Analysis Of Genes Related To Pullulan Synthesis And Clone,Expression Comp1961 Of Aureobasidium Pullulans

The Aureobasidium pullulans NG can synthesize and secret pullulan,but the molecular mechanism of the synthesis of pullulan is unknown.The aim of the experiment is to find out the key genes and preliminarily to explore the mechanism of pullulan biosynthesis in A.pullulans.In the previous study,we predicted that 15 glucose metabolism genes involved in pullulan synthesis from the transcriptome data.In this experiment,q RT-PCR was used to detect the transcription levels of these genes,and the results were consistent with the transcriptome data,which proved that the transcriptome data was reliable.This study was to detect the changes of biomass,intracellular glycogen,extracellular pullulan content and transcription levels of 15 candidate genes during the fermentation of pullulan.The gene comp1961 was analysed by bioinformatics software and the struction was predicted.The expression vector pPIC9K-1961/GS115 was constructed and heterologously expressed in Pichia pastoris.(1).In this experiment,the biomass,the production of pullulan,the change of glycogen content in cells at different time were measured.The results showed that A.pullulans grew rapidly and the biomass reached the maximum on the 3rd day then gradually decreased and stabilized in the later period.The content of pullulan reached the maximum on the 5th day then decreased gradually.The content of glycogen in cells reached the highest on the 2nd day and then gradually increased.We speculated that glycogen synthesis may be related to the synthesis of pullulan polysaccharides.(2).In this experiment,the transcription levels of these 15 genes in Aureobasidium pullulans were detected by q RT-PCR under the culture condition of YPD medium in different time and the relationship between these 15 gene and the synthesis of pullulan was analyzed.q RT-PCR results showed that the gene comp3609,comp9920,comp9549,comp7480,comp3824,comp3260 and comp1482 expressed lowly.The comp9219,comp1961 were relatively high in the whole process.The gene codes comp7877,comp7023,comp7120,comp6817,and comp5441 were relatively high in 36-72 h,while the gene comp4798 was relatively high after 72 h.(3).Bioinformatics software predicts that the protein is hydrophilic and has a alpha-amylase domain.The expression vector pPIC9K-1961 was constructed in this experiment.The gene was induced and expressed in Pichia pastoris.The molecular weight of the target protein was about 61 k D,but the activity of ?-amylase was not detected.The results need to be further analysis.It was found that ?-amylase played an important role in the synthesis of pullulan.The expression of comp1961 was significantly up-regulated in transcriptome data.It was speculated that comp1961 played an important role in the synthesis of pullulan.comp1961 gene was cloned and expressed successfully in vitro,but amylase activity was not detected.In this study,a possible synthesis pathway of pullulan was proposed based on previous studiesand transcriptome data analysis.we will knock out the comp1961 gene,detect the content of intracellular glycogen,and the production of extracellular pullulan in order to verify the correctness of the inference.