Construction And Analysis Of The Differences Rhizopus Stolonifer CDNA Genes By Suppression Subtractive Hybridization

With the continuous increasing of fossil fuel consumption and environmental issues, the focus of the global bioenergy had been transferred from unrenewable sources of carbon to renewable carbon source with the by-products of agriculture and forestry. However, in order to make full use of cellulose as the major renewable carbon source, it was necessary to learn the related enzyme in cellulose degradation. The research and application of cellulase had been continued many years, but had the great problem of them. The main reason was that the mechanism on degradation of cellulose, collaboration mechanism between different cellulase and cellulase synergistic mechanism itself have no exact theory. So the regulation of gene expression about related mechanism of cellulase was a hot topic. In this paper, the aim was to identify regulatory gene of cellulase expression in Rhizopus stolonifer, the ability of its degradation in cellulose had been improved. By suppression subtractive hybridization (SSH), after extracted mRNA, reversing transcription, enzyme digestion, two rounds of hybridization, two rounds of nested PCR, R. stolonifer cDNA library had been successful constructed with three parts of different expression. Finally expressed sequence tags (ESTs) had been found and analysised. The following results were obtained through research:(1)Using single nitrogen source in the culture medium of glucose, CMC and straw fermentation about R. stolonifer, the final determination that KNO3was the sole nitrogen source in glucose, CMC and straw fermentation was the best quality total RNA in extraction total RNA medium in R. stolonifer.(2)Through the research of R. stolonifer fermentation time on glucose, CMC and straw medium, the final determination of glucose medium R. stolonifer were fermented at36h, the CMC and rice straw medium R. stolonifer were fermented at60h, which the total RNA extracted with the best quality.(3) The known function of Expressed sequence tags (ESTs) was classified and analyzed, involving in various metabolic reactions. Differences in genes were related about stress response, signal transduction, energy metabolism, transcription regulation, protein synthesis and processing, RNA processing. BLASTn analysis showed that, in the cellulase gene expression regulation of these differences could be divided into four categories:structural genes, such as5.8s,18s,28s RNA genes, the unique expression of certain structural gene in different culture medium, should be the reason for the different mechanism of cellulase expression; metabolic pathways, such as (3-1,3-cellobiase, β-1,4-cellobiase, NAD dependent epimerase/dehydratase protein family,1-aminocyclopropane-l-carboxylic acid synthase, which involved in the formation of cellulase, amino acid, lipid and carbohydrate coenzyme metabolism; cell surface, such as semi cellulose-1,2-alpha mannosidase, actin, guanylate cyclase C type2, which involved in the regulation of gene expression in the mechanism of expression and cellular structure changes of cellulase; unknown gene, its function was unknown, which cellulase expression mechanism of function remained to be explored.