Application of tomato SlSPY gene in controlling ripening process of tomato fruits

A tomato, gene technology, applied in genetic engineering, application, plant genetic improvement and other directions

Active Publication Date: 2021-02-05
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the embodiment of the present application is to provide a kind of application of Sl

Method used

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  • Application of tomato SlSPY gene in controlling ripening process of tomato fruits
  • Application of tomato SlSPY gene in controlling ripening process of tomato fruits
  • Application of tomato SlSPY gene in controlling ripening process of tomato fruits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034]Example 1: Construction of SlSPY gene overexpression vector

[0035]In order to regulate the market time of tomato fruits, the SlSPY gene was cloned from the tomato genome. According to the sequence analysis of the coding region, specific primers SlSPY-F and SlSPY-R were designed, and restriction enzyme cleavage sites (Asc I and KpnI) were added to the primers. The sequences are shown in SEQ ID NOs: 3 and 4. The SlSPY fragment was amplified by PCR with PrimerSTAR high-fidelity enzyme, and then the PCR amplified fragment and vector were digested, and the SlSPY fragment was ligated to pFGC1008-HA to obtain the overexpression vector pFGC1008::SlSPY-HA. The recombinant plasmid was sent to Shangya Company for sequencing and confirmation, and the nucleotide sequence of the gene SlSPY obtained was shown in SEQ ID NO: 1; the amino acid sequence of the protein encoded by the gene was shown in SEQ ID NO: 2. The results showed that the cloned sequence was consistent with the sequence publis...

Embodiment 2

[0036]Example 2: Construction of SlSPY gene mutation vector

[0037]The SlSPY gene target sequence was designed using the CRISPR-P website. The specific sequence is shown in SEQ ID NO: 5, which is TGTAGTTCATGGCAAGTAAC. The synthesized target sequence is annealed and ligated to the Bbs I site of AtU6-sgRNA-AtUBQ-Cas9 vector, and then the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment is ligated to the Hind III / Kpn I site of pCAMBIA1301 vector to construct Tomato SlSPY gene CRISPR expression vector. Send the above recombinant plasmid to Shangya Company for sequencing and confirmation.

Embodiment 3

[0038]Example 3: Construction and detection of tomato SlSPY transgenic material

[0039]The overexpression vector pFGC1008::SlSPY-HA and the gene editing vector pCAMBIA1301::AtU6-sgRNA(SlSPY)-AtUBQ-Cas9 were used. Agrobacterium GV3101 was transformed, and tomato cotyledons were infected. Tissue cultured seedlings were obtained through callus induction, resistance induction differentiation and rooting culture. The T1 generation mutant seeds and overexpression seeds were respectively subjected to kanamycin resistance and chlorine For the test of mycin resistance, 3 / 4 of the strains with resistance and the remaining 1 / 4 were selected, indicating that the overexpression vector with the target gene was inserted in the form of a single copy in this strain. These plants are removed, and then a single plant is harvested. Western Blot was used to verify SlSPY overexpression positive transgenic plants, the results showed that the wild type did not have a protein band, while the overexpression li...

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Abstract

The invention discloses application of a tomato SlSPY gene in controlling the ripening process of tomato fruits. The expression level of the tomato SlSPY gene is increased through a gene overexpression technology; the nucleotide sequence of the tomato SlSPY gene is a nucleotide sequence shown in SEQ ID NO: 1. A tomato SlSPY overexpression plant and a gene knockout plant are constructed through a gene means, the expression level of the gene SlSPY is regulated and controlled to research the regulation and control mechanism of the tomato SlSPY gene on ripening of the tomato fruits, and the resultshows that SlSPY overexpression can improve the sensitivity of the tomato fruits in a green ripe stage to ethylene, accelerate color changing of the tomato fruits and promote fruit softening and accumulation of carotenoid such as lycopene. In the SPY gene knockout plant, the color changing and softening process of the tomato fruits is inhibited, and accumulation of the carotenoid such as the lycopene is less than that of wild type fruits in the same period.

Description

Technical field[0001]This application relates to the fields of genetic engineering, molecular biology and physiology, and more specifically, to the application of the SlSPY gene in promoting ripening of tomato fruits.Background technique[0002]Tomato (Solanum lycopersicum L.) belongs to the Solanaceae crop, an annual or perennial herb of the tomato genus, and is a widely cultivated vegetable in the world. Tomato fruit matures through a series of complex physiological and biochemical processes, in which the color, flavor, aroma, texture and nutrition will change drastically. This makes the tomato fruit rich in nutrition and becomes an important nutritious fruit in the human diet. Tomato fruit is a kind of climacteric respiration, and its ripening process is usually accompanied by a sharp increase in ethylene content. Therefore, ethylene plays an important role in the ripening process of tomato fruit. However, the role of each component of the ethylene signal transduction pathway in th...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N15/82A01H5/08A01H6/82
CPCC12N9/1051C12N15/8218C12N15/8249C12N15/825C12Y204/01094
Inventor 周艳虹徐进姜小春蔡溧聪喻景权
Owner ZHEJIANG UNIV
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