Cloning And E.coli Expression Of Anthocyanidin Reductase Gene From Tea Leaves

Proanthocyanidin is a kind of newly found antioxidants, especially, it's ability in scavenging free radicals is 50 times more effective than V_E and 20 times more effective than V_c. Catechins are the blocks that compose of PA, and also the initiate units. They play important roles in the formation of PA. Anthocyanidin reductase, which encoded by anr gene, transfer the substrates (cyanidin, pelargonidin, delphinidin) into their corresponding catechin and derivants. Thus ANR is critical to the biosynthesis of PA.For further research, the anr gene was cloned from Camellia sinensis cultival "1005", by using RACE technique. According to the sequencing result of RACE, the ORF of ANR was cloned, and expressed in E.coli after recombined with pET-28a expressing vector, then, analysed the fusion protein expressed by SDS-PAGE. The results were as following:1. Acquisition of the ANR full-length cDNA: Extracting total tea RNA with Trizol from fresh tea buds. Design primers according to highly conserved domains of different plant Anthocyanidin reductase genes. Then, amplify the 3'-sequence and 5'-sequence respectively. Splicing them to full length cDNA sequence (1260bp) and infer it's amino acid sequence with DNAman. Align results showed that the similarities between the target sequence with those of Camellia sinensis ANR, LAR and Viti vinifera ANR were 99%, 84% and 83% respectively.2. Prokaryotic expression: Design primers based on splicing and amplify the ORF of ANR gene and recombine with pET-28a expression vector. Then the recombinant was expressed in E.coli BL21, and SDS-PAGE was used to detect the molecular weight of fusion protein. The results indicated that the fusion protein had expected molecular weight.