CN113308479B - Application of SlNAC100 gene in improvement of low-temperature resistance of tomato - Google Patents
Application of SlNAC100 gene in improvement of low-temperature resistance of tomato Download PDFInfo
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Abstract
The invention discloses an application of a SlNAC100 gene in tomato low-temperature resistance, which is characterized in that the expression level of the tomato SlNAC100 gene is increased by a gene overexpression technology, and the nucleotide sequence of the tomato SlNAC100 gene is as follows: SEQ ID NO: 1. The invention provides gene resources for cultivating new species of low temperature resistant tomatoes, has potential application value, and lays a theoretical foundation for researching molecular mechanism of tomato responding to stress signals and mechanism for improving adverse environment tolerance.
Description
Technical Field
The invention relates to the field of genetic engineering and molecular biology, in particular to application of a SlNAC100 gene in improvement of low-temperature resistance of tomatoes.
Background
Tomato (Solanum lycopersicum L.) is native to south america, belongs to solanaceae, is a vegetable widely cultivated in the world, has an optimum growth temperature of about 20-25 ℃, and is sensitive to low temperature. However, as a facility cultivation country with the largest world area, cold damage in winter and spring in various parts of China and low temperature in the facility environment in the morning and evening greatly influence photosynthesis and growth and development of temperature-favored plants such as tomatoes, and the like, so that the yield and the quality of the plants are sharply reduced. Therefore, the research on the response mechanism and the molecular mechanism of the tomato to the low-temperature stress has important significance in digging the key gene for improving the low-temperature resistance of the tomato.
During low temperature stress response, activation of the stress signaling network can often induce the expression of various stress response genes efficiently and timely, and the expression of such a large number of differential genes needs to be co-regulated by different types of Transcription Factors (TFs) in a time and space manner in a synergistic manner. Generally, transcription factors act primarily as signal transmitters, transmitting external stress signals to the nucleus by specifically binding to cis-acting elements in the promoter regions of several genes, thereby affecting transcription of multiple genes associated with stress in plants and thus participating in various processes. NAC transcription factor is a class of transcriptional regulator that is unique in plants. The protein encoded by the NAC transcription factor is highly conserved at the N end and comprises A, B, C, D, E5 subdomains, wherein the A, C, D subdomain is highly conserved, B and E are not strongly conserved, and the C end of the protein has the characteristics of diversity and transcriptional activation. A large number of researches show that the NAC transcription factor plays an important role in various stresses such as plant growth and development, organ formation, hormone regulation, disease defense and the like. In the aspect of plant growth and development, Arabidopsis genes CUC1, CUC2 and CUC3 are related to apical meristem and floral organ formation, and genes NST1, NST2, SND1, SND2, VND7 and the like are related to secondary wall formation and thickening. However, the role of tomato NAC transcription factor in low temperature stress and its regulation mechanism are only rarely reported. Therefore, the tomato materials with different expression levels of SlNAC100 are cultivated by cloning and transgenic technology of the SlNAC100 gene, and the method has good application prospects in the aspects of improving the resistance of tomatoes to low-temperature stress and excavating adversity stress gene resources.
Disclosure of Invention
The purpose of the embodiments of the present application is to provide an application of a SlNAC100 gene in improving tomato low temperature resistance.
According to a first aspect of embodiments of the present application, a gene for improving tomato low temperature resistance is provided, the gene is a tomato SlNAC100 gene, and the tomato SlNAC100 gene has a nucleotide sequence of SEQ ID NO: 1.
According to a second aspect of embodiments of the present application, there is provided a use of a tomato SlNAC100 gene for improving the low temperature tolerance of tomato, wherein the expression level of the tomato SlNAC100 gene is increased by a gene overexpression technique, and the nucleotide sequence of the tomato SlNAC100 gene: SEQ ID NO: 1.
Further, the gene overexpression technology comprises:
extracting total RNA of the tomato, carrying out reverse transcription to obtain cDNA, amplifying SlNAC100 gene by using the cDNA as a template and F and R as primers, and constructing an amplification product onto a plant overexpression vector, wherein the nucleotide sequences of the F primer and the R primer are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
and (3) introducing the plant overexpression vector into a host cell, infecting a target plant by using the plant overexpression vector, and screening a positive transgenic plant to obtain a low-temperature-resistant transgenic tomato plant.
Further, the plant overexpression vector is an expression vector having a 35S promoter.
Further, the host cell is an escherichia coli cell or an agrobacterium cell.
Further, the agrobacterium cell is EHA 105.
Further, the plant overexpression vector is a vector pFGC1008-HA or a vector pCAMBIA 1301.
According to a third aspect of embodiments of the present application, there is provided a protein for improving the low temperature tolerance of tomato, wherein the protein is a tomato SlNAC100 protein, and the tomato SlNAC100 protein has an amino acid sequence shown in SEQ ID No. 2.
According to a fourth aspect of the embodiments of the present application, there is provided an application of a tomato SlNAC100 protein in improving low temperature tolerance of tomato, wherein the expression level of the tomato SlNAC100 protein is increased by a gene overexpression technique, and the tomato SlNAC100 protein has an amino acid sequence: SEQ ID NO:2, or a pharmaceutically acceptable salt thereof. The technical scheme provided by the embodiment of the application can have the following beneficial effects:
according to the embodiments, the tomato SlNAC100 overexpression plant or gene knockout plant is constructed by adopting a genetic means, so that the expression level of the tomato SlNAC100 gene is enhanced or inhibited, and the expression level of the gene SlNAC100 is regulated to study the regulation mechanism of the tomato low-temperature resistance. The result shows that SlNAC100 overexpression plants have no significant influence on tomato vegetative growth and reproductive growth, but can improve PSII maximum photochemical efficiency (Fv/Fm), and the cold resistance related genes COR47LIKE and COR6.6LIKE expression, reduce relative conductivity, and further improve the cold resistance of tomato plants.
The invention constructs a transgenic plant for overexpression and gene knockout of the tomato SlNAC100 gene for the first time and performs function research. The SlNAC100 gene provided by the invention provides gene resources for cultivating new low-temperature-resistant tomato varieties, has potential application value, and lays a theoretical foundation for researching a molecular mechanism of tomato responding to stress signals and a mechanism for improving adverse environment tolerance.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the application.
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The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the present application and together with the description, serve to explain the principles of the application.
FIG. 1 shows the Western Blot assay of the plant protein of tomato SlNAC100 overexpression line # 6 in the examples of the present application;
fig. 2 is the sequencing result of the sgRNA sequence of SlNAC100 knock-out tomato lines in the examples of the present application.
Fig. 3 is a phenotype of wild-type, SlNAC100 overexpressing plants and SlNAC100CRISPR/Cas9 knock-out plants after 7 days of cold treatment in the examples of the present application;
fig. 4 is the relative conductivities of wild-type, SlNAC100 overexpressing plants and SlNAC100CRISPR/Cas9 knock-out plants after 7 days of cold treatment in the examples of the present application;
fig. 5 shows the changes in the maximal photochemical efficiency (Fv/Fm) of PSII of wild-type, SlNAC100 overexpressing plants and SlNAC100CRISPR/Cas9 knock-out plants 7 days after cryopreservation in the examples of the present application.
FIG. 6 shows the change of cold-resistant gene expression level of wild-type, SlNAC100 overexpression plants and SlNAC100CRISPR/Cas9 knockout plants in 12h of low-temperature treatment in the examples of the application; wherein, A is the expression level of the tomato COR47like gene, and B is the expression level of the tomato COR6.6like gene.
Detailed Description
The invention will be further described with reference to the following examples, but the scope of the invention is not limited thereto.
Example 1: construction of SlNAC100 Gene overexpression vector
The SlNAC100 gene is derived from tomato, the number of the SlNAC100 gene in a tomato genome database is Solyc06g069710, and in order to investigate the influence of SlNAC100 overexpression on the low temperature resistance of tomato, the SlNAC100 gene is firstly cloned from a tomato genome. Specific primers SlNAC100-F and SlNAC100-R were designed based on coding region sequence analysis, and restriction sites (AscI and KpnI) were added to the primers, respectively, and the sequences are shown in SEQ ID NO:3 and 4. Amplifying an SlNAC100 fragment by PrimerSTAR high-fidelity enzyme PCR, then carrying out enzyme digestion on the PCR amplification fragment and the vector, connecting the SlNAC100 fragment to pFGC1008-HA, and obtaining a plant overexpression vector pFGC 1008-SlNAC 100-HA. The recombinant plasmid is sent to Shanghai company for sequencing confirmation, and the nucleotide sequence of the obtained gene SlNAC100 is shown as SEQ ID NO. 1; the amino acid sequence of the protein coded by the gene is shown in SEQ ID NO. 2. The results show that the cloned sequence is consistent with the sequence published in Solgenomics (Solyc06g069710), and a positive plasmid is extracted for later use and is named as pFGC1008:: SlNAC 100-HA.
Example 2: construction of SlNAC100CRISPR/Cas9 gene knockout vector
To explore the influence of SlNAC100 deletion on tomato low temperature resistance, a target gene sequence of SlNAC100 is designed, a pCAMBIA1301-U6-26-sgRNA1-SlNAC100-35S-Cas9SK vector is constructed by enzyme digestion and ligation, and SlNAC100 is knocked out by using a CRISPR/Cas9 technology for research.
Firstly, a target sequence of an SlNAC100 gene is designed by using a CRISPR-P website ((http:// cbi. hzau. edu. cn/cgi-bin/CRISPR), a specific sequence is shown as SEQ ID NO:5 and is sgRNA1: 5'-TGTTAAGGATGATGATCAGA-3'. a synthetic sgRNA1 sequence (single strand) is annealed to form a double-strand sgRNA1, and both ends of the sgRNA1 have BbsI restriction endonuclease cleavage sites, the formed sgRNA1 is connected with a AtU6-26SK vector which is cut by BbsI restriction endonuclease, a positive plasmid is extracted for standby, the positive plasmid is named as U6-26-sgRNA1-SlNAC 100-SK., the U6-26-sgRNA1-SLN 100-SK and the vector 35S-Cas9SK are subjected to double digestion by using KpnI and SalI restriction endonuclease at the same time, the respective digestion products are recovered, and the NACC 6-26-sgRNA-sln-25 vector is connected with a PCR bacterial liquid of a PCR 3884. the PCR vector 469-84, U6-26-F: 5'-GACGGCCAGTGAATTGTA-3' and U6-26-R: 5'-TATCTAAGCGATGTGGGACT-3', sequencing and verifying positive clones, extracting positive plasmids for later use, and naming the plasmids as U6-26-sgRNA1-SlNAC100-35S-cas9 SK. U6-26-sgRNA1-SlNAC100-35S-cas9SK and pCAMBIA1301 vectors were double digested with KpnI and XbaI restriction enzymes, and U6-26-sgRNA1-SlNAC100-35S-cas9SK recovered a band of about 6kb, i.e., the U6-26-sgRNA1-SlNAC100-35S-cas9 fragment, ligated to the digested pCAMBIA1301 vector. The ligation product was transformed into E.coli DH 5. alpha. competent cells, and a single colony was picked up and cultured overnight in liquid LB medium containing 50mg/L kanamycin (Kan) at 37 ℃ with shaking at 200 rpm. A primer is designed at the 5' end of a pCAMBIA1301 vector for carrying out bacterial liquid PCR detection (about 550bp), and the upstream primer and the downstream primer are respectively U6-26-Cas9-F:5'-GCTCGTATGTTGTGTGGAAT-3' and U6-26-Cas9-R: 5'-TATCTAAGCGATGTGGGACT-3'. Sequencing and verifying positive clones, extracting positive plasmids for later use, and naming the plasmids as pCAMBIA1301-U6-26-sgRNA1-SlNAC100-35S-cas 9.
Example 3: acquisition of SlNAC100 transgenic plants
SlNAC100-HA and a gene editing vector pCAMBIA1301-U6-26-sgRNA1-SlNAC100-35S-cas9 are used for transforming agrobacterium GV3101 by an electric shock method, wild type (Ailsa Craig) tomato cotyledon infection is carried out, tissue culture seedlings are obtained by inducing callus, resistance inducing differentiation and rooting culture, mutant seeds and over-expressed seeds of T1 generation are respectively tested for kanamycin resistance and chloramphenicol resistance, 3/4 resistant seeds and the rest 1/4 non-resistant seeds are selected, and the over-expressed vector connected with a target gene in the strain is inserted in a single copy form. These plants were removed and single harvest was performed. The result of verifying that SlNAC100 overexpresses a positive transgenic plant by using Western Blot shows that a wild type HAs no protein band, while an overexpression line HAs a SlNAC100-HA band (figure 1), and the result of verifying that the positive SlNAC100 mutates the transgenic plant by using PCR and sequencing technology shows that SlNAC100# 2 lacks 1 base and SlNAC100# 4 lacks 21 bases (figure 2).
Example 4: observation of low temperature resistance of SlNAC100 transgenic plants
The tomato varieties selected in the test are wild Ailsa Craig and SlNAC100 overexpression # 6 and SlNAC100CRISPR/Cas9 strains #2 and #4 obtained in example 3, seeds are sown in plastic pots filled with 3:1 turf and vermiculite composite culture substrates, after emergence of seedlings, watering is carried out according to the moisture condition of the substrates to keep the substrates wet, Hoagland nutrient solution is poured in the whole process, and low-temperature treatment is carried out when four leaves and one heart are treated, wherein the treatment temperature is constant at 4 ℃.
The experiment was set up with 8 treatments: 1) WT normal temperature group; 2) SlNAC100 overexpresses # 6; 3) SlNAC100CRISPR/Cas 9#2 normal temperature group; 4) SlNAC100CRISPR/Cas 9#4 normal temperature group; 5) WT low temperature group; 6) SlNAC100 overexpresses # 6; 7) SlNAC100CRISPR/Cas 9#2 cryogroup; 8) SlNAC100CRISPR/Cas 9#4 hypothermia. The low-temperature treatment time was 7 d. And after the low-temperature treatment is finished, phenotype shooting, the measurement of the maximum photochemical efficiency of a photosystem II and the measurement of relative conductivity are carried out.
The method for specifically measuring the maximum photochemical efficiency of the photosystem II comprises the following steps: after the plants were acclimated in a dark environment for 30 minutes, the minimum fluorescence Fo was measured by irradiating the detection light (< 0.5. mu. mol m-2s-1) with chlorophyll fluorescence imager (IMAG-PAM; Heinz Walz, Germany), and the maximum fluorescence Fm was measured by irradiating the detection light with saturated pulsed light (4000. mu. mol m-2 s-1).
Fluorescence parameter calculation method PS II maximum photochemical efficiency (Fv/Fm) ═ Fm-Fo)/Fm.
The method for measuring the relative conductivity of the plants comprises the following steps: uniformly cutting the treated tomato leaves into strips with proper length (avoiding main veins), quickly weighing 3 fresh samples (0.2 g per sample), respectively placing into a graduated centrifuge tube filled with 20ml of deionized water, covering with a cover, and placing into a shaking table at 28 ℃ for leaching for 1.5-2 h. Measuring conductivity of the leaching solution R1 with conductivity meter, heating in boiling water bath for 15min, cooling to room temperature, shaking, and measuring conductivity of the leaching solution R2. Relative conductivity R1/R2 100%.
The results show (fig. 3-6) that under low temperature conditions SlNAC100 overexpressing plants showed lower conductivity and higher Fv/Fm and COR gene expression compared to wild type, whereas SlNAC100CRISPR/Cas9 plants in contrast, indicating that the SlNAC100 gene has positive regulatory capacity for increasing tolerance to tomato low temperatures.
The SlNAC100 knockout can be realized by a CRISPR/Cas9 gene editing method, and can also be realized by methods such as T-DNA insertion, EMS mutagenesis and the like; and the vector introduction method is not limited to the method by Agrobacterium transformation, but includes plants obtained by introducing into crop cells, callus, tissues or organs through pollen tubes.
Sequence listing
<110> Zhejiang university
Application of <120> SlNAC100 gene in improvement of low-temperature resistance of tomato
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<170> SIPOSequenceListing 1.0
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actaatttcg ttgctattgc tattggtgat gttgatttga acaaagttga accttgggac 180
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Claims (7)
1. The application of the tomato SlNAC100 gene in improving the low temperature resistance of tomatoes is characterized in that the expression level of the tomato SlNAC100 gene is increased through a gene overexpression technology, and the nucleotide sequence of the tomato SlNAC100 gene: SEQ ID NO: 1.
2. The use according to claim 1, said gene overexpression technique comprising:
extracting total RNA of the tomato, carrying out reverse transcription to obtain cDNA, amplifying SlNAC100 gene by using the cDNA as a template and F and R as primers, and constructing an amplification product onto a plant overexpression vector, wherein the nucleotide sequences of the F primer and the R primer are respectively shown as SEQ ID NO. 3 and SEQ ID NO. 4;
and (3) introducing the plant overexpression vector into a host cell, infecting a target plant by using the plant overexpression vector, and screening a positive transgenic plant to obtain a low-temperature-resistant transgenic tomato plant.
3. The use according to claim 2, said plant overexpression vector being an expression vector with a 35S promoter.
4. The use according to claim 2, wherein the host cell is an Agrobacterium cell.
5. The use of claim 4, wherein said Agrobacterium cell is EHA 105.
6. The use of claim 5, wherein the plant overexpression vector is vector pFGC1008-HA or vector pCAMBIA 1301.
7. The application of tomato SlNAC100 protein in improving the low temperature resistance of tomatoes is characterized in that the expression level of the tomato SlNAC100 protein is increased through a gene overexpression technology, and the amino acid sequence of the tomato SlNAC100 protein is as follows: SEQ ID NO:2, or a pharmaceutically acceptable salt thereof.
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| CN114517202B (en) * | 2022-04-08 | 2024-04-19 | 山东农业大学 | Application of aquaporin gene SlPIP, 2 in improving resistance of tomatoes to facility continuous cropping soil |
| CN117069817B (en) * | 2023-10-13 | 2024-03-15 | 中国农业大学 | Method for forecasting low temperature stress and early prolonging low temperature resistance of tomatoes through overexpression of SlNAC3 gene |
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| CN113308479A (en) | 2021-08-27 |
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