Cloning Of SAD,FAD2 Of Vernicia Montana And Transformation In Fungus

Biodiesel is a reproducible and clean energy. Oil plants are the raw materials for making biodiesel and the resource is abundant in our country. But there are problems of high price,shortage of raw resource in using oil seeds as the materials for making Biodiesel. Microbe strains can produce oils and are easy to be industrialized. But present microbial oils are low production, complicated composition. With the development of molecular biology, people can use transgenetic technology to regulate fatty acid metabolism and get the required functional oils.In this work, we cloned cDNA of VeSAD and VeFAD2 genes from total RNA of Vernicia montana by RT-PCR, the coding region are 1191bp and 1152bp, and the two cDNA sequences had been submitted to the GenBank,the accession number are EU072353 and EF152993.We analyzed their structure and function by the software of biology. The SAD gene of Vernicia montana share above 80% identity with Jatropha curcas,Ricinus communes and Brassica campestris in deduced amino acid sequence. Function analysis show that VeSAD has the conservative region of Acyl–ACP desaturase enzymes, but does not have the region of membrane bound proteins. The FAD2 gene of Vernicia montana share 100% identity with Vernicia fordii, Jatropha curcas 90%, and 81.2% identity with other oil plants. It encodes a conservative protein. The enzyme has not only the conserved function region of fatty acid desaturases, but also the special region of△12 desaturase enzyme.Basing on the gene's different functions in fatty acid metabolism, we construct sense expression vector pCAM2300-sad of VeSAD gene and antisense expression vector pBI121-fad2 of VeFAD2 gene.Agrobacterium tumefaciens mediate-transformation of fungus has advantages of higher efficiency,more easy operation compareing to protoplast mediate methods, LiAc method, Electroporation. We studied the key factors in fungus transformation and have built the best system. The best cell concentration ratio of Rhizopus arrhizus and Agrobacterium tumefaciens is 1:50-100,We can distinguish the transformants easily when they co-culture eight hours under 21℃and will have the higher transformation ratio relatively. The ideal cell concentration ratio of Cryptococcus humicola and Agrobacterium tumefaciens is 1:200-300 and co-culture thirtyfive hours under 25℃will get the most transformants. This work has primarily established the methods of fungus transformation throught ATMT method and lays foundation for the study of oleaginous microorganism through molecular biological method.