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WRKY32 regulated YFT1 expression affected tomato fruit color and application of WRKY32 in tomato quality improvement

A technology of tomato, fruit, applied in the fields of molecular biology and genetics

Active Publication Date: 2021-03-02
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 regulated YFT1 expression affected tomato fruit color and application of WRKY32 in tomato quality improvement
  • WRKY32 regulated YFT1 expression affected tomato fruit color and application of WRKY32 in tomato quality improvement
  • WRKY32 regulated YFT1 expression affected tomato fruit color and application of WRKY32 in tomato quality improvement

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1: YFT1 efficient transcription factor screening

[0036] In order to screen the transcription factors that regulate the expression of YFT1, the present invention selects the transcription factors similar to the expression pattern of YFT1 (Solyc09g007870) based on the analysis of Tomato Expression Atlas (http: / / tea.solgenomics.net / ). At the same time, submit the YFT1 ATG upstream 3000bp sequence (pYFT1, SEQ ID NO: 6) to the PLACE website (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), and predict 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 dual luciferase assay (dual luciferase assay) to screen and select high-efficiency transcription factors of YFT1. Specific steps are as follows:

[0037] 1. Vector construction

[0038] Based on the upstream sequence of the YFT1 gene, primers were designed and synthesized [pYFT1-F: 5'-t...

Embodiment 2

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

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

[0053] 1. Yeast one-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:2) To the vector pB42AD vector (preserved in our laboratory) between EcoRI and XhoI restriction sites to form pB42AD-WRKY32. pYFT1 was segmented for three rounds by PCR method. In the first round, pYFT1 (SEQ ID NO: 5) was segmented into 12 sequences of about 300 bp (Table 3),...

Embodiment 3

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

[0064] In order to analyze the effect of WRKY32 on tomato fruit color formation, the WRKY32 RNAi vector (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, and RT-qPCR was used to analyze the expression changes of WRKY32 and YFT1 in cv.M82, yft1 mutant and WRKY32-RNAi and observe the fruit color changes of WRKY32-RNAi 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 a WRKY32 regulated YFT1 expression affected tomato fruit color and application of WRKY32 in tomato quality improvement. Specifically, the WRKY32 regulates the transcription expression of the YFT1 by combining a W-box (TTGAC, -784bp--780bp) motif (GTCTA, -1306bp--1302bp) in a YFT1 promoter region (defining the first nucleotide at the upstream of an initiation codon ATG of the YFT1 as -1bp). WRKY32 down-regulation expression tomatoes (WRKY32RNAi) are created through an RNAi technology, the fruit form and chemical phenotype of the WRKY32RNAi tomatoes are analyzed, and solid genetic evidences are provided for fruit ethylene release and signal transduction inhibition, carotenoid content reduction, chromosome development and fruit softening speed delay.

Description

technical field [0001] The invention belongs to the fields of molecular biology and genetics, and relates to a method of WRKY32 regulating YFT1 expression to affect tomato fruit color and its application in improving tomato quality; 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 Lycopersicon plant of Solanaceae (Solanaceae), and is the second largest vegetable crop in the world. Tomato is also an important model plant for berry research because of its small genome, short growth cycle, and easy genetic transformation. Fruit color is not only related to the commodity appearance of tomato, but also closely related to the nutritional quality, so fruit color is an important aspect of t...

Claims

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Application Information

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Patent Type & Authority Applications(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 JIAO TONG UNIV
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