Transcription factor csa5g156220 involved in regulating the synthesis of cucurbitacin c in cucumber and its application

A technology of csa5g156220 and transcription factor, applied in the field of genetic engineering and molecular biology, can solve the problems of unclear molecular mechanism and uncloned synthetic genes.

Active Publication Date: 2019-04-05
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

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

Method used

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  • Transcription factor csa5g156220 involved in regulating the synthesis of cucurbitacin c in cucumber and its application
  • Transcription factor csa5g156220 involved in regulating the synthesis of cucurbitacin c in cucumber and its application
  • Transcription factor csa5g156220 involved in regulating the synthesis of cucurbitacin c in cucumber and its application

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Mining and acquisition of transcription factors regulating the synthesis of cucumber cucurbitacin C

[0027] 1. Mining of candidate genes

[0028] A cucumber mutant (XY-3) with no bitter taste was found in Hunan, which was naturally mutated from bitter cucumber (XY-2) ( figure 1 , A). In the mutant XY-3, the expression level of the bitter taste synthesis gene cluster was very low in the mutant ( figure 1 , B), it is speculated that the gene that regulates the synthesis of bitter taste may be mutated in this mutant. The genomic DNA of XY-2 and XY-3 were resequenced. Using bioinformatics analysis, look for internal mutations that occur in transcription factors. This analysis found a SNP located inside the transcription factor Csa5G156220 ( figure 1 , C). Transcriptome data showed that the gene was only abundantly expressed in leaves, and the expression of the transcription factor was also greatly reduced in the mutant ( figure 1 , B). In addition, stress...

Embodiment 2

[0038] Example 2 Using the Agrobacterium-mediated transient expression method to verify the function of transcription factors that control the synthesis of cucumber bitterness

[0039] Since the stable cucumber transgenic system is immature, a transient expression system for cucumber cotyledon and fruit was established in this example to further verify the functions of Bf and Bt.

[0040] The Csa5G156220 and Csa5G157230 genes (SEQ ID No.3) were constructed on the binary vector pBIN-Plus. They were respectively introduced into Agrobacterium EA105 by chemical transformation. Agrobacterium containing Csa5G156220 gene was cultivated to OD 600 About 1.0, then diluted to OD with injection buffer 600 About 0.3 or so, use a syringe without a needle to inject Agrobacterium into the cotyledon of cucumber 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 r...

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Abstract

The invention provides a transcription factor Csa5G156220 participating in regulation and control of cucumber cucurbitacin C synthesis and an application of the transcription factor. The transcription factor is one of two bHLH (Basic Helix Loop Helix) transcription factors (Csa5G157230 and Csa5G156220) which are used for controlling the bitterness synthesis and found in cucumber genome for the first time, wherein the two bHLH transcription factors are respectively used for controlling the forming of the bitterness in fruits and leaves. Proved by yeast one-hybrid, gel tissue system and tobacco transient expression, the two transcription factors can be combined to a bitterness synthesis promoter region to activate the transcription of synthetic genes; in the meantime, abundant expression of corresponding transcription factors in bitterness-free cucumber leaves and fruits can restore the bitterness character of cucumber plant. The invention further discloses a molecular mechanism forming the cucumber bitterness so as to provide a theoretical basis and a molecular assisted breeding aim for the bitterness-free cucumber breeding.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a transcription factor Csa5G156220 involved in regulating the synthesis of cucumber cucurbitacin C and its application. Background technique [0002] The bitterness of cucumbers is caused by a class of triterpenoids called cucurbitacin C. The accumulation of cucurbitacin C in cucumber fruit will seriously affect the taste and quality of cucumber. Early classical genetic experiments found that the bitter taste of cucumber is controlled by two genes, Bi and Bt. A recent study found a gene cluster consisting of 8 genes involved in the synthesis of cucumber bitterness. Among them, the Bi gene encodes oxidized squalene cyclase, which catalyzes the formation of cucurbitacin 2-enol, which is the first step and the most critical step in the synthesis of cucurbitacin C. [0003] Although the molecular mechanism of bitter taste synthesis in cucumber has been bas...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84C12N1/21A01H5/00C12N15/11A01H6/34
CPCC07K14/415C12N15/8238C12N15/8243
Inventor 黄三文尚轶张慧明陈惠明张忠华
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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