Transcription factor participating into regulation of muskmelon bitter principle synthesis and application of transcription factor

A technology of transcription factor and bitterin, which is applied in the field of genetic engineering and molecular biology, can solve the problems that the gene has not been cloned and the molecular mechanism is unclear

Active Publication Date: 2017-05-31
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Transcription factor participating into regulation of muskmelon bitter principle synthesis and application of transcription factor
  • Transcription factor participating into regulation of muskmelon bitter principle synthesis and application of transcription factor
  • Transcription factor participating into regulation of muskmelon bitter principle synthesis and application of transcription factor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Mining and acquisition of transcription factors regulating the synthesis of cucurbitacin E in melon

[0038] 1. Mining of candidate genes

[0039] Through comparison, it was found that the bitter cucurbitacin B in melon and the bitter cucurbitacin C in cucumber have very similar structures ( 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 melon genome ( figure 2 ), the gene cluster is composed of 4 bHLH-type transcription factors, and the gene expression analysis by fluorescent quantitative PCR shows that the gene CmBr is specifically expressed in roots, and the CmBt gene is specifically expressed in wild fruits, and the expression trend of the two genes It is consistent with the content of bitterin in each tissue of melon ( image 3 ), we speculated that the CmBr gene should control the synthesi...

Embodiment 2

[0048] Example 2 Using the Agrobacterium-mediated transient expression method to verify the function of the transcription factor controlling the bitterness of melon

[0049] Since the stable muskmelon transgenic system is immature, the muskmelon cotyledon transient expression system was established in this example to further verify the functions of CmBr and CmBt.

[0050] The CmBr and CmBt genes were constructed on the binary 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 melon seedlings about 10 days old. After one week, the leaves were collected, the compounds in the leaves were extracted with methanol, and then UPLC-Q-TOF (Agilent) detection and related expression analysis were performed. The results are as foll...

Embodiment 3

[0051] Example 3 Melon CmBt gene belongs to domestication gene

[0052] 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 the nucleic acid diversity of wild and cultivated muskmelon materials, it was found that the CmBt gene of muskmelon is located in the domestication area, so there is a phenomenon of cooperative domestication of bitterness in Cucurbitaceae plants ( Figure 8 ).

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Abstract

The invention provides a transcription factor participating into regulation of muskmelon bitter principle synthesis and application of the transcription factor. A comparative genomics method is firstly utilized for finding two bHLH transcription factors CmBr and CmBt which control bitter synthesis in a muskmelon genome, the two bHLH transcription factors are respectively used for controlling the bitter formation at the root part and in the wild fruit. A yeast one-hybrid technology, a gel retardant experiment and a tobacco transient expression system are used for proving that the two transcription factors can be directly combined to a promoter region of a bitter principle synthesis gene, and can activate the expression of the synthesis gene; the muskmelon cotyledon transient expression proves that the overexpression of each of the CmBr and the CmBt can activate the expression of the bitter synthesis gene from the genetics, so that the bitter-free cotyledon can acquire the bitter phenotype. The CmBt gene is located in a domestication region. The invention further discloses a molecule mechanism of the muskmelon bitter formation, and provides the theoretical basis for the breeding of the bitter-free muskmelon.

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 melon bitterin and its application. Background technique [0002] The bitter taste of melons is caused by a class of triterpenoids called cucurbitacin B. The accumulation of cucurbitacin B in muskmelon fruit will seriously affect the taste and quality of muskmelon. Recently, using comparative genomics, biochemistry, and molecular biology methods, a gene cluster consisting of 8 genes was found to be involved in the synthesis of bitterness in melon. [0003] Although the molecular mechanism of bitter taste synthesis in melon has been basically determined, the molecular mechanism regulating bitter taste 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 factors CmBr and CmB...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/11C12N15/82A01H5/00
CPCC12N15/8205C12N15/8243
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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