Application of Tomato SlZF14 Gene in Improving Low Temperature Resistance of Plants

Abstract

The present invention discloses the application of tomato SlZF14 gene in improving the low-temperature tolerance of plants. The expression level of the tomato SlZF14 gene is increased by gene overexpression technology, and the gene has the nucleotide sequence shown in SEQ ID NO: 1. It is found that overexpression of SlZF14 can induce the expression of tomato low-temperature resistance genes under low temperature, thereby improving the low-temperature resistance of tomatoes. In SlZF14 gene-edited plants, the low-temperature resistance of tomatoes is significantly inhibited. The present invention provides gene resources for cultivating new tomato varieties with low-temperature tolerance, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants' response to stress signals and the molecular mechanism of tolerating adverse environments.

Description

Technical Field

[0001] This application relates to the fields of genetic engineering, molecular biology, physiology, etc. Specifically, it particularly relates to the application of the tomato SlZF14 gene in improving the low-temperature resistance of plants. Background Art

[0002] Tomato (Solanum lycopersicum L.) belongs to the Solanaceae crops and is an annual or perennial herbaceous plant of the genus Solanum. Crop yield reduction caused by low temperature is a common problem worldwide. As a widely cultivated warm-season vegetable, low temperature has an extremely serious impact on the growth and development of tomatoes. Improving the ability of tomatoes to resist low temperature and cultivating low-temperature tolerant varieties have important practical production significance.

[0003] After plants sense low-temperature stress signals, transcription factors play a crucial role in regulating the expression of stress-related genes. C2H2-type zinc finger proteins are a well-studied class of transcription factors in eukaryotes, containing a plant-specific QALGGH conserved sequence, which can regulate various physiological and biochemical reactions in plants and play an important role in plant growth, development, and stress resistance by recognizing target DNA or interacting with RNA. At present, there are few reports on the role of tomato C2H2-type zinc finger proteins in low-temperature stress. Some studies have overexpressed the tomato C2H2-type zinc finger protein gene SlZFP1 in Arabidopsis and rice by transgenic means and found that it can induce the expression of a series of downstream low-temperature response genes and enhance the cold resistance of Arabidopsis and rice. However, the method of directly constructing transgenic materials of the tomato C2H2-type zinc finger protein gene to change the low-temperature resistance of tomato plants has not been achieved.

[0004] Therefore, by cloning the SlZF14 gene and cultivating tomato materials with different expression levels of SlZF14 through transgenic technology, it has good application prospects in improving the resistance of tomatoes to low-temperature stress and exploring gene resources for adversity stress. Summary of the Invention

[0005] In view of this, an embodiment of this application provides an application of the tomato SlZF14 gene in improving the low-temperature resistance of plants.

[0006] According to an embodiment of this application, there is provided an application of the tomato SlZF14 gene in controlling the low-temperature resistance of tomatoes. The expression level of the tomato SlZF14 gene is increased through gene overexpression technology, and the gene is the nucleotide sequence shown in SEQ ID NO: 1.

[0007] Optionally, the gene overexpression technology is specifically as follows:

[0008] Extract total RNA from tomatoes, reverse transcribe to obtain cDNA, use cDNA as a template, F and R as primers to amplify the SlZF14 gene, and construct the amplified product onto an overexpression vector; the nucleotide sequences of the primers F and R are shown in SEQ ID NO: 3 and SEQ ID NO: 4; introduce the overexpression vector into host cells, and then use it to infect the target plants, and screen and obtain positive transgenic plants.

[0009] Optionally, the host cell is an Escherichia coli cell or an Agrobacterium cell.

[0010] Optionally, the Agrobacterium cell is GV3101.

[0011] Optionally, the overexpression vector is an expression vector with a 35S promoter.

[0012] Optionally, the overexpression vector plasmid is pFGC1008::SlZF14-HA.

[0013] The technical solutions provided in the embodiments of the present application may include the following beneficial effects:

[0014] In the present invention, the expression level of the tomato SlZF14 gene is increased through gene overexpression technology. Through low-temperature treatment, it is found that SlZF14 plays a positive regulatory role in the cold tolerance of tomatoes.

[0015] It should be understood that the above general description and the following detailed description are only exemplary and explanatory, and cannot limit the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] The accompanying drawings herein are incorporated into the specification and form a part of the specification, showing embodiments consistent with the present application, and are used together with the specification to explain the principles of the present application.

[0017] Figure 1 It is the detection result of plant protein Western Blot of the SlZF14 gene overexpressing tomato lines in Example 1 of the present invention.

[0018] Figure 2 It is the sequencing result of the sgRNA sequence of the SlZF14 gene knockout tomato lines in Example 2 of the present invention.

[0019] Figure 3 It is the phenotype of transgenic tomatoes SlZF14-OE#1 and SlZF14-OE#3, zf14#2, zf14#6 showing cold tolerance under normal temperature and low temperature conditions in Example 4 of the present invention.

[0020] Figure 4Conductivity changes of transgenic tomatoes SlZF14 - OE#1, SlZF14 - OE#3, zf14#2, and zf14#6 under normal temperature and low temperature conditions in Example 4 of the present invention.

[0021] Figure 5 Changes in the maximum photochemical quantum yield of PSII (Fv / Fm) of transgenic tomatoes SlZF14 - OE#1, SlZF14 - OE#3, zf14#2, and zf14#6 under normal temperature and low temperature conditions in Example 4 of the present invention. Detailed implementation mode

[0022] The present invention will be further described below in conjunction with specific embodiments, but the present invention is not limited to the following embodiments.

[0023] Example 1: Construction of SlZF14 over - expression vector

[0024] The SlZF14 gene was cloned from the tomato genome. According to the coding region sequence analysis, specific primers SlZF14 - F and SlZF14 - R were designed, and restriction enzyme cutting sites (Asc I and Kpn I) were added to the primers respectively. The sequences are shown in SEQ ID NO:3 and 4. The SlZF14 fragment was amplified by KOD high - fidelity enzyme PCR, and then the vector was digested. The SlZF14 fragment was homologous recombined onto pFGC1008 - HA to obtain the over - expression vector pFGC1008::SlZF14 - HA. The above - mentioned recombinant plasmid was sent to Shangya Company for sequencing confirmation. The nucleotide sequence of the obtained gene SlZF14 is shown in SEQ ID NO:1; the amino acid sequence of the protein encoded by this gene is shown in SEQ ID NO:2. The results show that the cloned sequence is consistent with the sequence (Solyc06g075780) published in Solgenomics.

[0025] Example 2: Construction of SlZF14 gene mutation vector

[0026] The target sequence of the SlZF14 gene was designed through the CRISPR - P website. The specific sequence is shown in SEQ ID NO:5. After annealing, the synthesized target sequence was ligated to the Bbs I site of the AtU6 - sgRNA - AtUBQ - Cas9 vector, and then the newly obtained AtU6 - sgRNA - AtUBQ - Cas9 fragment was ligated to the Hind III / Kpn I site of the pCAMBIA1301 vector to construct a CRISPR expression vector for the tomato SlZF14 gene. The above - mentioned recombinant plasmid was sent to Shangya Company for sequencing confirmation.

[0027] Example 3: Construction and detection of transgenic tomato SlZF14 materials

[0028] The overexpression vector pFGC1008::SlZF14-HA and the gene editing vector pCAMBIA1301::AtU6-sgRNA(SlZF14)-AtUBQ-Cas9 were transformed into Agrobacterium tumefaciens GV3101, and tomato cotyledons were infected. Through callus induction, resistant induction and differentiation, and rooting culture, tissue culture seedlings were obtained. The T1 generation mutant seeds and overexpression seeds were respectively tested for kanamycin resistance and chloramphenicol resistance. Lines with 3 / 4 resistance and the remaining 1 / 4 without resistance were selected, indicating that the overexpression vector linked with the target gene was inserted in a single-copy form in this line. These plants were removed, and then single-plant seeds were harvested. Western Blot was used to verify the SlZF14 overexpression positive transgenic plants. The results showed that there was no protein band in the wild type, while there was a band of SlZF14-HA in the overexpression line ( Figure 1 ). Positive SlZF14 mutant transgenic plants were verified by PCR and sequencing techniques. It was found that 4 bases and 7 bases were deleted, and mutations occurred at the 4th and 7th bases of the original adjacent motif (PAM), and translation stopped immediately ( Figure 2 ).

[0029] Example 4: Detection of the low-temperature tolerance of tomato SlZF14 transgenic materials

[0030] Wild-type tomato seedlings with five fully expanded leaves and the SlZF14 gene overexpression line and mutant line obtained in Example 3 were treated at 25°C and 4°C in an artificial incubator. After 7 days of low-temperature treatment, the low-temperature stress treatment group was compared with the control group that was not subjected to low-temperature treatment under the same conditions, and the phenotypes of wild-type, overexpression line, and mutant line tomato plants were observed ( Figure 3 ), electrical conductivity ( Figure 4 ), and the maximum photochemical quantum yield of PSII (Fv / Fm, Figure 5 ). The results showed that overexpressing tomato plants could significantly improve the low-temperature tolerance of tomatoes ( Figure 3 ), and the electrical conductivity was significantly lower than that of the wild type (WT) and the zf14 mutant line ( Figure 4 ). In addition, the Fv / Fm ( Figure 5 ) of overexpressing plants was higher than that of wild-type tomatoes (WT), while the mutant line was the lowest. Thus, it can be seen that tomato SlZF14 positively regulates the low-temperature tolerance of plants.

[0031] Although the present invention has been described in detail above with general descriptions and specific embodiments, this patent is not limited to the above embodiments and can have many variations or improvements, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of this patent fall within the scope of protection required by this patent.

Claims

1. Application of a gene in controlling low-temperature resistance of tomatoes, wherein the expression level of the gene in the tomatoes is increased by gene overexpression technology, and the gene has the nucleotide sequence shown in SEQ ID NO:

1. SlZF14 SlZF14 ​​ 2. The application according to claim 1, characterized in that The gene overexpression technology is specifically as follows: Extract total RNA from tomatoes, reverse transcribe to obtain cDNA, use cDNA as a template, and use F and R as primers to amplify SlZF14 the gene, and construct the amplified product onto an overexpression vector; the nucleotide sequences of the primers F and R are shown in SEQ ID NO: 3 and SEQ ID NO: 4; introduce the overexpression vector into Agrobacterium cells, and then use it to infect the target plant, screen and obtain positive transgenic plants.

3. The application according to claim 2, wherein The Agrobacterium cells are GV3101.

4. The application according to claim 2, wherein The overexpression vector is an expression vector with a 35S promoter.

5. The application according to claim 4, wherein The overexpression vector plasmid is pFGC1008::SlZF14-HA.

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