重度感染家蚕微孢子虫的家蚕丝腺cDNA文库构建及EST测序分析

用家蚕微孢子虫(Nosema bombycis,Nb)感染家蚕品种大造的3龄幼虫,于感染后第10天选择有Nb重度感染特征的家蚕丝腺组织无菌操作提取RNA,构建Nb和家蚕丝腺混合样品cDNA文库(文库滴度≥1.6×106PFU/mL)。对文库进行测序,获得EST序列5 026条,其中家蚕丝腺EST3 901条、家蚕微孢子虫EST970条。拼接后,分别获得家蚕丝腺和Nb的单一EST(unique EST)563和383条。对EST和unique EST进行分析发现,感染后第10天家蚕丝腺中Nb的组蛋白、细胞分裂激酶等与孢子分裂增殖相关的基因表达水平较高,一些可能与Nb侵染或寄生适应相关的基因,如Nb孢壁蛋白、极丝蛋白、转运体蛋白等基因亦在此时期较高水平表达,尤其是此期间有相对高水平的组蛋白脱乙酰基酶基因表达,说明Nb基因的表达受到组蛋白去乙酰化方式的调控。对家蚕微孢子虫uniqueEST的序列特征分析发现,Nb mRNA UTR的长度和GC含量与其它微孢子虫差异较小,但ORF区的AT含量较高,即家蚕微孢子虫基因ORF序列较其它微孢子虫发生了高AT变化。Nb unique EST的功能注释及分类结果表明感染后第10天家蚕丝腺中的Nb孢子仍处于多形期,还未完全成熟化。

cDNA Library Construction and EST Analysis of Bombyx mori Silk Gland Heavily Infected by Nosema bombycis

Ten days after the third instar larvae of the silkworm Bombyx mori were heavily infected by microsporidian Nosema bombycis,the silk glands were collected under sterile operation to extract RNA.cDNA library was constructed with titer 1.6×106 PFU/mL.After sequencing,we obtained 5 026 effective ESTs totally,including 3 901 ESTs from B.mori and 970 ESTs from N.bombycis.Upon assembly,563 and 383 unique ESTs were obtained from B.mori and N.bombycis,respectively.The analyses of total ESTs and unique ESTs from spores 10 days after infection displayed that genes of histone and cell division kinase were expressed in a higher level;some infection related genes,such as spore wall protein,polar tube protein and transporters,were also highly expressed.The abnormally higher expression of histone deacetylase indicated that the gene expression of spores was regulated by histone disacetylase.The mRNA sequence analysis showed that,relative to mRNAs of other microsporidia,the length and base composition of N.bombycis UTRs varied little while the ORF region changed to a higher AT content.Our results suggested that N.bombycis spores in B.mori 10 days after infection are still in multiform and have not transformed to mature spore.Meanwhile,the analysis of mRNA indicated a trend of high AT content in N.bombycis ORFs.