Transcriptome Analysis Of Aureobasidium Pullulans And Functional Research Of Genes Related To Pullulan Synthesis

Pullulan is a kind of linear polysaccharide produced by Aureobasidium pullulans is widely used in food,industry and medicine due to unique molecular structure and chemical properties.It's a complex process for Aureobasidium pullulans to produce pullulan.In order to study the mechanism of pullulan biosynthesis,this study determined transcriptome data of Aureobasidium pullulans and screened genes related to pullulan,and determine transcription level of genes to achieve further screening.In addition,the purified PI protein was obtained by constructing the expression vector of P1 to explore the interaction with genes related to pullulan synthesis.Finally,we constructed knockout system to verify PI function.Conclusions are as follows:(1)The results of transcriptome analysis of Aureobasidium pullulans NG which was deposited by our laboratory shows that there are 13,705 Unigenes in NG,The average length of the Unigenes is 1457 bp.In addition,Unigenes were predicted by using bioinformatics method,genes include seven glycosyltransferase genes and one pyrophosphorylase gene related to pullulan were screened by function annotation of GO,KOG and KEGG.It provides theoretical reference for the study of the synthesis and molecular mechanism of pullulan.(2)This study determined the transcription level of seven glycosyltransferase genes and one pyrophosphorylase gene under YAD,YPD and YND culture by RT-qPCR,verifying the reliability of transcriptome data.This study determined the transcription level of eight genes in different periods of UVMU3-1.Finally,five glycosyltransferase genes(comp5913,comp8271,comp8503,comp11697 and comp6663)and one pyrophosphorylase gene(comp 6817)positively correlated with the synthesis of pullulan were screened out.We speculate these six genes are related to the synthesis of pullulan.(3)This study constructed the expression vector of PI(compl4856,1164 bp)which associated with cell differentiation and synthesis of polysaccharides.This experiment successfully expressed P1 protein and obtained the purified P1 protein,It lays the foundation for exploring the interaction between P1 protein and six genes and confirming its function.(4)This study successfully obtained PI knockout vector by constructing the upstream and downstream homologous arm fragments of PI gene,but it's unsuccessful for Agrobacterium infection,probably the inaccurate plasmids and inappropriate conditions of infection lead to this result.Transcriptional data and candidate genes related to pullulan synthesis provide valuable data for the study of the regulatory regulation and synthesis mechanism of pullulan.The purified P1 provides important information for the study of cell differentiation and polysaccharide synthesis of Aureobasidium pullulans.The progress of above experiments lay the foundation for increasing the production of pullulan from the molecular level.