Transcriptome Analysis Of Aureobasidium Pullulans And Circadian Genes Involved In Pllulan Synthesis

Aureobasidium pullulans is a type of saprophytic fungi with typical cellular polymorphism.Their growth cycle includes yeast-like cells,spores,swollen cells,mycelial cells,and chlamydospore.Aureobasidium pullulans can anabolize different metabolites under different growth forms.Pullulan is an extracellular linear polysaccharide synthesized by Aureobasidium pullulans.pullulan polysaccharide has excellent physicochemical and biological characteristics such as film forming property,fibrillation property,oxygen barrier property,plasticity,cohesiveness,and natural degradation.It is non-toxic and harmless,and has no side effects on human body.It has a wide range of applications in biomedicine,food,etc.The process of biosynthesis of pullulan has not yet been fully understood.In order to study the characteristics of Aureobasidium pullulans in different forms,and the biosynthesis mechanism of pullulan.In this experiment,different forms of Aureobasidium pullulans cells were isolated by density gradient centrifugation.The transcriptome de novo assembly sequencing was performed on Aureobasidium pullulans cells under different culture conditions.The experiment obtained the following conclusions.(1)In 60% percoll cell separation solution,Aureobasidium yeast cells and swollen cells can be successfully isolated.Observation of cells of different morphologies by electron microscopy revealed that there were significant sugar accumulations in the swollen cells.(2)In summary,We generated 25,134,395 paired-end sequencing reads via mRNA-Seq on Illumina HiSeq 2000.Among them,23,843,614 clean reads(94.86%)with a GC content at 53.29% were assembled into 13,705 unigenes consisting of 19,965,178 bp.The length of these unigenes range from 201 to15116 bp,with the mean length at 1457 bp and the N50 at 2221 bp.70.25% unigenes were longer than 500 bp,and 54.38% were longer than 1000 bp.(3)To characterize genes involved in the production of pullulans in A.pullulans,we carried out a digital gene expression(DGE)analysis on YPD and YND.5649 unigenes exhibited significant variation in transcription level between YPD and YND.Compared toYND,2618 unigenes have higher expression level at YPD,while 3031 unigenes showed higher expression in YND.in the KEGG analysis,those unigenes were mapped to 137 pathways and 15 unigenes were involved in the pullulan biosynthesis pathway.De novo trandcriptome analysis provides an excellent plantform to generate a comprehensive resource of the gene space in an organism without whole genome sequencing and allows the discovery of novel genes,molecular markers and tissue-specific expression patterns.RNA-seq provide more meaningful reference for our future molecular biology experiments.