qRT-PCR检测柞蚕中肠热休克蛋白基因ApHSC70对微孢子虫入侵的响应  

Response Pattern of Heat Shock Protein Gene ApHSC70 in Antheraea pernyi Midgut to Nosem a pernyi Infection Detected by qRTPCR

作　　者：王勇[1] 刘微[1] 王斌赫 姜义仁[1] 孙影[1,2] 杨瑞生[1] 秦利[1] 

机构地区：[1]沈阳农业大学生物科学技术学院辽宁省昆虫资源工程技术研究中心,沈阳110866 [2]沈阳农业大学植物保护学院,沈阳110866

出　　处：《蚕业科学》2015年第2期300-307,共8页ACTA SERICOLOGICA SINICA

基　　金：现代农业产业技术体系建设专项(No.CARS-22);辽宁省农业领域青年科技创新人才培养计划项目(No.2014010);辽宁省重大科技公关项目;辽宁省教育厅一般项目(No.2014476)

摘　　要：为证实柞蚕热休克蛋白在柞蚕防御体系中的作用,利用RT-PCR技术在柞蚕5龄幼虫中肠组织克隆了一个热休克蛋白基因Ap HSC70（Gen Bank登录号：KJ437496）,并通过实时荧光定量PCR（qRT-PCR）检测该基因在感染柞蚕微孢子虫的柞蚕中肠中的表达变化。该基因的开放阅读框（ORF）长1 959 bp,编码652个氨基酸,预测蛋白质分子质量为71.49 k D,等电点（p I）为5.38,具有细胞质特征基序,推测为一种组成型热休克蛋白;Ap HSC70与斜纹夜蛾、棉铃虫同源蛋白质的序列相似度最高,分别达96.94%、96.33%。感染柞蚕微孢子虫后0-9 h的柞蚕5龄幼虫中肠组织中,Ap HSC70基因转录水平变化不大,但感染后12 h该基因的转录水平开始升高,至感染后21 h达到最高值。研究结果表明,柞蚕中肠组织的Ap HSC70基因在柞蚕微孢子虫入侵时的表达变化呈现出一定的应激响应,表达量最高可上调近5倍,推测该基因可能参与了柞蚕的免疫防御过程。In order to confirm the function of heat shock protein in the defense system of Antheraea pernyi,a heat shock protein gene Ap HSC70（ Gen Bank accession No. KJ437496） was cloned from midgut tissue of the fifth instar Antheraea pernyi larvae by using RT-PCR technology. Real-time fluorescent quantitative PCR（ qRT-PCR） was employedto further detect the expression variation of Ap HSC70 gene in midgut of Antheraea pernyi after Nosema pernyi infection. The open reading frame（ ORF） of Ap HSC70 is 1 959 bp and encodes a protein of 652 amino acids with predicted molecular mass of 71. 49 k D and theoretical isoelectric point of 5. 38. Ap-HSC70 contains cytoplasmic characteristic motif and it was predicted to be one kind of constitutive heat shock protein. Ap HSC70 has a high similarity to homologous proteins of Spodoptera litura and Heliocoverpa armigera,with sequence identity of96. 94% and 96. 33% respectively. qRT-PCR indicatedthat the expressional level of Ap HSC70 gene in midgut of Antheraea pernyi had little change from 0 h to 9 h after infection with Nosema pernyi,began to increase at 12 h and reached the maximum level at 21 h. These results showed that the expressional level of Ap HSC70 gene in midgut of Antheraea pernyi has a certain response to the infection of Nosema pernyi. The maximum expression level was up-regulated by 5 times. It is considered that Ap-HSC70 gene plays an important role in the immune and defense system of Antheraea pernyi.

关 键 词：柞蚕 热休克蛋白70 免疫防御 柞蚕微孢子虫 实时荧光定量PCR 

分 类 号：S884[农业科学—特种经济动物饲养]