Cloning And Expression Of Genes Related To Cordycepin In Paecilomyces Hepiali

Cordycepin has obvious pharmacological activities such as anti-inflammatory,anti-tumor, microbial growth inhibition, it has been produced a variety of medicines usingcordycepin as the main medicinal components, the prospect of its clinical application is very broad, but due to its low content in cordyceps militaris and synthetic difficulties, the prices of cordycepin is rising which limits its clinical application severely.So the research is to on biological method to make the expression of cordycepin in strains improve by themselves. It will solve the biggist problem that limitsthe application of cordycepin largely.This research uses Paecilomyces hepialias as the experimental strains.Combining with transcriptom and metabonomics to study the metabolic pathways of cordycepin.On the basis of completed the whole genome sequencing and the preliminary analysis of transcriptome information, the study analysedthe differentially expressed genes,and make the clustering analysis.Through the analysis, more than 140 target genes has been abtained, these genes are involved in nucleotide pathway of metabolism.Then,combining with STITCH compound interactions database, I selecte the gene AMI,IMP, GMP as proposed key genes in the subsequent experiment.And then, designing the specific primer of each key enzyme genes for PCR amplification, after cloningthe key enzyme genes, integrated the fragments to pMD18- T plasmid to sequencing,kept the segment which has the correct sequence and finally got the correct genes.Then, using the In- fusion recombinant technology, the target genes were integrated into the plasmid pCambia1300 containing a strong promoter called 35 s.Then,through the agrobacterium mediated process, transformed the recombinant plasmid into Paecilomyces hepiali. After several rounds of screening, obtaining the engineering strains which can be stable genetic marked as genetically modified AMI group, IMP group and GMP group. Then, the study uses the real-time fluorescent quantitativePCR technology to detect the expression of the key genes in the three groups of engineering bacteria. Results show that in the AMI group, the expression of AMI gene has a rising by about two times;Transgenic IMP group, compared with negative control group, the IMP gene expression increase and has the extremely significant difference, raising nearly 40 times; And the gene expression in transgenic GMP group,the GMP gene compared with negative control group also has obviously up-regulated and have significant difference, by about seven times.Finally,to confirm that the genes play a significant reaction in cordycepin metabolism, using liquid chromatography MS/MS techniques to detect cordycepin content of the genetically modified strains.Results show that the in the ninth day which its metabolic is the most actively, the cordycepin content of transgenic AMI group and transgenic IMP group increased compared with negative control group about two times, and the cordycepin content of genetically modified GMP group is three times more than the negative control group.These results not only confirmed the selected three key enzyme genes really play important roles in the cordycepin metabolic pathway, also provides a theoretical basis for the study of cordycepin metabolic pathways and lays the foundation of technology in cordycepin expression research.