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Method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene ANS and brassica carinata BAN

一种芥菜型油菜、大肠杆菌的技术，应用在芥菜型油菜中花色素合成酶ANS基因和埃塞俄比亚芥中花色素还原酶 BAN 基因共转化促进大肠杆菌产原花色素领域，能够解决未见有大肠杆菌原花色素等问题

Inactive Publication Date: 2014-04-30
HUNAN UNIV OF SCI & TECH
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Problems solved by technology

After searching the prior art literature, it was found that YanY J et al. (2008) reported on the "Biotechnol. Bioeng. 2008; 100: 126-140" by expressing Arabidopsis and Gerbera in Escherichia coli (Gerbera), Petunia (Petunia), Antirrhinum (Anthocyanin) synthesis pathway genes can increase the synthesis of E. coli anthocyanin, anthocyanin and proanthocyanin are flavonoids , but there is no report on using the proanthocyanidin synthesis gene of Brassica species to increase the synthesis of E. coli proanthocyanidin

Method used

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Embodiment Construction

[0016] The embodiments of the present invention are described in detail below: the present embodiment implements under the premise of the technical solution of the present invention, provides detailed implementation and specific operation process, but protection scope of the present invention is not limited to the following implementation .

[0017] The experimental method that does not indicate concrete condition in the embodiment, generally according to routine condition, for example " molecular cloning: laboratory handbook " (New York: Cold Spring Harbor Laboratory Press, 1989) such as Sambrook, " modern molecular biology such as Zheng Weijuan Experiment" (Beijing: Higher Education Press, 2010), or according to the conditions suggested by the manufacturer.

[0018] (1) Cloning of rapeseed ANS and BAN genes

[0019] (1) Extraction of total RNA from mustard rape seeds and Ethiopian mustard seeds

[0020] A small amount of seeds 20-25 days after pollination of mustard type r...

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Abstract

The invention discloses a method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene ANS and brassica carinata BAN. According to the invention, an anthocyanidin synthetase ANS gene is cloned from cDNA of brassica juncea seeds, and an anthocyanidin reductase BAN gene is cloned from cDNA of brassica carinata seeds; a prokaryotic expression vector pET-ANS-BAN containing the ANS and BAN genes is constructed; and the ANS and the BAN genes are introduced into a BL21 escherichia coli strain simultaneously. The transformed strain is cultured in an LB liquid medium at 28-38 DEG C at 150-250 rpm for 6-12 hours, transferred into an LB liquid medium containing grape skin extracts (0.2-0.8 g / L), and cultured under shaking at 28-38 DEG C at 150-250 rpm for 36-54 hours; the strain is collected by centrifugation; and the proanthocyanidin content in escherichia coli is determined by a DMACA-HCl method. The transgenic escherichia coli obtained in the invention has significantly increased proanthocyanidin content, and the maximum content can be 24.2 times higher than that of a non-transformed control strain.

Description

technical field [0001] The present invention relates to a method in the field of genetic engineering and microbial fermentation technology, in particular to a method for co-transformation of double key enzyme genes and adding crude extracts of key enzyme gene catalyzed reaction precursors in the culture medium to improve Escherichia coli Method for synthesizing proanthocyanidins. Background technique [0002] Proanthocyanidins (Proathocyanidins), also known as proanthocyanidins, are a type of flavonoids. Proanthocyanidin is one of the strongest antioxidants for scavenging free radicals, which can protect brain and nerve tissue, improve blood circulation, flexible joints and youthful skin. Long-term consumption of foods high in proanthocyanidins can alleviate cardiovascular and other diseases. Scientists have also discovered that proanthocyanidins and their derivatives also have anti-inflammatory, anti-adversity, anti-tumor, and immune-regulating functions. It can be seen t...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N1/21C12R1/19
Inventor 刘丽莉严明理向建华孙婵尹小明
Owner HUNAN UNIV OF SCI & TECH
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