Cloning Of Cellulase Genes And Their Expression During The Development Of Sclerotium In Inonotus Obliquus

Inonotus obliquus is the Hymenochaetaceae fungi which has important medical values.The main medicinal part of Lobliquus is sclerotium.At present,the sclerotium can be produced by artificial cultivation,however the yield of sclerotium is very low and lacking in economic benefits.In order to study the factors of sclerotium yield based on molecular level,degenerate PCR and RACE was adopted to clone two genes of BGL and EG from JLO1 strain in this study,and bioinformatics analysis was carried out on the two genes.Detecting the expression rule of two genes in five different sclerotium developmental periods by real-time fluorescent quantitative PCR method.Results are shown blow.1.The full length sequences of BGL and EG gene were successfully cloned by degenerate PCR and RACE technology.The results show that the full DNA sequences of the IO-BGL and IO-EG gene were 3382 bp and 1966 bp respectively,and the full cDNA sequences were 2853 bp and 1233 bp respectively,encoding 860 and 410 amino acids respectively.2.Bioinformatics analysis showed that the IO-BGL and IO-EG gene contained 14 exons and 13 introns.IO-BGL protein has not signal peptide sequence,which locate in the cytoplasm,endoplasmic reticulum lumen or cytoplasm and nuclei,most likely locate in cytoplasm.IO-BGL belongs to the third glycoside hydrolase family.IO-EG protein has a signal peptide sequence which is a secreted protein,belonging to the fifth glycoside hydrolase family.3.Encoding the amino acid sequences of IO-BGL and IO-EG gene were carried out on similarity search.The results show that the IO-BGL and IO-EG have high identity with other fungal BGL and EG IO-BGL protein has the highest identity with the BGL of Emediterranea which is 85%,and the identity values are 67%,66%,65%with G.trabeum,P.tinctorius,P.gigantean respectively.IO-EG protein has the highest identity with the BGL of F.mediterranea which is 82%.4.The results shown that IO-EG protein is closest relative with F.mediterranea by constructing the phylogenetic tree,and S.stellatus takes second place.IO-BGL protein with Kmediterranea is closest relative and other fungi takes second place.5.Detection by real-time fluorescent quantitative PCR method found that two cellulase genes are expressed continually in different developmental periods of sclerotium,however,the rules are different.IO-BGL gene in developmental period 4 reached maximum of expression quantity,then expression quantity decreased.The expression quantity of IO-EG gene is rising trend in detection of five times while there was not increase significantly about the yield of sclerotium.It shows that beta glycosidase may be the key enzyme of limiting the yield of sclerotium...