CN115786394A - Application of tomato SlZF14 gene in improving low-temperature resistance of plants - Google Patents

Abstract

The invention discloses an application of a tomato SlZF14 gene in improving the low temperature resistance of plants, wherein the expression level of the tomato SlZF14 gene is increased by a gene overexpression technology, and the gene is represented by SEQ ID NO: 1. The result shows that the expression of the tomato low-temperature resistance gene can be induced by the overexpression of the SlZF14 at low temperature, and the low-temperature resistance of the tomato is improved. In the SlZF14 gene editing plant, the low-temperature resistance of the tomato is obviously inhibited. The invention provides gene resources for cultivating new species of low temperature resistant tomatoes, has good potential application value, and lays a theoretical foundation for researching the mechanism of tomato plants responding to stress signals and the molecular mechanism of adverse environment resistance.

Description

Application of tomato SlZF14 gene in improving low-temperature resistance of plants

Technical Field

The application relates to the fields of genetic engineering, molecular biology, physiology and the like, in particular to application of a tomato SlZF14 gene in improving low-temperature resistance of plants.

Background

Tomatoes (Solanumlycopersicum l.) belong to the solanaceae family, annual or perennial herbs of the genus lycopersicon. Crop yield reduction caused by low temperature is a ubiquitous problem worldwide. Tomato is a kind of warm vegetable which is widely cultivated, and the growth and development of the tomato are seriously influenced by low temperature. Improving the low temperature resistance of the tomatoes and cultivating low temperature resistant varieties have important practical production significance.

After the plants are subjected to low-temperature stress signals, the transcription factors play an important role in the expression regulation of stress-related genes. The C2H2 type zinc finger protein is a transcription factor which is most clearly researched in eukaryotes, contains a specific QALGGH conserved sequence of a plant, can regulate and control various physiological and biochemical reactions in the plant body, and plays an important role in the growth and development of the plant and stress resistance by recognizing target DNA or interaction with RNA. At present, the research on the effect of tomato C2H2 type zinc finger protein in low temperature stress is rarely reported. The tomato C2H2 type zinc finger protein gene SlZFP1 is over-expressed in arabidopsis thaliana and rice by utilizing a transgenic means, and the expression of a series of low-temperature response genes at the downstream can be induced, so that the cold resistance of arabidopsis thaliana and rice is enhanced. However, a method for changing the low-temperature resistance of tomato plants by directly constructing transgenic materials of tomato C2H2 type zinc finger protein genes is not realized.

Therefore, tomato materials with different expression amounts of SlZF14 are cultivated by cloning and transgenic technology of the SlZF14 gene, and the method has good application prospects in the aspects of improving the resistance of tomatoes to low-temperature stress and excavating stress gene resources.

Disclosure of Invention

In view of the above, the embodiments of the present application provide an application of a tomato SlZF14 gene in improving low temperature resistance of plants.

According to the embodiment of the application, the application of the tomato SlZF14 gene in controlling the tomato low-temperature resistance is provided, the expression level of the tomato SlZF14 gene is increased through a gene overexpression technology, and the gene is SEQ ID NO: 1.

Optionally, the gene overexpression technology is specifically as follows:

extracting total RNA of the tomato, carrying out reverse transcription to obtain cDNA, amplifying an SlZF14 gene by using the cDNA as a template and F and R as primers, and constructing an amplification product on an overexpression vector; the nucleotide sequences of the primers F and R are shown as SEQ ID NO:3 and SEQ ID NO:4 is shown in the specification; and (3) introducing the over-expression vector into a host cell, infecting a target plant with the over-expression vector, and screening to obtain a positive transgenic plant.

Optionally, the host cell is an escherichia coli cell or an agrobacterium cell.

Alternatively, the agrobacterium cell is GV3101.

Optionally, the over-expression vector is an expression vector with a 35S promoter.

Optionally, the overexpression vector plasmid is pFGC 1008:SlZF14-HA.

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

according to the invention, the expression level of the SlZF14 gene of the tomato is increased through a gene overexpression technology, and the SlZF14 gene plays a role in forward regulation and control in low-temperature stress resistance of the tomato through low-temperature treatment.

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.

Drawings

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 detection result of the plant protein of the tomato line over-expressed by the SlZF14 gene in example 1 of the invention.

FIG. 2 shows the sequencing result of the sgRNA sequence of the SlZF14 gene knockout tomato line in example 2 of the present invention.

FIG. 3 shows that the transgenic tomatoes SlZF14-OE #1 and SlZF14-OE #3, zf14#2 and zf14#6 show low temperature resistant phenotypes under normal temperature and low temperature conditions in example 4 of the invention.

FIG. 4 shows the conductivity changes of SlZF14-OE #1 and SlZF14-OE #3, zf14#2 and zf14#6 of transgenic tomatoes under the conditions of normal temperature and low temperature in example 4 of the invention.

FIG. 5 shows the PSII maximum photochemical quantum yield (Fv/Fm) changes of transgenic tomatoes SlZF14-OE #1 and SlZF14-OE #3, zf14#2 and zf14#6 under normal temperature and low temperature conditions in example 4 of the invention.

Detailed Description

The present invention will be further described with reference to the following examples, but the present invention is not limited to the following examples.

Example 1: construction of SlZF14 overexpression vector

The SlZF14 gene was cloned from the tomato genome. Specific primers SlZF14-F and SlZF14-R are designed according to the sequence analysis of a coding region, restriction sites (Asc I and Kpn I) are respectively added on the primers, and the sequences are shown as SEQ ID NO. 3 and SEQ ID NO. 4. Amplifying an SlZF14 fragment by using KOD high-fidelity enzyme PCR, carrying out enzyme digestion on the vector, and carrying out homologous recombination on the SlZF14 fragment to pFGC1008-HA to obtain an overexpression vector pFGC1008, namely SlZF14-HA. The recombinant plasmid is sent to Shanghai company for sequencing confirmation, and the nucleotide sequence of the obtained gene SlZF14 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 showed that the cloned sequence was identical to the sequence published in Solgenomics (Solyc 06g 075780).

Example 2: construction of SlZF14 gene mutation vector

The SlZF14 gene target sequence is designed through a CRISPR-P website, the specific sequence is shown as SEQ ID NO:5, the synthesized target sequence is connected to the Bbs I site of an AtU6-sgRNA-AtUBQ-Cas9 vector after annealing, and then the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment is connected to the Hind III/Kpn I site of a pCAMBIA1301 vector, so that the tomato SlZF14 gene CRISPR expression vector is constructed. The recombinant plasmid was sent to Shanghai for sequencing confirmation.

Example 3: construction and detection of tomato SlZF14 transgenic material

An overexpression vector pFGC1008:: slZF14-HA and a gene editing vector pCAMBIA1301:: atU6-sgRNA (SlZF 14) -AtUBQ-Cas9 are added. Transforming agrobacterium GV3101, performing tomato cotyledon infection, obtaining tissue culture seedling through inducing callus, resistance inducing differentiation and rooting culture, testing kanamycin resistance and chloramphenicol resistance of T1 generation mutant seed and over-expressed seed separately, selecting 3/4 strain with resistance and 1/4 strain without resistance, and showing that the over-expression vector connected with target gene is inserted in single copy form. These plants were removed and single harvest was performed. Western Blot was used to verify that SlZF14 overexpresses positive transgenic plants, and the results showed that wild type HAs no protein band, while the overexpressing strain HAs a band of SlZF14-HA (FIG. 1). Positive SlZF14 mutant transgenic plants were verified by PCR and sequencing techniques, and found to lack 4 bases, 7 bases, mutations at the 4 th and 7 th bases of the original adjacent motif (PAM), respectively, and translation was immediately stopped (fig. 2).

Example 4: detection of low temperature resistance of tomato SlZF14 transgenic material

Five-leaf one-heart wild-type tomato seedlings and the SlZF14 gene overexpression strain and the mutant strain obtained in example 3 were subjected to 25 ℃ and 4 ℃ treatment in an artificial incubator, and after the low-temperature treatment for 7 days, the low-temperature stress treatment group was compared with a control group which was not subjected to the low-temperature treatment under the same conditions, and the phenotypes (FIG. 3), the conductivities (FIG. 4), the PSII maximum photochemical quantum yields (Fv/Fm, FIG. 5) of the wild-type, overexpression strain and mutant strain tomato plants were observed. The results show that overexpressing tomato plants can significantly improve the low temperature resistance of tomatoes (fig. 3), with a conductivity significantly lower than that of the wild-type (WT) and zf14 mutant lines (fig. 4). Furthermore, the Fv/Fm of the over-expressed plants (FIG. 5) was higher than that of wild type tomato (WT), while the mutant lines were the lowest. Thus, tomato SlZF14 positively regulates the low temperature tolerance of plants.

Although the invention has been described in detail hereinabove with respect to specific embodiments thereof, it will be apparent to those skilled in the art that it is not limited to the above examples, but is susceptible to numerous variations and modifications. Accordingly, such modifications and improvements are intended to be within the scope of this patent, as claimed.

Claims (6)

1. The application of the tomato SlZF14 gene in controlling the low-temperature resistance of tomatoes is characterized in that the expression level of the tomato SlZF14 gene is increased by a gene overexpression technology, and the gene is SEQ ID NO: 1.

2. The method of claim 1, wherein the gene overexpression technique is specifically as follows:

extracting total RNA of the tomato, carrying out reverse transcription to obtain cDNA, amplifying an SlZF14 gene by using the cDNA as a template and F and R as primers, and constructing an amplification product on an overexpression vector; the nucleotide sequences of the primers F and R are shown as SEQ ID NO:3 and SEQ ID NO:4 is shown in the specification; and (3) introducing the over-expression vector into a host cell, infecting a target plant with the over-expression vector, and screening to obtain a positive transgenic plant.

3. Use according to claim 2, wherein the host cell is an E.coli cell or an Agrobacterium cell.

4. The use of claim 3, wherein the Agrobacterium cells are GV3101.

5. Use according to claim 2, wherein the overexpression vector is an expression vector with a 35S promoter.

6. The use as claimed in claim 5, wherein the overexpression vector plasmid is pFGC1008:: slZF14-HA.

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