Study On The Ability Of Lignocellulose Degradation And The Pathway Of Scleroglucan Biosynthesis In Sclerotium Rolfsii

Lignocellulosic biomass can be fermented from mixed sugar to bio-energy or other types of biomaterials,which has long been considered as a potential resource for sustainable use.However,the resource has not been fully utilized,the main reason is that it is difficult to be degraded and transformed.Filamentous fungi Sclerotium rolfsii as white rot fungus,it was found that it has strong ability of lignocellulose degradation in my schoolmate's experiment,it could made use of biomass directly and has a charming future on lignocellulosic resources.At the same time,it is also a kind of carrier for the production of scleroglucan which is an excellent biorefinery product with high additional value,it has been widely used,such as industry and pharmaceutical industry and has rapidly become an important application prospect in industrial resources.However,the mechanism of Sclerotium rolfsii biosynthesis is not available.The study provides a theoretical basis for the realization of high efficiency scleroglucan industrialization by Sclerotium rolfsii.Detailed works as followings: Research on the robust lignocellulose degradation ability of Sclerotium rolfsii.1.Based on my schoolmate's experiment,it was found that Sclerotium rolfsii had strong ability of lignocellulose degradation.Then we got did genomic sequencing on S.rolfsii.From the phylogenetic tree of Sclerotium rolfsii and some relative strains,we found that the S.rolfsii had close relationship with many lignocellulose degrading model fungi.Analyzed the key genes relevant to lignocellulose-degrading enzymes.The total number of CAZy genes in Sclerotium rolfsii is 1558 while the number of the lignocellulose-degrading model strain Dichomitus squalens is only 391.And also it has 21 genes related to lignin degradation which is more than 12 in Dichomitus squalens.we calculated that S.rolfsii may has special ability to degrading lignocellulose.2.Did lots of physiological experiments to identifying the ability of lignocellulose degradation of S.rolfsii.Mainly by comparison with other fungi special in lignocellulose degradation(Tv: Trametes versicolor,Ds: Dichomitus squalens,Tr: Trichoderma reesei).Cultivated these homologous fungi in different lignocellulose mediums including filter paper,alkaline lignin and wheat straw,observed the phenomenon: 20 days later,the fungi had degradation phenomenon to some extent,while the phenomenon of filter paper collapse in Sclerotium rolfsii medium was the most obvious,the filter paper degradation efficiency was as high as 65%,the cellulase activity was 2500U/g.After 30 days of cultivation,the wheat straw was decomposed by the fungi in different degrees,the degradation effect of wheat straw by Sclerotium rolfsii was the similar with Trametes versicolor(a well-known model strain to degrading lignocellulose),the components of hemicellulose,cellulose and lignin all had different obvious reduction compared with the control group,the cellulase activity was the highest in the Sclerotium rolfsii medium.These phenomena fully validated of Sclerotium rolfsii's high degradation ability again.The results of this part provided the possibility of more efficient lignocellulose degradation candidates,and provide a powerful guarantee for the further exploration on degradation mechanism.Research on the mechanism of scleroglucan biosynthesis in Sclerotium rolfsii.1.Based on the reported hypothetical biosynthesis pathway of scleroglucan,we annotated the key genes involved in the metabolic pathway.2.Established two different scleroglucan-producing conditions(high scleroglucan production and low scleroglucan production),did RNA-seq data respectively,analyzed the key genes relative expression level and did enrichment analysis of GO and KEGG.3.Designed experiments independently to select appropriate reference genes for qRT-PCR expression analysis in S.rolfsii: Based on the RNA-seq data and the information from previous reports,16 candidate reference genes were selected.Did qRT-PCR on these candidate reference genes under 8 different culture conditons,comprehensive analyzed the results of all quantification using several analysis programs and finally the 3 most stable candidate genes DUF?HYP and ?-tub were selected,specially,the first two genes were the novel ones we selected from our RNA-seq data.To validate our screening results,we did qRT-PCR on the target genes ZinP and DimL under different culture conditions,using DUF,HYP and ?-tub as housekeeping genes,the results of quantification was the same with RNA-seq data which validate the feasibility of the selected results.It was the first time to selected new internal gene in s.rolfsii,it not only provides guarantee for the subsequent gene quantification,but also support candidate reference genes for other homologous white-rot fungus.4.Verified the key genes in scleroglucan biosynthesis pathway of S.rolfsii using qRT-PCR under two different culture conditions.Comprehensive analyzed the qualification results and the literature's results,finally determined all the key genes in scleroglucan metabolic pathway.