Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof

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

Active Publication Date: 2014-04-23
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Although the molecular mechanism of bitter taste synthesis in cucumber has been basically determined, the molecu

Method used

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  • Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof
  • Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof
  • Transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof

Examples

Experimental program
Comparison scheme
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] Csa5G156220 (SEQ ID No. 2) 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) det...

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Abstract

The invention provides a transcription factor Csa5G157230 participating in regulation of synthesis of cucumber cucurbitacine C and application thereof. The transcription factor is one of two bHLH transcription factors (Csa5G157230 and Csa5G156220) which are found in cucumber genome firstly and used for controlling synthesis of bitter taste; the two bHLH transcription factors are used for controlling the forming of the bitter taste in fruits and leaves respectively; simple crossing of yeast, a gel tissue system and instantaneous expression of tobacco prove that the two transcription factors can be combined to a bitter taste synthesis promoter region and used for activating transcription of a synthesis gene; meanwhile, the bitter taste characteristics of cucumber plants can be restored by expressing the corresponding transcription factors in the leaves or fruits of the cucumber free of bitter taste abundantly. The invention further discloses a molecular mechanism for forming bitter taste of cucumber and provides theoretical basis for breeding cucumber free of bitter taste and molecular assisted breeding goals.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a transcription factor Csa5G157230 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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IPC IPC(8): C07K14/415C12N15/29C12N15/84C12N1/21A01H5/00C12N15/11
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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