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Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster

A gene cluster, cucurbitacin technology, applied in the fields of genetic engineering and molecular biology, to achieve the effect of speeding up the breeding process

Inactive Publication Date: 2015-11-11
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

Cucurbitacin has anti-insect effect, and medical research has proved that cucurbitacin can inhibit the growth of various cancer cells. At present, cucurbitacin is mainly extracted from plants and cannot be synthesized through heterologous biosynthesis

Method used

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  • Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster
  • Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster
  • Gene cluster participating in synthesis of cucurbitacin E of watermelon and application of gene cluster

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Experimental program
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Effect test

Embodiment 1

[0017] Example 1 Mining and acquisition of gene clusters involved in the synthesis of watermelon cucurbitacin E

[0018] 1. Mining of gene clusters

[0019] Based on the research on the synthesis gene cluster of cucumber bitter substance cucurbitacin C, a gene cluster similar to the synthesis of cucumber cucurbitacin C was found in the watermelon genome by using the method of comparative genomics. A total of 8 genes were found, including 1 cucurbitadiene Alcohol synthase gene Bi (Cla007080), 6 P450 genes (Cla007077, Cla007078, Cla007079, Cla007082, Cla008354, and Cla008355) and 1 acetyltransferase gene (Cla007081), except for Cla008354 and Cla008355 genes, other genes are located on chromosome 6 Within the range of 57kb, a structure similar to a gene cluster was formed, and these eight genes were specifically co-expressed in watermelon roots, and were positively correlated with the content of cucurbitacin E in each tissue ( figure 1 ), so that the gene cluster is involved ...

Embodiment 2

[0027]Example 2 Using the yeast expression system to verify the function of the cucurbitacin E synthesis candidate gene

[0028] The Cla007077, Cla007078, Cla007079, Cla007080, Cla007081, Cla007082, Cla008354 and Cla008355 genes and the reductase CPR gene 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 petroleum ether. After the samples were prepared, they were detected by UPLC-Q-TOF. The results were Such as figure 2 , image 3 shown.

[0029] figure 2 It is the result of using UPLC-Q-TOF to detect the second and third steps of bitterin synthesis; where A is the specific product of Cla008354 and Cla008355 detected by UPLC-Q-TOF, and B is the MS / MS detection results, C is nuclear magnetic resonance NMR structure analysis results.

[0030] image 3 It is the result of using UPLC-Q-TOF to detect t...

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Abstract

The invention relates to a gene cluster participating in synthesis of cucurbitacin E of watermelon and an application of the gene cluster. According to the invention, the gene cluster participating in synthesis of cucurbitacin E is found in the watermelon genome for the first time, the gene cluster comprises eight genes, wherein six of the eight genes are positioned within the range of the No.6 chromosome 57kb. A yeast system is adopted for analyzing the reaction from the step 1 to the step 4 of the synthesis of cucurbitacin E. The invention further discloses a molecular mechanism for forming the bitter taste of the watermelon, so that a theoretical basis and a molecule assistant breeding objective are provided for the breeding of the watermelon without the bitter taste, and valuable experiences are provided for heterologous biosynthesis and development and utilization of cucurbitacine. The technology provided by the invention can replace the traditional method for extracting bitter principle from a plant material, so that synthesis of the bitter principle in vitro is realized by utilizing synthetic biology.

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 watermelon cucurbitacin E 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. The previous research used genomics, molecular biology, biochemistry and other means to reveal the synthesis pathway of cucurbitacin C, a bitter substance in cucumber, involving 9 genes, including 1 oxidative squalene cyclase Bi, 7 cytochrome P450 gene and an acetyltransferase gene, and fully elucidated the four-step reaction by using biochemical methods, which has important guiding significance for the research of other types of cucurbitacin biosynthetic pathways in cucurbitaceae plants, and also for the other three types of cucurbi...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N9/10C12N9/90C12N15/61C12N15/54C12N15/53C12N15/81C12N1/19
CPCC12N9/0042C12N9/1029C12N9/90C12Y106/02004
Inventor 黄三文尚轶马永硕周渊张忠华
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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