Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof

A gene cluster and cucurbitacin technology, applied in the fields of genetic engineering and molecular biology, can solve the problems that the synthetic gene has not been cloned, the synthetic route is not clear, and cannot be directly synthesized in vitro, so as to speed up the breeding process

Active Publication Date: 2014-01-01
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

However, their synthetic pathways are not yet clear, and related synthetic genes have not been cloned
Therefore, cucurbitacin is mainly extracted from plants and cannot be directly synthesized in vitro

Method used

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  • Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof
  • Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof
  • Gene cluster participating in synthesis of cucumber cucurbitacine C and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Example 1 Mining and acquisition of gene clusters involved in the synthesis of cucumber cucurbitacin C

[0023] 1 Gene cluster mining

[0024] Bi genes are abundantly expressed in cucumber leaves. Taking this as a clue, the cucumber transcriptome data (RNA-Seq) was analyzed to find P450 genes and acetyltransferase genes co-expressed with Bi, and a total of 6 P450 genes were found in the cucumber genome ( Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, and Csa1G044890) and one acetyltransferase gene (Csa6G088700). The expression status of these seven genes is very similar to that of Bi(Csa6G088690) in various tissues of cucumber, and they are only abundantly expressed in leaves ( figure 1 ). In addition, except for the three P450 genes (Csa3G903540, Csa3G903550, and Csa1G044890), other genes and Bi genes are located within 35kb of chromosome 6, forming a gene cluster-like structure ( figure 1 ). The distribution of cucurbitacin C in various tissues o...

Embodiment 2

[0033] Example 2 Using Yeast Expression System to Verify the Function of Candidate Genes for Bitterness Synthesis

[0034] Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 genes and coenzyme CPR genes were respectively constructed on the expression vector pYES2 (Invitrogen), and transformed into yeast. Induce the yeast to express the protein and collect the yeast. After cracking, the compound was extracted with n-hexane. After the samples were prepared, they were detected by GC-MS (Agilent) or UPLC-Q-TOF. The results are as follows Figure 2-Figure 4 shown.

[0035] The CPR gene (SEQ ID No.20) is a coenzyme gene of the P450 gene in the oxidation substrate, adding CPR is beneficial to improve the oxidation reaction efficiency.

Embodiment 3

[0036] Example 3 Using Agrobacterium-mediated Tobacco Transient Expression Method to Verify the Function of Cucumber Bitter Gene Cluster

[0037] The Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700 genes were constructed on the binary vector pBIN-Plus. They were respectively introduced into Agrobacterium EA105 by chemical transformation. Agrobacteria containing Bi(Csa6G088690), HMGR, FPS, Csa6G088160, Csa6G088170, Csa6G088710, Csa3G903540, Csa3G903550, Csa1G044890 and Csa6G088700 genes were cultured to OD 600 About 1.0, and then mix equal amounts into one piece, and inject Agrobacterium into tobacco about 6 weeks old with a syringe. Tobacco leaves were collected one week later, and the compounds in the leaves were extracted with methanol, followed by UPLC-Q-TOF (Agilent) detection. The result is as Figure 5 shown.

[0038] Since the HMGR gene (SEQ ID No.21) and the FPS gene (SEQ ID No.22) are involved in the synthesis of the su...

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Abstract

The invention discovers a gene cluster participating in synthesis of cucurbitacine C in a cucumber genome for the first time. The gene cluster totally consists of 8 genes, wherein 5 genes are located in a 35kb range of No. 6 chromosome. The 1st to 4th steps of reaction for synthesizing cucurbitacine C are analyzed in a yeast system, and the 8 genes are co-expressed in a tobacco system, so that synthesis of cucurbitacine C can be realized. The invention further discloses a molecular mechanism of cucumber bitter formation, which provides a theoretical basis and a molecular aided breeding goal for breeding bitter-free cucumber; meanwhile, precious experience is provided for in-vitro artificial synthesis of cucurbitacine.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to a gene cluster involved in 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. It is found in CN103013966 that 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. The synthesis of cucurbitacin C requires a series of catalytic modifications in addition to the first step to generate cucurbitacin 2-enol. However, how cucurbitacin 2-enol is modified in plants to finally synthe...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/82
CPCC12N15/52
Inventor 黄三文尚轶张忠华马永硕张慧敏李颖
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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