Cloning And Expression Of Key Enzyme Gene Of Cordyceps Militaris Polysaccharide Synthesis By Agrobacterium

Cordyceps militaris is an edible fungus with superio nutritional value.It is extensively applied in the pharmaceutical industry because it contains a variety of effective components.In the midst of its miscellaneous effective ingredients,polysaccharides of Cordyceps militaris is one of the prime active chemical constituents.Cordyceps militaris polysaccharide plays a variety of biological functions,such as immunoregulation,anti-tumor,anti-inflammation,anti-oxidation,anti-aging,regulate blood sugar level and other biological activities.For the time being,polysaccharides are chiefly extracted from the fermentation broth,mycelia and fruit bodys.Most of the researches focuses on how to improve the culture conditions and extraction process to increase the yield,but the research on the regulation of polysaccharide biosynthesis and metabolism by molecular biological means is still lacking.The purpose of this study is to study the role of these enzymes in polysaccharide synthesis and metabolism by regulating the expression of related enzymes in the process of polysaccharide synthesis and metabolism at the molecular level.In this thesis,the recombinant plasmids were constructed and the key enzymes,galactokinase(Galk1)and UDP glucose pyrophosphorylase(GALT)genes,in the metabolic pathways of Cordyceps militaris Polysaccharide,were overexpressed in the protoplasm body of Cordyceps militaris by Agrobacterium tumefaciens transformation(ATMT).Western Blot was used to detect the expression of the target genes in the recombinant strains.and resultant quantity of Cordyceps militaris polysaccharide,monosaccharide composition and Cordyceps militaris polysaccharides in synthetic pathways related enzyme activity were studied.The research findings principaly involve:1.KEGG database was used to find the gene sequence encoding galactokinase and UDP-glucose pyrophosphorylase in Cordyceps militaris strain.Primers were devised with CDS,and Genomic DNA was applied for the template,and the target genes gallk1 and galt were amplified with polymerase chain reaction.Plasmid vector p CAMBIA1302 was linearized.The recombinant plasmids PJW-GALK1 and PJWGALT were constructed by homologous recombination,after that transformed into Agrobacterium tumefaciens and filtrated positive strains.Then the protoplast of Cordyceps militaris was transformed under agrobacterium-mediated transformation,and the reassembled Cordyceps militaris strain was screened from the plate containing hypomycin and kanamycin.2.The biomass of PJW-GALK1 and PJW-GALT of the recombinant strains of Cordyceps militaris was significantly increased,and the reducing sugar depletion rate of them was accelerated.Over all the whole primary metabolic course,the polysaccharide yield of the recombinant strain was significantly higher than that of the wild-type strain.In the study of polysaccharide composition and proportion changes in the fermentation process,it was found that the composition of galactose increased significantly due to the overexpression of gallk1 and galt genes.3.Western blot was used to detect the expression of important enzymes involved in nucleotide donors.It was found that the overexpression of gallk1 and galt genes increased the expression of galactokinase and UDP glucose pyrophosphorylase,and the specific activity of the two enzymes was significantly higher than that of wild type.In this study,galactokinase and UDP glucose pyrophosphorylase,the key enzymes of polysaccharide biosynthesis of Cordyceps militaris,were synthesized by Agrobacterium tumefaciens,and their differences in polysaccharide yield,specific enzyme activity and wild-type Cordyceps militaris were verified,so as to study their important role in polysaccharide biosynthesis of Cordyceps militaris,and provide a new research idea for the research of polysaccharide biosynthesis of Cordyceps militaris...