摘要
柞蚕微粒子病的病原为柞蚕微孢子虫（Nosemapernyi）。采用SMART（switchingmechanismat5＇-endofRNAtranscript）技术构建柞蚕微孢子虫全长cDNA文库，初始文库滴度4．2×10^pfu／mL，文库容量1．0×10^7pfu，重组率为98．39％。随机挑取32个克隆，经PCR检测插入的片段长度在750—3000bp之间，平均长度〉1000bp。挑选文库中864个克隆进行表达序列标签（EST）测序，得到626条高质量的EST，拼接后得到197条单一基因（unigenes），包含64条重叠群（contigs）和133条单一EST，冗余度为68．53％。将这些Unigenes与NCBI数据库进行比对，146条Unigenes在蜜蜂微孢子虫（Nosemaceranae）、家蚕微孢子虫（Nosemabombycis）、兔脑炎微孢子虫（Encephalitozooncuniculi）等的基因组中比对到同源基因。在随机EST测序中克隆获得一个孢子形成蛋白（spomlationprotein）基因，命名为脚s尸一1。该基因ORF长度为1014bp，编码337个氨基酸，蛋白质的理论分子质量为39．2kD，等电点5．71。柞蚕微孢子虫cDNA文库的构建及EST测序的完成，为进一步发现和研究柞蚕微孢子虫的功能基因奠定了基础。
Nosema pernyi is a lethal pathogen of microsporidiosis in Antheraea pernyi. Using SMART （switching mecha- nism at 5＇-end of RNA transcript） technology, we constructed a full-length cDNA library of Nosema pernyi. The titer of primary library was 4.2×10^8 pfu/mL, and the library capacity was 1.0×10^ pfu with a recombination rate of 98.39%. PCR amplification of 32 random clones reveared that the inserted cDNA fragments ranged from 750 to 3 000 bp with an aver- age length over 1 000 bp. 864 clones were randomly selected from the library for expressed sequence tag （EST） se-quencing. As a result, 626 high-quality ESTs were obtained. These ESTs were assembled into 197 Unigenes including 64 Contigs and 133 unique ESTs with a redun- dancy rate of 68.53%. Among these Unigenes, 146 Uni- genes showed similarity with correspondent genes of Nosema ceranae, Nosema bombycis, and Encephalito- zoon cuniculi in Blast analysis against NCBI databases. A full-length gene encoding sporulation protein （named Np- SP-1） was obtained from randomly sequenced EST se- quences. This gene contained an open reading flame （ORF） of 1 014 bp which encoded a protein of 337 aminoacids. The predicted molecular weight and isoelectric point of this protein was 39.2 kD and 5.71 respectively. This cDNA library construction and EST sequencing would greatly facilitate molecular cloning and characterization of functional genes in Nosema pernyi.
出处
《蚕业科学》
CAS
CSCD
北大核心
2014年第2期265-271,共7页
ACTA SERICOLOGICA SINICA
基金
现代农业产业技术体系建设专项(No.CARS-22)
沈阳市发改委科研项目(No.2011154)
沈阳农业大学国家自然科学基金启动基金项目(No.20112002)
沈阳农业大学校青年基金项目(No.201010002)
