CN114736911B

CN114736911B - Tomato SlBTB19 gene, protein and application thereof in improving low temperature resistance of plants

Info

Publication number: CN114736911B
Application number: CN202210220012.XA
Authority: CN
Inventors: 周艳虹; 刘斯迪; 徐进; 胡璋健; 齐振宇; 喻景权
Original assignee: Anqing Mayor's Triangle Future Industry Research Institute; Zhejiang University ZJU
Current assignee: Anqing Mayor Triangle Future Industry Research Institute; Zhejiang University ZJU
Priority date: 2022-03-08
Filing date: 2022-03-08
Publication date: 2023-09-26
Anticipated expiration: 2042-03-08
Also published as: CN114736911A

Abstract

The application discloses a tomato SlBTB19 gene, a protein and application thereof in improving low temperature resistance of plants, wherein the nucleotide sequence of the gene SlBTB19 is SEQ ID NO.1, and the corresponding amino acid sequence is shown as SEQ ID NO. 2. A tomato SlBTB19 gene knockout plant is constructed by a gene means, the expression level of the gene SlBTB19 is regulated to research a regulation mechanism of low-temperature resistance of the tomato, and as a result, the low-temperature resistance of the tomato can be improved by the SlBTB19 gene silencing plant. The SlBTB19 gene provided by the application provides gene resources for cultivating new low-temperature-resistant tomato varieties, has a good potential application value, and lays a theoretical foundation for researching a mechanism of a tomato plant responding to stress signals and a molecular mechanism of a harmful environment tolerance.

Description

Tomato SlBTB19 gene, protein and application thereof in improving low temperature resistance of plants

Technical Field

The application relates to the fields of genetic engineering and molecular biology, in particular to a tomato SlBTB19 gene, protein and application thereof in improving low temperature resistance of plants.

Background

The temperature is critical to the growth and development of crops, and directly affects the yield and quality of crops. The cold injury in winter and spring of China and the threat of low temperature in the morning and evening in facility environment greatly influence photosynthesis of crops like tomatoes with happiness temperature, and limit production and supply of vegetables in China. The cold damage can cause the growth of plants to be inhibited, the membrane lipid to change phase and the metabolism to be disturbed, so that the yield and the quality of fruits are reduced, and serious economic loss is caused. Therefore, the method is of great significance in exploring key genes of vegetable crops in the low-temperature response and signal transmission processes and improving the low-temperature resistance of the vegetable crops by using modern molecular technology means.

Tomatoes (Solanum lycopersicum l.) originate from the andes mountain in south america, and it has been demonstrated that, in the first generation, tomatoes cultivated in china were introduced from europe by american tutorials, initially as ornamental foods by people, and later on, their fruits were gradually consumed. Since the beginning of the 50 s of the last century, the tomato industry has developed rapidly and is now a stock of fruit and vegetable in the home. From the research point of view, tomatoes are warm-loving plants, are sensitive to low temperatures, have small genomes and are easy to transform, and are widely used in researches of functional genomics, molecular biology and the like. The tomato is used as an object for research, so that not only can the development of the tomato industry be directly promoted, but also a theoretical basis can be provided for exploring the injury mechanism and response mechanism of plants under cold injury and improving the cold resistance of the plants.

BTB proteins are a class of conserved domain-containing proteins found earliest in drosophila and are involved in multiple stages of eukaryotic growth and development processes such as phototropic growth, stress resistance, ubiquitin, 26S proteasome degradation processes, ion channels, and cell cycle regulation. The BTB-TAZ protein (BT protein) belongs to a subfamily of BTB proteins, is taken as a framework protein, can interact with different substrate proteins, and participates in different signal pathways to regulate the growth and development of plants. At present, the research on BT protein is mostly focused on Arabidopsis thaliana and apples, for example, atBT2 can interact with auxin related kinase PID1, negatively regulate the kinase activity of the protein and influence auxin signals in plants; in apples, BTB-TAZ protein BT2 significantly reduced anthocyanin biosynthesis induced by ethylene, ABA, intense light, drought stress and mechanical injury by inhibiting expression of MYB1 gene. These all indicate that BT proteins may be involved in plant response to stress.

However, few studies on tomato BT protein are carried out, and the action and the regulation mechanism of BT protein in tomato under low temperature stress are not reported.

Disclosure of Invention

In view of the above, the embodiment of the application provides a tomato SlBTB19 gene, a protein and application thereof in improving low temperature resistance of plants.

According to a first aspect of the embodiment of the application, a gene for improving the low temperature resistance of tomatoes is provided, wherein the gene is a tomato SlBTB19 gene, and the tomato SlBTB19 gene has a nucleotide sequence shown as SEQ ID NO. 1.

According to a second aspect of the embodiment of the application, the application of the tomato SlBTB19 gene in improving the low temperature resistance of tomatoes is provided, the expression level of the tomato SlBTB19 gene is reduced by a gene CRISPR/Cas9 technology, and the nucleotide sequence of the SlBTB19 is shown as SEQ ID NO. 1.

Further, the gene SlBTB19 is mutated by gene CRISPR/Cas9 technology; the gene CRISPR/Cas9 technology is specifically as follows:

designing a target sequence of a SlBTB19 gene by using a CRISPR-P website, connecting the synthesized target sequence to a Bbs I site of a U6-26-sgRNA1-SlBTB19-SK vector after annealing, and then connecting a newly obtained U6-26-sgRNA1-SlBTB19-SK fragment to a HindIII/Kpn I site of a pCAMBIA1301 vector to construct a tomato SlBT19 gene CRISPR expression vector; wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 3;

introducing a tomato SlBT19 gene CRISPR expression vector into a host cell, infecting a target plant by using the expression vector, and screening a positive transgenic plant to obtain a low-temperature-resistant transgenic plant.

Further, the tomato SlBT19 gene CRISPR expression vector is an expression vector with a 35S promoter.

Further, the expression vector of the 35S promoter is a vector pCAMBIA1301.

Further, the gene CRISPR expression vector plasmid is pCAMBIA1301-U6-26-sgRNA1-SlBTB19-35S-cas9SK.

Further, the host cell is an E.coli cell or an Agrobacterium cell.

Further, the agrobacterium is GV3101.

According to a third aspect of the embodiment of the application, a protein for improving the low temperature resistance of tomatoes is provided, wherein the protein is tomato SlBTB19 protein, and the tomato SlBTB19 protein has an amino acid sequence shown as SEQ ID NO. 2.

The technical scheme provided by the embodiment of the application can comprise the following beneficial effects:

according to the embodiment, the tomato SLBTB19 gene knockout plant is constructed by a genetic means, the expression level of the gene SLBTB19 is regulated to research the regulation mechanism of the gene SLBTB19 on the low-temperature resistance of the tomato, and the result shows that the low-temperature resistance of the SLBTB19 gene knockout plant is enhanced. The application provides gene resources for cultivating new low-temperature-resistant tomato varieties, has better potential application value, and lays a theoretical foundation for researching the mechanism of the tomato plants for responding to stress signals and the molecular mechanism of the adverse environment.

The application constructs a transgenic plant with the tomato SlBT19 gene knocked out for the first time, and performs functional research. Through low-temperature treatment experiments, the SlBTB19 gene is found to play a negative regulation role in low-temperature stress resistance of tomatoes.

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 as claimed.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments consistent with the application and together with the description, serve to explain the principles of the application.

FIG. 1 shows the sequencing result of sgRNA sequence of the SlBTB19 knockout tomato strain in the example of the present application;

FIG. 2 shows the phenotype of wild type and SlBTB19Crispr/Cas9 knockout plants after 6 days of low temperature treatment in examples of the present application;

FIG. 3 shows the relative conductivities of wild-type and SlBTB19Crispr/Cas9 knockout plants after 6 days of low temperature treatment in examples of the application;

FIG. 4 shows the PSII maximum photochemical efficiency (Fv/Fm) change after 6 days of low temperature treatment of wild type and SlBTB19Crispr/Cas9 knockout plants in examples of the application.

FIG. 5 shows the variation of the expression level of cold-resistant gene of wild-type and SlBTB19Crispr/Cas9 knockout plants treated at low temperature for 6 hours in the examples of the present application, wherein A is the expression level of tomato CBF1 gene, B is the expression level of tomato CBF2 gene, and C is the expression level of tomato CBF3 gene.

Detailed Description

Reference will now be made in detail to exemplary embodiments, examples of which are illustrated in the accompanying drawings. When the following description refers to the accompanying drawings, the same numbers in different drawings refer to the same or similar elements, unless otherwise indicated. The implementations described in the following exemplary examples do not represent all implementations consistent with the application. Rather, they are merely examples of apparatus and methods consistent with aspects of the application as detailed in the accompanying claims.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this specification and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. It should also be understood that the term "and/or" as used herein refers to and encompasses any or all possible combinations of one or more of the associated listed items.

Example 1:

construction of a SLBTB19CRISPR/Cas9 gene knockout vector and acquisition of a SLBTB19CRISPR/Cas9 gene knockout plant: in order to explore the influence of the deletion of the SlBTB19 on the low temperature resistance of tomatoes, a target gene sequence of the SlBTB19 is designed, a pCAMBIA1301-U6-26-sgRNA1-SlBTB19-35S-Cas9SK vector is constructed through enzyme digestion connection, and the SlBTB19 is knocked out by using a CRISPR/Cas9 technology for research.

First, a CRISPR-P website ((http): the target sequence sgRNA1 of the SlBTB19 gene was designed by// cbi.hzau.edu.cn/cgi-bin/CRISPR): 5'-GGGAAGGGTGTACGAGCGTA-3' annealing the synthesized sgRNA1 sequence (single strand) to form double strand sgRNA1 with BbsI restriction sites at both ends, ligating the formed sgRNA1 with the BbsI restriction enzyme-digested AtU-26 SK vector, extracting positive plasmid for later use, and naming U6-26-sgRNA1-SlBTB19-SK. as U6-26-sgRNA1-SlBTB19-SK and 35S-Cas9SK vectors by Kpn I and Sal I restriction enzyme, double digestion of U6-26-sgRNA1-SlBTB19-SK and 35S-Cas9SK vectors, recovering the digested fragments of U6-26-sgRNA1-SlBTB19 and ligating the digested fragments of U6-26-SbRNA 1-SlBTB19 onto the same digested 35S-Cas9SK vector, and bacterial liquid PCR detection primers: 5'-GACGGCCAGTGAATTGTA-3', U6-26-R5'-TACGCTCGTACACCCTTCCC-3', sequencing to verify positive clones, extracting positive plasmid for later use, named U6-26-sgRNA1-SlBTB19-35S-Cas9SK, double digestion of U6-26-sgRNA1-SlBTB19-35S-Cas9SK and pCAMBIA1301 vector with Kpn I and Xba I restriction enzyme, recovering about 6kb band of U6-26-sgRNA1-SlBTB19-35S-Cas9SK, i.e. fragment U6-26-sgRNA-SlBTB19-35S-Cas9, connecting to digested pCAMBIA1301 vector, transforming E.coli DH 5. Alpha. Competent cells with the ligation product, picking single colony in liquid LB medium containing 50mg/L kanamycin (Kan), shaking culture was carried out at 37℃and 200rpm overnight. Primers are designed at the 5' end of the pCAMBIA1301 vector for bacterial liquid PCR detection (about 550 bp), and the upstream and downstream primers are U6-26-Cas9-F:5'-GCTCGTATGTTGTGTGGAAT-3', U6-26-Cas9-R:5'-TACGCTCGTACACCCTTCCC-3'. The positive clones were sequenced and the positive plasmids were extracted for use and designated pCAMBIA1301-U6-26-sgRNA1-SlBTB19-35Scas9SK. The positive plasmid was transferred into Agrobacterium GV3101 by electric shock method, mixed with glycerol 1:1 and stored at-80 ℃.

The vector uses a leaf disc method to infect wild type (Ailsa Craig) tomato cotyledons by GV3101 agrobacterium to obtain a resistant bud system of the vector knocked out by transforming pCAMBIA1301-U6-26-sgRNA1-SlBTB19-35S-cas9SK, and the corresponding plant is obtained after transplanting. Finally, 13 SLBTB19CRISPR/Cas9 gene knockout lines are obtained. Sequencing of the DNA extracted from lines #1 and #2 revealed that line #1 deleted the 10bp mutation and line #2 increased the 1bp mutation (FIG. 1).

Example 2: detection of low temperature resistance of SlBTB19CRISPR/Cas9 gene knockout plant

Tomato varieties selected for testing were wild type Ailsa Craig and the SlBTB19CRISPR/Cas9 lines #1 and #2 obtained in example 1, seed was sown at full 3:1, watering the seedling according to the moisture condition of the substrate to keep the substrate moist after the seedling emerges in a plastic basin of the turf and vermiculite composite cultivation substrate, watering Hoagland nutrient solution in the whole process, and carrying out low-temperature treatment when four leaves are concentric, wherein the treatment temperature is 4 ℃.

The test had a total of 6 treatments: 1) WT normal temperature group; 2) SlBTB19 CRISPR/Cas9#1 normal temperature group; 3) SlBTB19 CRISPR/Cas9#2 normal temperature group; 4) WT low temperature group; 5) The SlBTB19 CRISPR/Cas9#1 low temperature group; 6) SlBTB19 CRISPR/Cas9#2 low temperature group. The low temperature treatment time was 6d. Phenotype photographing, relative conductivity measurement and maximum photochemical efficiency measurement of the optical system II are carried out after the low-temperature treatment is finished.

The specific determination method of the maximum photochemical efficiency of the optical system II comprises the following steps: after the plants were acclimatized in a dark environment for 30 minutes, they were irradiated with detection light using a chlorophyll fluorescence imager (IMAG-PAM; heinz Walz, germany)<0.5μmol m ^-2 s ^-1 ) The minimum fluorescence Fo was measured and saturated pulse light (4000. Mu. Mol m) ^-2 s ^-1 ) The maximum fluorescence Fm was measured.

The fluorescence parameter calculation method comprises the following steps: PS II maximum photochemical efficiency (Fv/Fm) = (Fm-Fo)/Fm.

The method for measuring the relative conductivity of the plants comprises the following steps: cutting the treated tomato leaves into strips with proper length (avoiding main pulse), rapidly weighing 3 parts of fresh samples, respectively placing each part of fresh samples into a graduated centrifuge tube filled with 10ml of deionized water, covering a cover, and placing the solution in a shaking table at room temperature for leaching for 2 hours. The conductivity R1 of the extract is measured by a conductivity meter, then the extract is heated in a boiling water bath for 15min, cooled to room temperature and shaken uniformly, and the conductivity R2 of the extract is measured again. Relative conductivity = R1/R2 x 100%.

The results show that, under low temperature conditions, the SlBTB19CRISPR/Cas9 plants show a cold resistant phenotype (fig. 2), lower conductivity (fig. 3), higher Fv/Fm (fig. 4) and CBF gene expression (fig. 5) compared to the wild type, indicating that the SlBTB19 gene knockout can significantly increase the tolerance of tomato to low temperatures.

Other embodiments of the application will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure herein. This application is intended to cover any variations, uses, or adaptations of the application following, in general, the principles of the application and including such departures from the present disclosure as come within known or customary practice within the art to which the application pertains. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the application being indicated by the following claims.

It is to be understood that the application is not limited to the precise arrangements and instrumentalities shown in the drawings, which have been described above, and that various modifications and changes may be effected without departing from the scope thereof. The scope of the application is limited only by the appended claims.

Sequence listing

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Claims

1. Tomato (tomato)SlBTB19Application of gene in improving low temperature resistance of tomato, and gene CRISPR/Cas9 technology is adopted to enable tomato to be subjected to low temperature resistanceSlBTB19The expression level of the gene is reduced, whichSlBTB19The nucleotide sequence of (2) is shown as SEQ ID NO. 1.

2. The use according to claim 1, the gene being caused by gene CRISPR/Cas9 technologySlBTB19Mutation occurs; the base isThe CRISPR/Cas9 technology is specifically as follows:

design of website by CRISPR-PSlBTB19Gene target sequence sgRNA 5'-GGGAAGGGTGTACGAGCGTA-3', synthetic target sequence annealed and connected to U6-26-sgRNA-SlBTB19-SK carrierBbs I site, then the newly obtained U6-26-sgRNA-SlBTB19-SK fragment was ligated into pCAMBIA1301 vectorHind III/KpnI site, tomato was constructedSlBTB19A gene CRISPR expression vector; wherein the nucleotide sequence of the sgRNA is shown as SEQ ID NO. 3; the U6-26-sgRNA-SlBTB19-SK vector is obtained by connecting double-stranded sgRNA formed after sgRNA annealing with AtU6-26SK vector cut by BbsI restriction enzyme, and extracting positive plasmid;

tomato is preparedSlBTB19The gene CRISPR expression vector is led into host cells, and then the target plant is infected by the gene CRISPR expression vector, and positive transgenic plants are screened to obtain low-temperature-resistant transgenic plants.

3. The use according to claim 2, the gene CRISPR expression vector plasmid is pCAMBIA1301-U6-26-sgRNA-SlBTB19-35S-cas9SK, wherein:

by means ofKpnI andSali restriction endonuclease carries out double digestion on U6-26-sgRNA-SlBTB19-SK and 35S-Cas9SK vectors simultaneously, the digestion products are recovered, and the digested U6-26-sgRNA-SlBTB19 fragment is connected to the same digested 35S-Cas9SK vector, and bacterial liquid PCR detection primers are U6-26-F:5'-GACGGCCAGTGAATTGTA-3', U6-26-R:5'-TACGCTCGTACACCCTTCCC-3' sequencing and verifying positive clones, and extracting positive plasmids for later use, wherein the positive plasmids are named U6-26-sgRNA-SlBTB19-35S-cas9SK; by means ofKpnI andHindIII restriction enzyme double-enzyme cutting U6-26-sgRNA-SlBTB19-35S-cas9SK and pCAMBIA1301 vector, recovering 6kb band from U6-26-sgRNA-SlBTB19-35S-cas9SK, namely U6-26-sgRNA-SlBTB19-35S-cas9 fragment, connecting to the enzyme-cut pCAMBIA1301 vector; e.coli DH5 alpha competent cells are transformed by the connection product, single colony is selected, the mixture is cultured overnight in liquid LB culture medium containing 50mg/L kanamycin at 37 ℃ and 200rpm in an oscillating way, and primers are designed at the 5' end of pCAMBIA1301 vector to carry out bacterial liquid PCR detection of 550bpThe upstream and downstream primers are U6-26-Cas9-F:5'-GCTCGTATGTTGTGTGGAAT-3', U6-26-Cas9-R:5'-TACGCTCGTACACCCTTCCC-3' the positive clones were sequenced and the positive plasmids were extracted for use and designated pCAMBIA1301-U6-26-sgRNA-SlBTB19-35Scas9SK.

4. The use according to claim 2, wherein the host cell is an e.

5. The use according to claim 4, wherein the agrobacterium is GV3101.