基于第三代PacBio测序技术的家蚕微孢子虫浙江株全基因组及其比较分析

Genome-Wide Analysis of Nosema bombycis Zhejiang Strain Based on PacBio’s Third-Generation Sequencing Technology

家蚕微孢子虫(Nosema bombycis)是危害家蚕养殖业的主要病原微生物,已在不同养蚕地区发现多种株型。本研究采用第三代基因组测序技术PacBio RSⅡ对分离自浙江地区的家蚕微孢子虫浙江株(N.bombycis Zhejiang)进行基因组测序,并对其基因组序列特征和基因功能注释进行分析。该微孢子虫基因组测序数据量为2.34 Gb,基因组大小为17.3 Mb,共拼接获得95个scaffolds,基因组覆盖深度达到138倍。比较基因组分析结果显示,N.bombycis Zhejiang与N.bombycis CQ1的基因组大小和GC含量相似,并且具有较好的基因组共线性关系。GO注释热图分析表明,不同微孢子虫基本功能分类基因丰度相似,但是浙江株在代谢过程等方面具有更高丰度。基于GLUT3氨基酸序列的系统进化分析显示,家蚕微孢子虫GLUT3蛋白主要分为2个亚家族,浙江株有3个GLUT3蛋白与CQ1株的亲缘关系最近,其余8个则与柞蚕微孢子虫形成进化支。利用实时荧光定量PCR对葡萄糖诱导后的浙江株GLUT3基因表达模式进行分析,结果表明,50 mmol/L葡萄糖诱导1 h后,A3834和A5849基因表达显著升高。各微孢子虫的GLUT3保守基序片段构成转运体的核心α螺旋结构,分子对接分析表明该片段氨基酸残基可能是葡萄糖结合和蛋白质稳定的关键位点。此外,家蚕微孢子虫2个GLUT3亚家族的蛋白序列长度、跨膜次数和平均疏水性均存在不同,推测2株家蚕微孢子虫在葡萄糖的摄取转运方面可能有所差异。研究结果为今后深入研究家蚕微孢子虫不同株型的进化机制和与宿主的相互作用提供了支撑。

Nosema bombycis is an important pathogenic microorganism endangering the silkworm industry. Many N. bombycis strains have been reported in different regions. In this paper, genomic sequence features, as well as structural and functional annotations of N. bombycis Zhejiang, were analyzed based on the third-generation sequencing PacBio RSⅡ. We generated 2.34 Gb of raw data from se-quencing, with a coverage of more than 138 fold to the genome, and assembled them into a 17.3 Mb genome of N. bombycis including 95 scaffolds. Comparative gen-omic analysis showed that the genome size and GC content of the Zhejiang strain and CQ1 strain were similar. They have good genomic collinearity. Heat map of gene ontology(GO) analysis showed that the gene abundance of different Nosema microsporidian genomic basic functional classifications was similar. The relative abundance of metabolic process related genes was higher in the Zhejiang strain. Phylogenetic analysis based on GLUT3 amino acid sequences showed that GLUT3 proteins of N. bombycis were divided into two subfamilies. Three GLUT3 proteins of the Zhejiang strain were closest to those of the CQ1 strain, and the other 8 proteins formed evolutionary branches with Nosema antheraeae. The expression profiles of GLUT3 genes induced by different concentrations of glucose in the Zhejiang strain were detected by the qRT-PCR method. The results showed that the expressions of the A3834 and A5984 genes significantly increased after being treated with 50 mmol/L glucose for 1 h. Conserved motif fragment of Microsporidian GLUT3 constitutes the core of the transporter α-helix structure. And molecular docking analysis showed that the amino acid residue of this fragment may be the key site for glucose binding and protein stability. In addition, the protein sequence length, the number of transmembrane structures, and the average hydrophobicity of the two N. bombycis GLUT3 subfamilies were different. We speculated that the two N. bombycis strains may have different glucose uptake and transport mode. These results provide support for the further study of the evolutionary mechanism of different N. bombycis and its interaction with the host.