Tomato Sl1PIF4 gene, protein and application thereof to improving low temperature resistance of plant

A gene, tomato technology, applied in the application of SlPIF4 gene in improving plant low temperature resistance, in the field of tomato transformation, to achieve the effect of enhancing low temperature resistance, good application value, and improving low temperature resistance

Active Publication Date: 2019-03-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These all indicate that PIFs are involved in the process of plant response to adversity stress, but the role of tomato PIFs in low temperature stress and its regulatory mechanism are rarely reported.

Method used

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  • Tomato Sl1PIF4 gene, protein and application thereof to improving low temperature resistance of plant
  • Tomato Sl1PIF4 gene, protein and application thereof to improving low temperature resistance of plant
  • Tomato Sl1PIF4 gene, protein and application thereof to improving low temperature resistance of plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Construction of SlPIF4 gene overexpression vector

[0042] To understand the molecular mechanism of plant response to low temperature, the SlPIF4 gene was cloned from the tomato genome. According to the sequence analysis of the coding region, design specific primers SlPIF4-F and SlPIF4-R, and respectively add restriction enzyme cutting sites (Asc I and Kpn I) on the primers, the sequences are shown in SEQ ID NO: 3 and 4 . The SlPIF4 fragment was amplified by PrimerSTAR high-fidelity enzyme PCR, and then the PCR amplified fragment and the vector were digested, and the SlPIF4 fragment was connected to pFGC1008-HA to obtain the overexpression vector OE. The above-mentioned recombinant plasmid was sent to Qingke Company for sequencing and confirmation. The nucleotide sequence of the obtained gene SlPIF4 is shown in SEQ ID No: 1; the amino acid sequence of the protein encoded by the gene is shown in SEQ ID No: 2. The results showed that the cloned sequence wa...

Embodiment 2

[0043] Embodiment 2: Construction of SlPIF4 gene mutation carrier

[0044] The SlPIF4 gene target sequence was designed using the CRISPR-P website, and the specific sequence is shown in SEQ ID No: 5, which is AGGTCATCCAATGTGCAGCT. The synthetic target sequence was annealed and 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. The above-mentioned recombinant plasmids were sent to Qingke Company for sequencing and confirmation.

Embodiment 3

[0045] Example 3: Construction and detection of tomato SlPIF4 transgenic material

[0046] The overexpression vector pFGC1008::SlPIF4-HA and the gene editing vector pCAMBIA1301::AtU6-sgRNA(SlPIF4)-AtUBQ-Cas9 were used. Transform Agrobacterium EHA105, and infect tomato cotyledon, through induction of callus, resistance-induced differentiation and rooting culture, to obtain tissue culture shoots, T2 generation mutant seeds and overexpression seeds were tested for kanamycin resistance and chlorine For the test of mycin resistance, 3 / 4 of the strains with resistance and the remaining 1 / 4 without resistance were selected, indicating that the overexpression vector with the target gene was inserted in a single copy form in this strain. These plants were removed and harvested individually. Using Western Blot to verify SlPIF4 overexpression positive transgenic plants, the results showed that the wild type had no protein band, while the overexpression line had a band of SlPIF4-HA ( fi...

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Abstract

The invention discloses a tomato S1lPIF4 gene, protein and application thereof to improving the low temperature resistance of plant. The nucleotide sequence of the S1lPIF4 gene is SEQ ID NO.1, and thecorresponding amino acid sequence is as shown in SEQ ID NO. 2. A tomato SlIPIF4 overexpression plant or a gene knockout plant through the gene means to regulate the expression level of the S1lPIF4 gene in order to do research on the regulatory mechanism of the S1lPIF4 gene on the low temperature resistance of tomato. According to the result, overexpression of S1lPIF4 at the lower temperature canpromote accumulation of plant hormone abscisic acid (ABA) and jasmonic acid (JA), inhibits accumulation of gibberellin (GA), further induces expression of the low temperature resistant gene of the tomato, and finally improves the low temperature resistance of the tomato. Therefore, the S1lPIF4 gene of the tomato enhances the low temperature resistance by inducing formation of hormones ABA and JA in the body. The tomato S1lPIF4 gene provides the gene resource for breeding new varieties of low temperature resistant tomato, has the good potential application value, and lays theoretical basis fordoing research on tomato plant distress response signal mechanism and molecular mechanism of being tolerant to adverse environment.

Description

technical field [0001] The present invention relates to the fields of genetic engineering, molecular biology, physiology and the like, in particular to the cloning of the tomato SlPIF4 gene, the construction of a transgene vector and the transformation of tomato, and in particular to the application of the SlPIF4 gene in improving the low temperature tolerance of plants. Background technique [0002] The origin of tomato is the tropical region of South America. It is one of the most widely cultivated vegetable crops in the world. It is a thermophilic plant, and its growth and development are extremely sensitive to environmental changes, especially temperature changes. When subjected to external abiotic stresses such as low temperature, due to the immobility of plants, their growth and crop yield are severely limited. In recent years, with the frequent occurrence of global extreme climate phenomena, low temperature has become one of the bottlenecks restricting the development...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8273
Inventor 周艳虹王峰陈笑笑喻景权
Owner ZHEJIANG UNIV
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