Study On The Mechanism Of Efficient Utilization Of Lignocellulose By Higher "Fungus-growing" Termite

So called "fungus-growing" termites belong to Termitidae,Macrotermitinae.The special name is got from the symbiosis between termite and fungi of genus Termitomyces. For its ability of consuming more than 90%of dry wood in some arid tropical areas and mineralizing up to 20%of the net primary products in wetter savannas,they aroused extensive interests of many researchers.Under the preliminary study of this symbiosis,we surveyed the diversity of symbiotic organisms using molecular biology techniques and constructed one cDNA library in order to screening for genes related to the degradation of lignocelluloses.The main results were listed as below.Firstly,termites samples were widely collected in South China(e.g.Anhui Province, Hunan Province,Jiangsu Province and Yunnan Province),which were well identified through traditional morphological classification and molecular method.The genetic diversity of five major peregrinuses was analyzed by inter-simple sequence repeat(ISSR) markers.10 primers,which generated bright,polymorphic and reproducible amplification products were selected.145 discernible DNA fragments were produced,of which 144 (99.31%) were polymorphic loci.The observed number of alleles(n_a) was 1.9931±0.0830, the effective number of alleles(n_e) was 1.5661±0.3043,the Nei's gene diversity(h) was 0.3355±0.1363 and the Shannon's information index(I) was 0.5065±0.1645.These data suggested high genetic diversity in the overall populations.Some population genetic diversities were severally affected by geographic separation,which blocked intercourses of genes.The ISSR-PCR pattern was uniform between different castes of one colony and the amplification bands of different individuals in one caste were also stable.So,when soldiers were absent,it is feasible to use workers as objects in molecular identification and survey of diversity.Phylogenetic trees were constructed on sequence of cytochrome oxidase subunitâ…¡(COâ…¡) and 0/1 matrix generated by bands of ISSR-PCR electrophoresis.The results showed that topology of two trees partially coincided with each other and they were also congruous with the result of morphologic identification.Next,enzyme activities of 4 kinds of cellulases were compared in 17 colonies,5 species of higher termite.As a result,Odontotermes yunnanensis,which was widely distributed in Xishuangbanna,was chosen as our specimen for the highest enzymes activity of the symbiotic system.In order to explore the possible role of prokaryote in the symbiosis,two bacterial 16S rDNA gene clone libraries of microbial community from termite's gut and fungus combs were constructed respectively.Analysis of library from the gut showed 623 usable sequences were divided into 187 phylotypes.Among these representative phylotypes,most were affiliated to Firmicutes(38%) and 94.4%of this phylum were regarded as the pure cultured or uncultured microorganisms from Clostridiales.Most of the rest phylotypes were mainly consisted of Bacteroidetes(26.7%), Proteobacteria(11.8%),Spirochaetes(8%) and Bacteroidales,Deltaproteobacteria, Treponema were corresponding dominant colonies.Only a few phylotypes belonged to Planctomycetes,Actinobacteria,TM7 phylum,Deferribacteres,Chlorobi and TG 1 phylum. Results showed lower bacterial diversity appeared in fungus comb than that in termite flora,and the combs were also consisted mostly of phylotypes belonging to Firmicutes.A series of fundamental research were carried out after pure cultured strain was isolated from fungus comb.Microexamination of one nodule collected in the wild showed many typical infllated conidiogenous structures and abundant conidia with the shape of long oval.It suggested that conidia were comparted from sphere cells as protoplasm concentrating.The ITS sequences of fungus nodule(GenBANK No.:FJ769409) and cultured strain(FJ769410) were the same and on the status of classification,they were also near to Termitomyces sp.Group 8(AB073529).But it was still hard to give the accurate species name due to the absence of fruiting bodies formed in the field.Analysis of fungal diversity revealed that only Termitomyces sp.fungi were cultivated on fungus comb by termites and any other non-Termitomyces fungi cannot be detected.Uniform fungus appeared in intestinal tract and that combs also indicated that Termitomyces sp.was partial constituent of termites' food.Considering the significant role of symbiotic fungi in the degradation of lignocelluloses,a cDNA library of Termitomyces sp.collected directly from fungus combs was constructed via SMART^TM cDNA library construction kit,which provided a valuable library for screening of cellulase genes and laid a foundation for basic biology research of this fungi.The library has a high titer of 7×10⁶ Pfu/μl and the capacity was 3.5×10⁶ Pfu. The recombination rate was 97%.The size of average insert cDNAs was 1.33 Kb.These experiment data showed the good quality of this library.A positive clone containing xylanase gene,named as Xyl_C5,was obtained using PCR screening method from library successfully.This gene coded 226 amino acids and had an obvious polyA structure existing at the 3' end.The molecular weight of protein coded by this gene was about 24 KDa and from 43 to 223 amino acids was functional domain of glycoside hydrolase family 11.The 129 and 220 amino acids were two active sites,made up of glutamate.The amino acids sequence was 58%-60%identical to some other xylanase proteins from Aspergillus,indicating the first report of xylanase from Termitonyces.Last,a recombinant plasmid,pET22b-XylC5 was constructed and induced.SDS-PAGE showed two insoluble target proteins expressed with sizes of 17 KDa and 15 KDa,respectively.The difference between this result and the prediction perhaps was due to the unsuitable prokaryotic expression system or the folding and modification of the proteins.