美国白蛾丝氨酸蛋白酶基因HcSP1的克隆、时空表达及对取食不同寄主植物的表达响应  

Cloning and spatio-temporal expression of serine protease gene HcSP1 and its expression in response to feeding on different host plants in Hyphantria cunea (Lepidoptera: Arctiidae)

作　　者：赵旭东 孙宇航 陈昌宇[1,2] 田朔 陶蓉 郝德君 ZHAO Xu-Dong;SUN Yu-Hang;CHEN Chang-Yu;TIAN Shuo;TAO Rong;HAO De-Jun(Coinnovation Center for the Sustainable Forestry in Southern China,Nanjing Forestry University,Nanjing 210037,China;College of Forestry,Nanjing Forestry University,Nanjing 210037,China)

机构地区：[1]南京林业大学南方现代林业协同创新中心,南京210037 [2]南京林业大学林学院,南京210037

出　　处：《昆虫学报》2019年第2期160-169,共10页Acta Entomologica Sinica

基　　金：江苏省研究生培养创新工程(KYCX18_0957);江苏省六大人才高峰资助项目(NY-013);江苏省高校"青蓝工程"资助项目

摘　　要：【目的】为探究美国白蛾Hyphantria cunea在寄主转换过程中的消化生理机制奠定基础。【方法】通过筛选美国白蛾cDNA文库,克隆美国白蛾丝氨酸蛋白酶基因。荧光定量PCR检测该基因在美国白蛾不同发育阶段的表达特性;半定量RT-PCR和荧光定量PCR分别检测该基因在美国白蛾5龄幼虫体内不同组织中的分布及表达特性;荧光定量PCR检测取食不同寄主植物(美洲黑杨Populus deltoides,日本晚樱Cerasus serrulata var.lannesiana,山樱花Cerasus serrulata,喜树Camptotheca acuminata和法国梧桐Platanus orientalis)叶片后美国白蛾4龄幼虫中该基因的表达量。【结果】克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1(GenBank登录号:MH663425),开放阅读框长882 bp,编码293个氨基酸,预测分子量为30.5 kD,理论等电点预测为9.86。编码蛋白N末端疏水区包含15个氨基酸组成的信号肽;具有丝氨酸蛋白酶的典型特征,即氨基酸序列中具有组氨酸(His)、天门冬氨酸(Asp)以及丝氨酸(Ser)残基组成的酶活性催化中心三元件;具有明显的胰蛋白酶前体的特征,即具有信号肽、激活肽以及胰蛋白酶N末端保守的起始氨基酸序列(IVGG)。NCBI BLAST比对结果表明美国白蛾HcSP1与其他鳞翅目昆虫丝氨酸蛋白酶的氨基酸序列一致性在50%～70%之间。荧光定量PCR结果显示,HcSP1在美国白蛾幼虫不同发育阶段的相对表达量呈现动态的变化,并随着幼虫虫龄的增长呈现上升趋势。半定量RT-PCR及荧光定量PCR结果显示,HcSP1在美国白蛾5龄幼虫头部、唾液腺、中肠、脂肪体、表皮、马氏管和血淋巴等组织中均有表达且在幼虫中肠中表达量极高。与取食其他寄主植物叶片相比,美国白蛾取食喜树叶片后HcSP1的相对表达量明显升高,并显著高于取食其他寄主植物。【结论】本研究克隆获得美国白蛾丝氨酸蛋白酶基因HcSP1,检测了其在美国白蛾不同发育阶段、不同组织以及取【Aim】To lay a foundation for exploring the mechanism of digestive physiology of Hyphantria cunea(Drury)in host transformation.【Methods】We first cloned a serine protease gene by screening the H.cunea cDNA library.We then analyzed the expression patterns of this gene in different developmental stages of H.cunea by qPCR.The distribution and expression patterns of this gene in different tissues of the 5th instar larvae of H.cunea were analyzed by semi-quantitative RT-PCR and qPCR,respectively,and the expression patterns in the 4th instar larvae of H.cunea feeding on leaves of different host species(Populus deltoids,Cerasus serrulata var.lannesiana,Cerasus serrulata,Camptotheca acuminata and Platanus orientalis)were detected by qPCR.【Results】A serine protease gene HcSP 1(GenBank accession no.:MH663425)was successfully cloned from H.cunea.The open reading frame(ORF)of HcSP 1 is 882 bp in length,encoding 293 amino acids,with the predicted molecular weight(MW)of 30.5 kD and the theoretical isoelectric point(pI)of 9.86.The predicted N-terminal hydrophobic region contains signal peptide consisting of 15 amino acid residues.The protein encoded by HcSP 1 contains the enzyme activity catalytic center formulated with His,Asp and Ser residues,which is a typical feature of serine proteases.HcSP1 also possesses some typical features of putative trypsin precursors,such as containing a signal peptide,activation peptide and conserved N-terminus(IVGG).The NCBI BLAST alignment revealed that the amino acid sequence identities between HcSP1 of H.cunea and other lepidopteran serine proteases are between 50%and 70%.The qPCR results indicated that the expression of HcSP 1 in H.cunea in different developmental stages showed dynamic change,increasing with the larval instar.Semi-quantitative RT-PCR results showed that HcSP 1 was expressed in various tissues of the 5th instar larva of H.cunea including head,salivary gland,midgut,fat body,cuticle,Malpighian tubules and hemolymph,and qPCR results further revealed that this gene had t

关 键 词：美国白蛾 丝氨酸蛋白酶 胰蛋白酶 序列分析 时空表达 寄主植物 

分 类 号：S763.42[农业科学—森林保护学]