家蝇几丁质酶基因MDCⅡ重组表达质粒的构建及表达模式研究

摘要

从家蝇EST测序数据库中筛选获得家蝇几丁质酶基因MDCⅡ,对该基因进行克隆及分子特性分析,探讨其在家蝇不同组织、不同发育时期及经不同微生物诱导后的时空表达模式.利用EST测序技术从已构建的家蝇幼虫cDNA质粒文库筛选出MDCⅡ基因,运用生物信息学方法分析该基因序列及其编码蛋白的理化特性,采用邻接法构建系统进化树;PCR技术扩增目的基因,构建pEASY-E1-MDCⅡ重组质粒,转化到Trans1-T1克隆感受态细胞中;采用实时荧光定量PCR(Real Time PCR)技术,检测MDCⅡ基因在不同发育时期和不同组织部位的表达差异;采用注射法将不同微生物导入到家蝇3龄幼虫体内,Real Time PCR检测诱导后不同时间点MDCⅡ基因表达水平的变化.结果显示,MDCⅡ基因的ORF框全长1 374 bp,编码457个氨基酸,理论分子量51.6 kD,进化树分析比对与果蝇成虫盘生长因子的遗传距离较近.构建了具有正确基因序列的pEASY-E1-MDCⅡ重组质粒.MDCⅡ基因在家蝇不同发育阶段中均有不同程度的表达,在3龄幼虫中,以唾液腺和脂肪体中的表达水平较高;在白色念珠菌、金黄色葡萄球菌、大肠埃希菌诱导后3h,MDCⅡ基因均强现明显的表达上调.MDCⅡ基因属于几丁质酶中成虫盘生长因子,参与了家蝇的生长发育,在免疫防御过程中也发挥了一定作用.%The aims of this study are to isolate the chitinase gene MDCH from Muscadomestica EST sequencing database,to clone the gene and analyze its molecular characteristic,and to explore the temporal-spatial expression patterns of the gene in different tissues and different developmental stages as well as after induced by different microorganism.Firstly,EST sequencing technology was employed to screen the chitinase gene MDCH from the constructed cDNA plasmid library of M.domestica larvae,the bioinformatics method to analyze the gene sequence and physicochemical properties of the encoded protein,and the neighbor joining to build phylogenetic tree.Then,the target gene amplified by PCRthen was constructed into the recombinant plasmid pEASY-E1-MDCⅡ,the recombinant plasmid was transformed in clonal cell Trans1-T1,and the real-time PCR technology was to detect the expression difference of the MDCⅡ gene in different developmental stages and different tissues Finaly,injection method was used to irduce different microorganisms into the 3 instar larvae ofM.domestica,and realtime PCR to detect the changes of expression level in different time points after inducing.The results indicated that the ORF length of the MDCⅡ gene was 1 374 bp,encoding 457 amino acids,and the molecular weight 51.6 kD.The alignment analysis of the phylogenetic tree revealed that the genetic distance of MDCⅡ was close to the adult growth factor of Drosophila.The recombinant plasmid pEASY-E1-MDCⅡ with correct gene sequence was successfully constructed.The MDCⅡ gene expressed in different developmental stages of M.domestica at different exprssion levels,and the highest in salivary glands and fat body of 3 instar larva.The MDCⅡgenepresented up-regulated expression at 3 hafter induction of Candida albicans,Staphylococcus aureus,and Escherichia coli.In conclusion,the MDCⅡ gene is the adult growth factor in chitinase,participating in growth and development ofM.domestica,and also playing a certain role in immune defense process.