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wrky32 regulates yft1 expression to affect tomato fruit color and its application in tomato quality improvement

A technique for tomato, fruit color, applied in the fields of molecular biology and genetics

Active Publication Date: 2022-06-28
SHANGHAI JIAOTONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In view of the important position of EIN2, the research on its upstream regulation is rarely reported. Therefore, screening the upstream genes that regulate the expression of YFT1 and analyzing the mechanism of regulation of tomato fruit ripening and fruit color formation through YFT1 mediation will be helpful for constructing tomato fruit color. Forming a regulatory network and precise regulation and genetic improvement of tomato fruit color formation will be of great significance

Method used

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  • wrky32 regulates yft1 expression to affect tomato fruit color and its application in tomato quality improvement
  • wrky32 regulates yft1 expression to affect tomato fruit color and its application in tomato quality improvement
  • wrky32 regulates yft1 expression to affect tomato fruit color and its application in tomato quality improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: YFT1 high-efficiency transcription factor screening

[0036] In order to screen transcription factors that regulate the expression of YFT1, the present invention selects transcription factors with similar expression patterns to YFT1 (Solyc09g007870) based on Tomato Expression Atlas (http: / / tea.solgenomics.net / ) analysis. At the same time, the upstream 3000bp sequence of YFT1 ATG (pYFT1, SEQ ID NO: 6) was submitted to the PLACE website (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), and the pYFT1 cis-regulatory element ( Table 1), among the above transcription factors, those with binding sites in pYFT1 were selected as candidate transcription factors. And use the dual luciferase assay to select the high-efficiency transcription factor of YFT1. Specific steps are as follows:

[0037] 1. Vector construction

[0038] Based on the upstream sequence of the YFT1 gene, primers [pYFT1-F: 5'-tccatcgatatattgatacgatatc-3' (SEQ ID NO: 7), pYFT1-R: 5'-tatttgattt...

Embodiment 2

[0051] Example 2: Determination of WRKY32 at the pYFT1 binding site

[0052] In order to determine the binding site of WRKY32 in pYFT1, pYFT1 (3000bp upstream of YFT1 ATG sequence, the first nucleotide upstream of which is defined as -1bp) was subjected to three rounds of segmentation, and subjected to yeast one-hybrid and EMSA (electrophoreticmobility shift assay) experiments. verify. The specific experimental steps are as follows:

[0053] 1. Yeast single hybrid vector construction

[0054] Design specific primers and clone the CDS sequence of WRKY32 (WRKY32-EcoRI-F: 5′-gattatgcctctcccgaattcatggatgacgaaaacgagagc-3′, SEQ ID NO: 21; WRKY32-XhoI-R: 5′-agaagtccaaagcttctcgagttagcaaggcttaatttcaaatc-3′, SEQ ID NO: 22) To the vector pB42AD vector (preserved in our laboratory) between the two restriction sites of EcoRI and XhoI to form pB42AD-WRKY32. pYFT1 was divided into three rounds by PCR method. In the first round, pYFT1 (SEQ ID NO:5) was divided into 12 sequences of about ...

Embodiment 3

[0063] Example 3: WRKY32 regulates tomato fruit ripening development and fruit color formation

[0064] In order to analyze the effect of WRKY32 on tomato fruit color formation, the RNAi vector of WRKY32 (WRKY32-RNAi) was constructed and transformed into Agrobacterium EHA105 competent (prepared and preserved in our laboratory) for genetic transformation of tomato cv.M82. 35 days past anthesis (35dpa), 47dpa and 54dpa tomato fruits were extracted, total RNA was extracted, reverse transcribed into cDNA, RT-qPCR analysis of WRKY32 and YFT1 expression changes in cv.M82, yft1 mutant and WRKY32-RNAi ; And observe the change of WRKY32-RNAi fruit color at 35dpa, 47dpa and 54dpa. Specific steps are as follows:

[0065] Design specific primers (WRKY32-RNAi-F: 5'-gattcagagttttctccctcttat-3', SEQ ID NO: 51; WRKY32-RNAi-R: 5'-gcgagttggtctctttagaactg-3', SEQ ID NO: 52) to amplify WRKY32-specific fragments 345bp, cloned into the RNAi vector pHELLSGATE 12 (preserved in our laboratory) to ...

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Abstract

The invention discloses that WRKY32 regulates the expression of YFT1 to affect tomato fruit color and its application in tomato quality improvement; specifically relates to WRKY32 by combining with the YFT1 promoter region W-box (TTGAC, -784bp~-780bp) and W-box Like (GTCTA , -1306bp~-1302bp) motif (defining the first nucleotide upstream of the YFT1 initiation codon ATG as -1bp), which regulates the transcription and expression of YFT1. Create WRKY32 down-regulated expression tomato (WRKY32‑RNAi) by RNAi technology, analyze the morphology and chemical phenotype of WRKY32‑RNAi tomato fruit, for fruit ethylene release and signal transduction inhibition, carotenoid content decline, chromoplast development and fruit softening speed The delay provides solid genetic evidence.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetics, and relates to a kind of WRKY32 regulating YFT1 expression to affect tomato fruit color and its application in tomato quality improvement; in particular, it relates to a tomato transcription factor WRKY32 regulating ethylene signal core component gene YFT1 / EIN2 (YELLOW FRUITED TOMATO 1 / ETHYLENE INSENSITIVE 2) expression to control tomato fruit color and its application in tomato quality improvement. Background technique [0002] Tomato (Solanum lycopersicum, 2n=24) belongs to the Solanaceae (Solanaceae) genus Lycopersicon and is the second largest vegetable crop in the world. Tomato is also an important model plant for berry research due to its advantages of small genome, short growth cycle and easy genetic transformation. Fruit color is not only related to the commercial appearance of tomatoes, but also closely related to nutritional quality, so fruit color is an important aspect ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00A01H5/08A01H6/82
CPCC07K14/415C12N15/825C12N15/8249
Inventor 赵凌侠黎宇航赵伟华李文贞温腾健
Owner SHANGHAI JIAOTONG UNIV
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