新生隐球菌毒力差异菌株的全基因组测序及毒力相关基因的筛选

Genomic sequencing analysis of Cryptococcus neoformans var grubii strains of two genotypes with dif- ferent virulence and selection of virulence-associated genes

目的了解及分析新生隐球菌格鲁比变种（Cryptococcus neoformans var grubii）两组毒力差异明显的多位点微卫星型（multilocus microsatellite typing，MLMT）菌株的全基因组序列，筛选出引起其毒力差异的相关基因。方法选取毒力差异最为明显的不同基因型菌株各1株进行全基因组重测序，运用比较基因组学方法全面分析测序结果，使用非同义SNPs（non-synonymous SNPs，nsSNPs）、无义突变SNPs、产生移码突变InDels的策略广泛筛选差异基因。对筛选出的基因在所有实验菌株中进行DNA测序验证，并提取总RNA，采用快速扩增5’和3’cDNA末端（rapidamplificationof5’and3’cDNAends，RACE）技术，克隆后测序获得对应基因的完整cDNA全长序列信息。结果通过全基因组重测序，环境株IFM56731和临床株IFM56800均分别获得127倍和111倍覆盖率的有效数据。分别对SNPs和InDels数据进行统计分析，应用无义突变SNPs和产生移码突变的InDel分别筛选出3个差异基因。对筛选出的6个基因在所有实验菌株中进行扩增测序，并对其中3个基因进行cDNA的测序分析，最终确定基因CNAG_01032的转录序列位置和结构，并验证了预测的无义突变位点存在于实际的mRNA中。结论本研究通过全基因重测序数据分析证明，应用无义突变SNPs和产生移码突变的InDels进行差异基因筛选的策略具有较好针对性，并筛选出6个潜在与毒力相关的基因，通过对所有实验菌株的测序和对全长cDNA的测序验证，最终筛选出基因CNAG一01032为可能引起菌株毒力差异的基因。

Objective To analyze the genomic sequences of Cryptococcus neoformans var grubii strains of two genotypes with different virulence and to screen out the virulence-associated genes. Methods A clinical strain （IFM56800） with the strongest virulence and an environmental strain （IFM56731） with the weakest virulence were screened out for whole genome sequencing analysis. The results of sequencing analy- sis were comprehensively analyzed by using the method of comparative genomics. Genetic variations were ex- tensively screened by using the strategies of non-synonymous single nucleotide polymorphisms （nsSNPs） , nonsense SNPs and the insertions or deletions （InDels） causing frameshift mutations. The filtered genes were sequenced in 20 experimental strains. The whole RNAs were extracted and then the full-length cDNAs were sequenced by using the rapid amplification of 5＇ and 3＇ cDNA ends （RACE） method. Results By whole genome sequencing, valid data with high coverage （127 times and 111 times） was obtained in both the environmental strain IFM56731 and the clinical strain IFM56800. The data of lnDels and SNPs were statisti- cally analyzed, respectively. Six genes were chosen for further analysis based on the strategies of nonsense SNPs and the InDels causing frameshift mutations. The six genes were amplified and sequenced in all of the experimental strains, three of which were further analyzed with cDNA sequencing. Ultimately, the location and structure of CNAG_01032 gene were determined. The predicted nonsense mutation locus was verified to present in the actual mRNA. Conclusion The strategies of nonsense SNPs and the InDels causing frame- shift mutations showed high-efficiency in screening potential virulence-associated genes. The CNAG_01032 gene was screened out as a novel virulence-associated gene.