Application of tomato SlCLE10 gene in improving drought tolerance and salt tolerance of plants

Abstract

The invention discloses an application of a tomato SlCLE10 gene in improving drought tolerance and salt tolerance of plants. A tomato SlCLE10 overexpression plant or a gene knockout plant is constructed through a gene means, and results show that the SlCLE10 overexpression tomato under salt stress and drought stress can promote the plant root length, the mature zone root hair length and the root diameter elongation, meanwhile, the accumulation of soluble sugar is improved, the proline content is higher, and the SlCLE10 overexpression tomato can be applied to the field of salt stress and drought stress. Meanwhile, the enzyme activity of scavenging enzymes such as SOD, CAT and POD related to an anti-oxidation defense scavenging system is higher than that of scavenging enzymes related to an anti-oxidation defense scavenging system, such as SOD, CAT and POD, and the tomato material with overexpressed SlCLE10 is more resistant to salt stress and drought stress. The invention provides a gene resource for cultivating a salt-stress and drought-stress tomato new variety, has a relatively good potential application value, and lays a theoretical foundation for researching a stress signal response mechanism and an adverse environment tolerance molecular mechanism of a tomato plant.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and particularly relates to application of a tomato SlCLE10 gene in improving drought and salt tolerance of plants. Background Art

[0002] Plants living in the natural environment are subject to various stresses from the external environment. Since plants are rooted in the soil and cannot move to seek benefits or avoid harm, they must be able to adapt to local environmental conditions and evolve tolerance and resistance to adverse environments. In crop production, they often encounter a variety of abiotic stresses such as drought and salt stress, as well as biotic stresses such as pests and diseases. These stresses affect the physiological and biochemical processes, growth and development stages, and final yield and quality of plants. Therefore, in agricultural production, it is necessary to take reasonable irrigation and fertilization measures, as well as select varieties with strong stress resistance, to reduce the adverse effects of drought and salt stress on plants. The study of the effects of drought and salt stress on crops is helpful for us to understand how plants resist external stress, and then to explore the genes of plant disease resistance and stress resistance and apply them to actual crop molecular breeding. It is of great significance.

[0003] The effects of drought and salt stress on plant growth and development are mainly reflected in the following aspects. When drought is severe, plants will lose water and nutrients, resulting in restricted growth or even death. Drought stress inhibits plant growth and tillering by reducing water supply and limiting photosynthesis. When salt stress is severe, plants will be damaged by tissue dehydration and energy depletion, resulting in restricted growth or death. Salt stress causes excessive accumulation of sodium ions in cells, interferes with normal metabolism, and leads to cell dehydration, membrane damage and oxidative stress. Drought and salt stress can cause imbalance in the metabolism of active oxygen in plants, produce oxidative stress, and cause damage to the structure of biological macromolecules such as proteins, lipids, and nucleic acids, affecting the normal physiological and biochemical functions of plants. In short, drought and salt stress have significant negative effects on plant growth and development.

[0004] Tomato (Solanum lycopersicum) is a vegetable crop that is widely grown around the world and is also one of the most consumed vegetables in my country. During the cultivation process, it is also affected by salt damage or drought stress, resulting in a decline in tomato quality and a decrease in yield. Analyzing the drought and salt stress resistance regulatory mechanism of tomatoes, cloning related genes, and systematically and in-depth studying the expression and regulation of drought and salt stress-related genes under salt damage or drought stress conditions can provide a molecular basis for breeding new drought-resistant / high-salt tomato varieties, open up a new way to improve the drought / high-salt resistance of tomatoes through genetic engineering, and also provide genetic resources for the drought / high-salt resistance molecular breeding and variety improvement of other crops. Summary of the invention

[0005] The object of the present invention is to provide the application of tomato SlCLE10 gene in improving the salt tolerance and drought tolerance of plants. The application of the present invention has important production significance for improving the salt tolerance and drought tolerance of crops, screening germplasm materials and directional breeding, etc.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The application of tomato SlCLE10 gene in improving the salt tolerance and drought tolerance of plants, wherein the nucleotide sequence of the tomato SlCLE10 gene is as shown in SEQ ID NO.1.

[0008] Furthermore, the application is to prepare the tomato SlCLE10 gene into a recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus for application in improving the salt tolerance and drought tolerance of plants.

[0009] A method for cultivating salt-tolerant and drought-tolerant tomatoes, the method comprising overexpressing the tomato SlCLE10 gene in the tomato gene to obtain salt-tolerant and drought-tolerant tomatoes.

[0010] In previous experiments, the inventors performed salt stress treatment on wild-type tomato seedlings with 175 mM NaCl and detected the relative expression levels of tomato CLE family genes within 0 - 3 h. The results showed that SlCLE10 among them could rapidly respond to salt stress and the transcriptional level was significantly increased, as Figure 1 shown.

[0011] In the present invention, by genetic means, tomato SlCLE10 overexpression plants (OE-SlCLE10) or gene knockout plants (CR-slcle10) were constructed to regulate the expression level of the gene SlCLE10 to study its regulatory mechanism on the salt stress and drought stress resistance of tomatoes. The results showed that under salt stress and drought stress, SlCLE10 overexpression tomatoes could promote the elongation of plant root length, root hair length in the mature zone and root diameter, and at the same time increase the accumulation of soluble sugar, the proline content was also higher. At the same time, the enzyme activities of scavenging enzymes such as SOD, CAT and POD related to the antioxidant defense scavenging system were also higher than those of knockout mutants and wild-type materials. Tomato materials overexpressing SlCLE10 were more salt-tolerant and drought-tolerant.

[0012] The present invention provides gene resources for cultivating new tomato varieties with salt stress and drought stress tolerance, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to stress signals and the molecular mechanism of tolerating adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 Results of the expression response of SlCLE10 under salt stress treatment.

[0014] Figure 2 Expression changes of SlCLE10 in proCLE10-GUS transgenic tomato seedlings under osmotic treatment.

[0015] Figure 3 Phenotypic analysis of plants overexpressing the SlCLE10 gene (OE-SlCLE10) and knockout mutant materials (CR-slcle10) under drought / salt stress treatment. Among them, A shows the phenotypes of OE-SlCLE10 overexpression and mutant materials under drought / salt stress treatment; B shows the comparison of hypocotyl length and above-ground fresh weight of plants after drought / salt stress treatment of OE-SlCLE10 overexpression and mutant materials.

[0016] Figure 4 Phenotypes and root length statistics of tomato seedlings with different backgrounds under osmotic stress treatment. Among them, A and B show that there are no significant differences in the roots of WT, OE-SlCLE10, and CR-slcle10 materials under the condition of 1 / 2MS; C and D show that there are significant differences in the roots of WT, OE-SlCLE10, and CR-slcle10 materials after 5 days of osmotic stress treatment with 300 mM Mannitol.

[0017] Figure 5 Phenotypes, mature zone root hair length, and root diameter statistics of tomato seedlings with different backgrounds under osmotic stress treatment. Among them, A and B show that there are no significant differences in root diameter and root hair length of WT, OE-SlCLE10, and CR-slcle10 materials under the condition of 1 / 2MS; C and D show that there are significant differences in root diameter and root hair length of WT, OE-SlCLE10, and CR-slcle10 materials after 5 days of osmotic stress treatment with 300 mM Mannitol.

[0018] Figure 6 Determination results of CAT (A), POD (B), SOD (C), and soluble sugar (D) in WT, OE-SlCLE10, and CR-slcle10 materials treated with salt and drought tolerance for 4 days.

[0019] Figure 7 Determination results of Pro (A), MDA (B), O 2- (C), and H2O2 (D) in WT, OE-SlCLE10, and CR-slcle10 materials treated with salt and drought tolerance for 4 days. Specific implementation manner

[0020] The present invention provides the application of tomato SlCLE10 gene or protein or recombinant vector, expression cassette, or recombinant bacterium containing the gene in improving plant salt and drought tolerance. The nucleotide sequence of the tomato SlCLE10 gene is shown in SEQ ID NO.1.

[0021] The sequence shown in SEQ ID NO.1 of the present invention is a CDS sequence, derived from the sequence published in Solgenomics (Solyc07g053370.1.1), and the amino acid sequence encoded by SEQ ID NO.1 is preferably as shown in SEQ ID NO.2.

[0022] SEQ ID NO.1:

[0023] ATGGCTAATTCATCATCAAAAATGTTCATAATCCTCTTCATGATTATTTTCCTTTTTTGTCACTTTATTTCTTTGGAGGGTCGTATTCTTGATGATCTTCAAGTTGTTCAAAACAAATATGATAGTCATTTTGTTCTTAGCAAGGCTGGATTTAGCCCTAGGGAGATTGAGGAGTATACGAGGCGATCTTTAAAAGGCGGTTCAGATAGAGTTGTACCTGGTGGACCTGACTCTCAACATCACTCTTCGCCTCCGACTAACTAG.

[0024] SEQ ID NO.2:

[0025] MANSSSKMFIILFMIIFLFCHFISLEGRILDDLQVVQNKYDSHFVLSKAGFSPREIEEYTRRSLKGGSDRVVPGGPDSQHHSSPPTN*.

[0026] In the embodiments of the present invention, the tomato SlCLE10 gene is preferably obtained by PCR amplification. The primer pair used for cloning preferably includes SlCLE10-ORF-F and SlCLE10-ORF-R. The nucleotide sequence of SlCLE10-ORF-F is preferably as shown in SEQ ID NO.3: GGCTTAAUATGGCTAATTCATCATC, and the nucleotide sequence of SlCLE10-ORF-R is preferably as shown in SEQ ID NO.4: GGTTTAAUGTTAGTCGGAGGCGAAG. The system of the PCR amplification of the present invention is calculated as 50 μl, and preferably includes template DNA (2 μl), 2×KOD FX buffer (25 μl), dNTP (5 μl), Primer-F (2 μl), Primer-R (2 μl), KOD FX (1 μl) and deionized water (13 μl); the amplification program preferably includes: pre-denaturation at 95°C for 3 min; denaturation at 94°C for 30 s, annealing at 55-60°C for 30 s, extension at 68°C (1 kb / min), 35 cycles; extension at 68°C for 10 min; and preservation at 16°C.

[0027] The plant of the present invention preferably includes dicotyledonous plants, more preferably includes tomato, eggplant or Arabidopsis thaliana, and the physiological indexes of the salt tolerance and drought tolerance of the plant preferably include: the enzyme activities of catalase, peroxidase and superoxide dismutase, and the contents of soluble sugar, proline, malondialdehyde, superoxide anion and hydrogen peroxide.

[0028] In the present invention, it is preferably to verify the mechanism of the tomato gene in response to salt and drought by constructing overexpression transgenic plants and gene knockout plants. When constructing the overexpression transgenic plants of the present invention, the tomato SlCLE10 gene is preferably constructed into the plant overexpression vector pCambia1300-35S, the obtained overexpression vector is used to transform Agrobacterium tumefaciens, and then the cotyledons of tomato are infected and plant tissue culture is carried out to screen positive transgenic tomato plants to obtain transgenic tomatoes with salt tolerance and drought tolerance. When constructing the gene knockout plants of the present invention, it is carried out by using the gene editing knockout vector pTX041. The present invention has no special limitation on the sources of the plant overexpression vector pCambia1300-35S and the gene editing knockout vector pTX041, and the conventional commercially available vectors in the art can be used.

[0029] The host bacteria of the recombinant bacteria of the present invention preferably include Escherichia coli cells or Agrobacterium tumefaciens cells, more preferably Agrobacterium tumefaciens cells, and most preferably Agrobacterium tumefaciens GV3101.

[0030] Using the overexpressing transgenic plants and gene knockout plants of the present invention, the same salt and drought tolerance verification was carried out. Under salt and drought stress, overexpression of SlCLE10 can promote the accumulation of soluble sugars in plants, and the proline content is also higher. At the same time, the enzyme activities of scavenging enzymes such as SOD, CAT, and POD related to the antioxidant defense scavenging system are also higher than those of the knockout mutants and wild-type materials. Therefore, the tomato materials overexpressing SlCLE10 are more salt- and drought-tolerant.

[0031] The present invention also provides the application of the overexpressed tomato SlCLE10 gene in improving the salt and drought tolerance of plants, and the nucleotide sequence of the tomato SlCLE10 gene is shown in SEQ ID NO.1.

[0032] The present invention does not specifically limit the method of overexpression, and preferably the method is the same as that of the above-mentioned overexpressing transgenic plants, which will not be elaborated here.

[0033] The present invention also provides a method for cultivating salt- and drought-tolerant tomatoes, which includes overexpressing the tomato SlCLE10 gene in the tomato gene, and the nucleotide sequence of the tomato SlCLE10 gene is shown in SEQ ID NO.1.

[0034] The method of the present invention is preferably the same as the above-mentioned overexpression method, which will not be elaborated here.

[0035] The following combines examples to detail the application of the tomato SlCLE10 gene provided by the present invention in improving the salt and drought tolerance of plants, but they cannot be understood as limiting the protection scope of the present invention.

[0036] In the present invention, unless otherwise specified, the reagents and methods used are all conventional commercially available reagents, reagent kits and methods for supporting reagent kits in the art. For example, all consumables in the RNA extraction process are RNAase-free products, and the RNA extraction of tomatoes refers to the instruction manual of Biospin Plant Total RNA Extraction Kit (DNA-free) of Bioer Technology; when reverse transcription is carried out, it is carried out according to the Ⅱ1st Strand cDNA Synthesis SuperMix for qPCR (gDNA digester plus) reagent kit of YEASEN; when real-time fluorescence quantitative PCR is carried out, the ratio and program setting of the reaction solution are all configured and set with reference to the instruction manual of TB Premix Ex Taq TM II reagent, and the calculation method of relative expression level adopts 2 -△△CTMethod. Moreover, in the embodiments of the present invention, for the determination of plant physiological indicators, such as the determination principles and methods of soluble sugar, proline, malondialdehyde, superoxide anion, catalase, peroxidase, superoxide dismutase, and hydrogen peroxide content are carried out according to the kit instructions of Suzhou Keming Biotechnology Co., Ltd.

[0037] After extracting the RNA of different materials, reverse transcribe it into cDNA, prepare the qRT-PCR reaction system, and perform real-time fluorescence quantitative PCR. Using the SlACTIN gene as the internal reference gene, the sequence is shown in Table 1:

[0038] Table 1 Primers required for real-time fluorescence quantitative PCR

[0039]

[0040] Using an Eppendorf Mastercylcer ep realplex real-time fluorescence quantitative PCR instrument, prepare a 20 μl reaction system: 10 μl Premix Ex Taq TM (2×), 0.4 μl Forward Primer (10 μM), 0.4 μl Reverse Primer (10 μM), 2 μl cDNA template, 7.2 μl deionized water.

[0041] The reaction program uses a two-step amplification method: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 30 s, 40 cycles; melting curve analysis. Use the 2 －△△CT method to analyze the relative gene expression level.

[0042] Example 1

[0043] Construction and transformation of SlCLE10 overexpression and knockout vectors

[0044] Using the pTX041 and pCambia1300-35S vectors, different cloning constructs of the SlCLE10 gene were carried out, and a knockout vector and an overexpression vector were respectively constructed. After correct sequencing, it was transferred to Agrobacterium tumefaciens GV3101, and the tomato material Ailsa Craig was transformed by the Agrobacterium-mediated method. For the obtained SlCLE10 overexpression and knockout lines, first, DNA-level identification was carried out, and primers for SlCLE10 overexpression and knockout were designed for identification. Among them, the SlCLE10 knockout mutant material was sent for sequencing, and after 3 generations of purification to obtain homozygous plants, the expression of the SlCLE10 gene in the overexpression and knockout mutant materials was detected by qPCR, and overexpression and knockout mutant materials were screened and obtained. The constructed vectors were transferred to Agrobacterium tumefaciens GV3101, and the tomato material Ailsa Craig was transformed by the Agrobacterium-mediated method.

[0045] Primers in the construction of SlCLE10 overexpression and knockout vectors

[0046]

[0047]

[0048] Example 2

[0049] Construction and transformation of SlCLE10 promoter fused to reporter gene

[0050] By PCR method, the promoter of SlCLE10 gene was linked to pCambia1301 - GUS vector. The constructed vector was transferred into Agrobacterium tumefaciens GV3101, and tomato material Ailsa Craig was transformed by Agrobacterium - mediated method.

[0051] Primers in the construction of SlCLE10 promoter fused to reporter gene

[0052] Primer Name Sequence SEQ ID NO Promoter1-F GGCTTAAUTTGACATGTGAAAGTCGGCG 9 Promoter1-R GGTTTAAUAGGTGAAGATTAATTAATTA 10

[0053] Example 3

[0054] Expression status of SlCLE10 induced by osmotic stress

[0055] Two - week - old proSlCLE10 - GUS transgenic tomato seedlings with 1 - 2 true leaves just emerging were used as experimental materials. The roots were immersed in 1 / 2MS solution containing 300 mM Mannitol for treatment, and samples were taken for staining at 0.5, 1, 2, 3, and 4 h respectively. As Figure 2 shown, with the increase of treatment time, SlCLE10 was gradually expressed in roots and true leaves, and the expression level of SlCLE10 reached the highest at 2 h of treatment, and then the expression level gradually decreased.

[0056] Example 4

[0057] Growth status of seedlings under salt stress and drought stress treatments

[0058] One - week - old seedlings grown on 1 / 2MS were transplanted into culture soil, and the weight of culture soil used for each seedling was 25 ± 0.5 g. When they grew to 21 days old, drought and salt stress treatments were carried out. Salt stress was treated with an aqueous solution of 175 mM NaCl, and 1 L of the solution was poured every 3 days, and the control was watering. For drought treatment, watering was stopped. After 18 days of treatment, 2 L of water was poured into the drought - treated pots for re - watering treatment, and the length and fresh weight of the above - ground parts were photographed and recorded 2 days later. As Figure 3As shown, under normal growth conditions, there were no significant differences in the above-ground fresh weight and length among WT, CR-slcle10 mutants, and OE-SlCLE10; under drought stress, the CR-slcle10 mutants showed obvious wilting and could not recover to the normal growth state after rewatering. The above-ground fresh weight and length were significantly lower than those of the wild type, while the overexpressing plants could recover to the normal state after rewatering, and the above-ground fresh weight and length were significantly higher than those of the wild type; after salt stress treatment, the new leaves of the plants showed curling and yellowing. The above-ground height and fresh weight of OE-SlCLE10 were significantly higher than those of the wild type and mutants, showing a more salt-tolerant phenotype.

[0059] Example 5

[0060] Growth status of roots of different tomato materials under osmotic stress treatment

[0061] After WT, OE-SlCLE10, and CR-slcle10 mutants grew on 1 / 2MS for 2 days, when the seedlings showed white tips, they were transferred to osmotic plates. After 5 days, photos were taken for observation, and the root length, root hair length in the mature zone, and root diameter were quantitatively counted respectively, as Figure 4 and Figure 5 shown. Under the condition of 1 / 2MS, there were no significant differences in the root length, root diameter in the mature zone, and root hair length among the WT, OE-SlCLE10, and CR-slcle10 mutant materials; when tomato seedlings were treated with 300 mM Mannitol osmotic stress for 5 days, the root length, root hair length, and root diameter in the mature zone of the OE-SlCLE10 material were significantly higher than those of the wild type and mutants, while the root length and root hair in the mature zone of the CR-slcle10 mutant material were significantly shorter than those of the wild type.

[0062] Example 6

[0063] Determination of soluble sugars and stress-resistant related enzyme activities in tomatoes under drought and salt stresses

[0064] The SlCLE10 overexpressing material and knockout mutant material obtained in Example 1 were subjected to short-term drought and salt stress treatments during the seedling stage, and relevant indicators were measured.

[0065] The results are as Figure 6As shown, under drought and salt stress adversity treatments, the soluble sugar content increased in wild-type, SlCLE10 overexpression materials, and mutant materials. However, the content was lower in the mutant materials than in the wild-type, while the soluble sugar content was significantly higher in the overexpression lines than in the wild-type. After salt and drought treatments, the activity of SOD in the overexpression materials was always higher than that in the wild-type; the SOD activity in the mutant materials was always lower than that in the wild-type after salt and drought treatments. After salt and drought adversity treatments, the activities of catalase (CAT) and peroxidase (POD) were significantly higher in the overexpression lines than in the wild-type, thus affecting the stress resistance of plants. The above results indicate that changes in the expression level of the SlCLE10 gene in plants lead to different responses of plants to salt and drought environments.

[0066] Example 7

[0067] Determination of Pro (A), MDA (B), O 2- (C) and H2O2 in materials under drought and salt stress

[0068] The proline content of the SlCLE10 overexpression materials and knockout mutant materials obtained in Example 1 was measured, and the results are as Figure 7 shown. After salt and drought treatments, the proline content of different materials tended to increase, but the overexpression materials accumulated more proline. The increase in proline content can enhance the stress resistance of plants. This may indicate that the change in the expression level of SlCLE10 in tomatoes affects the salt and drought tolerance of tomato seedlings, which may be related to the accumulation of proline content.

[0069] The level of lipid oxidation can be detected by measuring malondialdehyde (MDA). After salt and drought treatments, the increase in the MDA content in plants can reflect the severity of plant damage. After salt and osmotic treatments, MDA accumulated significantly, and its content increased significantly in the mutant materials. This indicates that the mutant materials suffered more severe membrane damage caused by salt and drought stress.

[0070] There was no significant difference in the O 2- content in the control, but after salt and drought adversity treatments, the O 2-The content increased significantly, while in the overexpression materials, the increase in its content was not obvious. In the control treatment, there was no significant difference in the content of H2O2 between the wild type and the overexpression lines. After salt and drought treatments, the enhanced stress resistance of the overexpression materials at the seedling stage of tomatoes might be due to the fact that a large amount of ROS was not accumulated in the cells, thus reducing the degree of cell damage. After salt and drought treatments, the content of H2O2 in the mutant lines showed a significant upward trend. From this, it can be found that the reduced stress resistance of the mutant lines at the seedling stage of tomatoes might be due to the large accumulation of ROS, which led to damage to plant cells.

[0071] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. Use of the tomato SlCLE10 gene in improving plant drought and salt tolerance, wherein the nucleotide sequence of the tomato SlCLE10 gene is as shown in SEQ ID NO.

1.

2. The application according to claim 1, wherein Prepare the tomato SlCLE10 gene into a recombinant expression vector, expression cassette, transgenic cell line, recombinant bacterium or recombinant virus for use in improving the salt and drought tolerance of a target plant.

3. The application according to claim 2, characterized in that, The target plant is tomato.

4. A method for cultivating salt-tolerant and drought-tolerant plants, characterized in that, The method includes overexpressing the tomato SlCLE10 gene in the genes of the target plant to obtain a target plant with salt and drought tolerance.

5. The method according to claim 4, wherein Construct a recombinant expression vector with the tomato SlCLE10 gene, then transform the recombinant expression vector into a strain to obtain a recombinant strain carrying the tomato SlCLE10 gene, then infect the plant leaves with the recombinant strain to make the plant carry the tomato SlCLE10 gene, and finally regulate the salt and drought tolerance of the plant through the expression of the tomato SlCLE10 gene.

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