantly lower than that of wild-type plants. The results of chlorophyll fluorescence parameters (Fv / Fm) and total chlorophyll content detection are as follows: Figure 4 and Figure 5 As shown, after high-temperature treatment, the Fv / Fm and total chlorophyll content of SlMT5-overexpressing plants were significantly higher than those of wild-type plants, indicating that SlMT5 overexpression helps maintain photosynthetic capacity under high-temperature stress. The results of reactive oxygen species (ROS) accumulation detection are shown below. Figure 6 As shown, after high-temperature treatment, the wild-type plants... and The SlMT5 overexpression plants accumulated more and less. The results of antioxidant enzyme activity detection are shown in Figure 6 The SlMT5 overexpression plants showed increased antioxidant enzyme SOD, POD and CAT activities and reduced membrane damage (as indicated by reduced malondialdehyde content) under high temperature stress. The results of osmoprotectant and non-enzyme antioxidant substance content detection are shown in Figure 7 The proline, ascorbic acid and soluble sugar contents of the SlMT5 overexpression plants were significantly higher than those of the wild type. The above results indicate that the SlMT5 gene positively regulates tomato heat resistance.

[0044] Example 5, Analysis of Heat Response Gene Expression Amounts in SlMT5 Gene Overexpression Plants

[0045] Wild type tomato Micro-Tom and SlMT5 gene overexpression plants (OE-3, OE-4) grown to four-leaf one-heart were subjected to high temperature treatment in a light incubator, and the relative expression amounts of heat response related genes (including SlHSP70, SlHSP90, SlHSFA1a and SlHSFA2, etc.) in the leaves were detected at different time points using qRT-PCR technology. The primer sequences are shown in Table 3 as SEQ ID NOs: 12 to 19.

[0046] The heat response gene expression amount detection results are shown in Figure 8 Under the control condition, there was no significant difference in the expression amounts of all detected heat response genes between the wild type and the SlMT5 overexpression plants, and they were maintained at a low basic level. However, after high temperature stress, the expression amounts of the heat response genes (such as SlHSP70, SlHSF90, etc.) of the overexpression lines were significantly up-regulated at an early stage, and the response speed was faster than that of the wild type; at the peak expression period, the expression amounts of the SlHSP70, SlHSP90, SlHSFA1a and SlHSFA2 genes of the overexpression lines were significantly higher than those of the wild type; in the late stage after stress, the expression amounts of these genes of the overexpression lines were still maintained at a higher level than that of the wild type. All detected heat response genes (including heat shock protein genes and heat shock transcription factor genes) showed a consistent expression rule of "higher expression intensity in overexpression lines than in wild type". The above results indicate that the SlMT5 gene positively regulates tomato heat resistance by promoting the expression of SlHSP70, SlHSFP90 and other heat response genes.

[0047] Table 3 Heat response gene detection primers

[0048] Name Sequence No. Hsp70-F 5'-GGATCAACACGAATACCGGC-3' SEQ ID NO: 12 Hsp70-R 5'-CAGCCAAGATACCAGCCTGAA-3' SEQ ID NO: 13 Hsp90-F 5'-CAAGGAAGACCAGTTGGAGT-3' SEQ ID NO: 14 Hsp90-R 5'-TCACTGATTTCCTTCTCAGTTGTCT-3' SEQ ID NO: 15 HsfAla-F 5'-TCAACAACAGCAGCAGCCACAT-3' SEQ ID NO: 16 HsfAla-R 5'-TCAGCCTCTCAACCTCTTCTTCAAG-3' SEQ ID NO: 17 HSFA2-F 5'-GAGCAGCAGAGGAAGTAGTG-3' SEQ ID NO: 18 HSFA2-R 5'-TCTGCATCTTAACCTCTAAAGGAA-3' SEQ ID NO: 19

[0049] While the application has been described and illustrated with reference to specific preferred embodiments, it is not intended that it be limited to these particulars. Various changes in form and detail can be made without departing from the spirit and scope of the application as defined by the appended claims.

Claims

1. Application of a tomato SlMT5 gene in improving plant resistance to high temperature stress, wherein the amino acid sequence of the tomato SlMT5 gene is shown as SEQ ID NO.

3.

2. Use according to claim 1, characterized in that: The full-length DNA sequence of the tomato SlMT5 gene is shown as SEQ ID NO:

1.

3. Use according to claim 1, characterized in that: The full-length CDS sequence of the tomato SlMT5 gene is shown as SEQ ID NO:

2.

4. Use according to claim 1, characterized in that: The tomato SlMT5 gene is prepared into an overexpression vector to be applied to improve the high temperature resistance of a target plant.

5. Use according to claim 4, characterized in that: The target plant is a tomato.

6. A method for cultivating heat-resistant plants, characterized in that: The method comprises overexpressing the tomato SlMT5 gene in a target plant gene to obtain a target plant resistant to high temperature.

7. The method of claim 6, wherein, The tomato SlMT5 gene is constructed into a recombinant expression vector, and then the recombinant expression vector is transformed into a bacterial strain to obtain a recombinant bacterial strain carrying the tomato SlMT5 gene, and then the recombinant bacterial strain is used to infect a plant leaf, so that the plant carries the tomato SlMT5 gene, and finally the high temperature resistance, photosynthesis capacity and antioxidant level of the plant are regulated through the expression of the tomato SlMT5 gene.

2. A recombinant expression vector comprising a tomato SlMT5 gene, wherein the amino acid sequence of the tomato SlMT5 gene is shown as SEQ ID NO.

3. The full-length DNA sequence of the tomato SlMT5 gene is shown as SEQ ID NO:

1. The full-length CDS sequence of the tomato SlMT5 gene is shown as SEQ ID NO:

2. The tomato SlMT5 gene is prepared into an overexpression vector to be applied to improve the high temperature resistance of a target plant. The target plant is a tomato. The method comprises overexpressing the tomato SlMT5 gene in a target plant gene to obtain a target plant resistant to high temperature. The tomato SlMT5 gene is constructed into a recombinant expression vector, and then the recombinant expression vector is transformed into a bacterial strain to obtain a recombinant bacterial strain carrying the tomato SlMT5 gene, and then the recombinant bacterial strain is used to infect a plant leaf, so that the plant carries the tomato SlMT5 gene, and finally the high temperature resistance, photosynthesis capacity and antioxidant level of the plant are regulated through the expression of the tomato SlMT5 gene.

3. A recombinant bacterial strain carrying a tomato SlMT5 gene, wherein the amino acid sequence of the tomato SlMT5 gene is shown as SEQ ID NO.

3. The full-length DNA sequence of the tomato SlMT5 gene is shown as

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