Cloning Of Saccharopine Reductase Gene From Fungal Endophyte Of Oxytropis Embellisia And Constructing Of The Gene Deletion Vector

Oxytropis glabra is a kind of locoweeds distributed in Inner Mongolia which produces a toxic alkaloid, swainsonine. Swainsonine inhibits lysosomal mannosidase in animal cells. Livestocks which are feed Oxytropis lead to poisoning, that cause heavy losses to grassland animal husbandry. The toxicity of swainsonine is caused by the Embellisia fungal endophyte from Oxytropis glabra. However, the biosynthetic pathway of the swainsonine in this fungal endophyte is still unknown. There was a report in which found that lacking saccharopine reductase in a strain of Undifilum oxytropis extrated from other locoweed species in United States resulted in increased levels of swainsonine and its precursor with decreased levels of saccharopine and lysine in2012.In this research the fungal endophyte from Oxytropis Embellisia endophyte OW7.8was investigated. The middle fragment of saccharopine reductase gene was amplified and sequenced by degenerate PCR, and the cDNA of the saccharopine reductase was identified by RACE. According to the cDNA sequence, primers were designed to amplify the saccharopine reductase gene. Afterwords the gene deletion vector was developed by using gene engineering techniques in order to investigate the role of saccharopine reductase gene in swainsonine metabolism from Embellisia fungal endophyte. The main results show as follows:1. An875bp fregment of the saccharopine reductase gene from Embellisia fungal endophyte genomic DNA sequence was amplified by using degenerate PCR. BLAST comparisons revealed99%of sequence similarity between the deciphered Embellisia fungal endophyte and that of U. oxytropis. 2. An873bp fragment of the saccharopine reductase cDNA from Embellisia fungal endophyte which contained the stop codon and3’untranslated region (UTR) was amplified by using3’RACE. Then a836bp fregment of the cDNA which contained start codon was obtained using5’RACE. The cDNA sequence length of saccharopine reductase from Embellisia fungal endophyte was1562bp including the143bp of3’UTR. The two sequences were aligned to form the complete cDNA. Afterwords the sequence identity between this cDNA and that of cDNA from U. Oxytropis by BLAST which revealed82.3%.3. Primers based on the cDNA sequence the above saccharopine reductase were designed to amplify the saccharopine reductase gene sequence, taking the genomic DNA extracted from OW7.8as the tamplet. The1750bp of the gene sequence was then obtained, which began from ATG start codon the3’UTR.4. An OW7.8saccharopine reductase gene deletion vector was constructed with pUC19as start plasmid, which contained the hygromycin resistance gene as a selection marker gene. The middle part of the target DNA was the hph (hygromycin resistance gene), and the two end of it were the5’end and3’end sequences of saccharopine reductase gene respectively.