Cloning And Sequence Analysis Of Cellulase Gene From Thermootlerant Fungus

Heat tolerated fungi is a kind of microbial groups, that can show high viability in ahigh-temperature environment, including thermophilic fungi and thermotolerant fungi. Heattolerated fungus are widely distributed in the world. Currently, over130species had beenreported. Heat tolerated fungi is closely related to human life. They produce cellulase,amylase, fatty acid synthase, etc, show higher thermal stability. In the actual production andlife, cellease has a great application prospect and research value. In recent years, researchesaround the heat tolerated fungi have important strategic significance.Cellulase refers to the degradation of cellulose into glucose of a group of enzymes. Inrecent years, a growing number of studies have found cellulase molecules with diversity.Micro-organisms don’t just produce a kind of cellulase components in general. Even if onlyproduce one cellulase components, they also tend to have a variety of different forms anddifferent molecular weight of cellulose enzyme. The diversity phenomenon of cellulasemolecular make people to explore the glucan enzymes molecular diversity and theevolutionary origins of diversity by molecular biology techniques. In this experiment, we usedthermophilic fungi and thermotolerant fungi as experimental materials, and cloned thecellulase gene and the fatty acid synthase gene. Then we analysed this sequences. The mainresults are summarized as following:We downloaded many different family cellulase sequences from NCBI and designed thedegenerate primers based on conserved regions of the other reported glycoside hydrolasefamily proteins. We cloned eleven cellulase genes from eight thermotolerant fungus. Theywere four6family genes, which were named gh-1、gh-5、gh-7、gh-9, and seven7family genes,which were named gh-2、gh-3、gh-4、gh-6、gh-8、gh-10、gh-11. We registered those genesin GenBank.We used DNAMAN and SignalP4.1to analyze of the obtained genes about thenucleotide sequence and amino acid sequence. Judging from the nucleotide sequence, wefound that the full-length of the longest gh-8DNA and cDNA gene were1662bp and1593bp.The full-length of the shortest gh-5DNA and cDNA gene were1207bp and1140bp. Differentgenes in aspects such as length, number of introns had certain difference. Looking from theamino acid sequence, amino acids sequences have high homology of the same family.Cellulase gene of different family had different molecular weight and isoelectric point. Aminoacid sequence analysis showed that11genes have1-6glycosylation sites, and the signalpeptides of these genes were predicted and found that some gene signal peptide are not obvious. To compare the obtained genes by NCBI Blast, we found that gh-1gene had thehighest similarity of86%with Myceliophthora thermophila ATCC424641,4-beta-cellobiosidase in submitted sequences. gh-5、gh-7、gh-9had higher similarity withMyceliophthora thermophila ATCC42464glycoside hydrolase family6protein. Thesimilarities were79%,79%and81%, respectively. In cellulase gene of family7, gh-4had thehighest similarity of94%with Hypocrea rufa1,4-beta-cellobiohydrolase in submittedsequences. gh-2、gh-3、gh-6、gh-8had higher similarity with Myceliophthora thermophilaATCC424641,4-beta-cellobiosidase. The similarities were87%、77%、75%and79%in turns.gh-10and gh-11had higher similarity with Thielavia terrestris NRRL8126glycosidehydrolase family7protein. The similarities were86%and87%.We used Clustalx and MEGA4software to construst phylogenetic tree and homologytree. According to the amino acid sequence homology tree and phylogenetic tree analysis, wefound that cellulase genes gh-5, gh-7, gh-9belonging to the same family6are in one branchof the high homology. gh-1of family6showed high homology with Myceliophthorathermophila ATCC42464. The cellulase genes gh-2, gh-3, gh-8of family7had highhomology with the gene gh-6. gh-10and gh-11showed up to90%high homology withThielavia terrestris NRRL8126. gh-4gene of family7is alone one.In this paper,11cellobiohydrolase genes were cloned from eight kinds of thermotolerantfungus. Then we analyzed those sequences. By cellulose genes cloning, we laid a solidfoundation for development and efficient utilization for cellulose in the future.