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Transcription factor participating in regulating watermelon bitter principle and application thereof

A transcription factor and bittern technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of unclear molecular mechanism and uncloned genes.

Active Publication Date: 2017-03-22
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Although the molecular mechanism of bitterness synthesis in watermelon has been basically determined, the molecular mechanism regulating bitterness formation is still unclear, and the related genes have not been cloned.

Method used

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  • Transcription factor participating in regulating watermelon bitter principle and application thereof
  • Transcription factor participating in regulating watermelon bitter principle and application thereof
  • Transcription factor participating in regulating watermelon bitter principle and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Mining and acquisition of transcription factors regulating the synthesis of cucurbitacin E in watermelon

[0042] 1. Mining of candidate genes

[0043] By comparison, it was found that the bitter cucurbitacin E in watermelon was very similar to the bitter cucurbitacin C in cucumber ( figure 1 ), so it is speculated that its synthesis mechanism should be relatively similar, so we used comparative genomics to find a gene cluster similar to the regulation of cucumber bitterness synthesis in the watermelon genome ( figure 2 ), the gene cluster is composed of 5 bHLH-type transcription factors, and the gene expression analysis by fluorescent quantitative PCR shows that the gene ClBr is specifically expressed in roots, and the ClBt gene is specifically expressed in wild fruits, and the expression trend of the two genes Consistent with the content of bitterin in each tissue of watermelon ( image 3 ), we speculated that the ClBr gene should control the synthesis of...

Embodiment 2

[0055] Example 2 Using the Agrobacterium-mediated transient expression method to verify the function of transcription factors that control the synthesis of watermelon bitterness

[0056] Since a stable watermelon transgenic system is immature, a transient expression system for watermelon cotyledons was established in this example to further verify the functions of ClBr and ClBt.

[0057] The ClBr, ClBt and ClBtdel genes were constructed on the binary expression vector pBIN-Plus. They were respectively introduced into Agrobacterium EA105 by chemical transformation. Cultivate Agrobacterium containing the target gene to OD 600 About 1.0, then diluted to OD with injection buffer 600 About 0.4, use a syringe without a needle to inject Agrobacterium into the cotyledons of watermelon seedlings about 10 days old. One week later, the leaves were collected, and the compounds in the leaves were extracted with methanol, followed by UPLC-Q-TOF (Agilent) detection and related expression ...

Embodiment 3

[0058] Example 3 Watermelon ClBt gene belongs to domestication gene

[0059] In the previous studies on the synthesis, regulation and domestication of cucumber bittern, it was found that the transcription factor Bt, which controls the bitterness of cucumber fruit, is a domestication gene. Through the analysis of nucleic acid diversity of wild and cultivated watermelon materials, it was found that the ClBt gene of watermelon is located in the domestication area, so there is a phenomenon of cooperative domestication of bitterness in Cucurbitaceae plants ( Figure 10 ).

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Abstract

The invention provides a transcription factor participating in regulating watermelon bitter principle and an application thereof. Two bHLH transcription factors ClBr and ClBt controlling bitterness synthesis are discovered in watermelon genome for the first time by use of comparative genomics, wherein the two bHLH transcription factors control the formation of bitterness in the root and wild fruits respectively. The yeast one-hybrid technology, gel retardation experiment and tobacco transient expression system prove that the two transcription factors can be directly combined to the promoter area of the bitter principle synthesis gene and activate the expression of the synthesis gene; and meanwhile, through the transient expression of watermelon cotyledon, the overexpression of ClBr and ClBt is genetically proven to activate the expression of the bitterness synthesis gene so that the bitterless cotyledon obtains a bitterness phenotype. The ClBt gene encodes the mutation of an SNP so that the bitter fruit loses bitterness; and moreover, the gene positioned in an acclimation area is an acclimation gene. The invention further discloses a molecular mechanism forming watermelon bitterness and provides a theoretical basis for bitterless watermelon breeding.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a transcription factor involved in regulating the synthesis of watermelon bittern and its application. Background technique [0002] The bitter taste of watermelon is caused by a class of triterpenoids called cucurbitacin E. The accumulation of cucurbitacin E in watermelon fruit will seriously affect the taste and quality of watermelon. Recently, using methods of comparative genomics, biochemistry, and molecular biology, a gene cluster consisting of 10 genes was found to be involved in the synthesis of watermelon bitterness. [0003] Although the molecular mechanism of bitterness synthesis in watermelon has been basically determined, the molecular mechanism regulating bitterness formation is still unclear, and the related genes have not been cloned. Contents of the invention [0004] The purpose of the present invention is to provide transcription fac...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/82C12Q1/68A01H5/00
CPCC07K14/415C12N15/8205C12N15/8243C12Q1/6895C12Q2600/13C12Q2600/156
Inventor 黄三文马永硕尚轶张忠华
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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