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

Abstract

The invention discloses a method for increasing proanthocyanidin content in escherichia coli by cotransformation of brassica juncea gene BAN and DFR. According to the invention, an anthocyanidin reductase BAN gene and a 4-flavanonol reductase DFR gene are cloned from cDNA of brassica juncea purple-leaf mustard seed coat; a prokaryotic expression vector pET-BAN-DFR containing the BAN and DFR genesis constructed; and the BAN and the DFR genes are introduced into DH5alpha escherichia coli simultaneously. The transformed strain is cultured in an LB liquid medium at 25-37 DEG C at 150-250 rpm for6-12 hours, then cultured under shaking in an LB liquid medium containing black soybean seed coat extracts (0.5-1.0 g / L) at 25-37 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 19.8 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 medium to improve the synthesis of Escherichia coli Proanthocyanidin method. 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 that proan...

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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) in the anthocyanin synthesis pathway genes can increase the synthesis of E. coli anthocyanin, anthocyanin and proanthocyanin are flavonoids , but there is no report on the use of rapeseed proanthocyanidin synthesis gene to increase the synthesis of E. coli proanthocyanidin