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
发布时间：2018-08-30 19:19
【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c DNA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p EASY-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 k D,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p EASY-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3 h,MDCII基因均出现明显的表达上调。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。
[Abstract]:The chitinase gene MDCII, was selected from the EST sequencing database of housefly to clone the gene and analyze its molecular characteristics. The expression patterns of chitinase gene in different tissues, different developmental stages and induced by different microorganisms were discussed. The MDCII gene was screened by EST sequencing from the constructed cDNA library of housefly larva c DNA, and the physicochemical properties of the gene and its encoded protein were analyzed by bioinformatics. The recombinant plasmid of p EASY-E1-MDCII was constructed and transformed into the competent Trans1-T1 cells by using the method of contiguous construction of phylogenetic tree-PCR technique, and real-time fluorescence quantitative PCR (Real Time PCR) technique was used to amplify the target gene, and the recombinant plasmid was transformed into the competent cells of Trans1-T1. The expression of MDCII gene was detected in different developmental stages and different tissues, and the changes of MDCII gene expression at different time points after induction were detected by injecting different microorganisms into the third instar larvae of Musca domestica. The results showed that the ORF frame of MDCII gene encoded 457 amino acids with theoretical molecular weight of 51.6 kD, and the genetic distance between phylogenetic tree analysis and adult disk growth factor of Drosophila melanogaster was close. The recombinant plasmid of p EASY-E1-MDCII. MDCII with correct gene sequence was constructed and expressed in different developmental stages of Musca domestica. In the third instar larva, the expression level was higher in salivary gland and fat body, while in Candida albicans, the expression level was higher in the third instar larva, and in Candida albicans. The expression of MDCII gene in Staphylococcus aureus and Escherichia coli were obviously up-regulated. MDCII gene belonged to adult disk growth factor in chitinase, which participated in the growth and development of housefly and played a certain role in immune defense.
【作者单位】： 贵州医科大学;贵州医科大学附属医院肿瘤生物治疗中心;
【基金】：国家自然科学基金项目(81560337,81160204)
【分类号】：Q78;Q966
，
本文编号：2214045
[Abstract]:The chitinase gene MDCII, was selected from the EST sequencing database of housefly to clone the gene and analyze its molecular characteristics. The expression patterns of chitinase gene in different tissues, different developmental stages and induced by different microorganisms were discussed. The MDCII gene was screened by EST sequencing from the constructed cDNA library of housefly larva c DNA, and the physicochemical properties of the gene and its encoded protein were analyzed by bioinformatics. The recombinant plasmid of p EASY-E1-MDCII was constructed and transformed into the competent Trans1-T1 cells by using the method of contiguous construction of phylogenetic tree-PCR technique, and real-time fluorescence quantitative PCR (Real Time PCR) technique was used to amplify the target gene, and the recombinant plasmid was transformed into the competent cells of Trans1-T1. The expression of MDCII gene was detected in different developmental stages and different tissues, and the changes of MDCII gene expression at different time points after induction were detected by injecting different microorganisms into the third instar larvae of Musca domestica. The results showed that the ORF frame of MDCII gene encoded 457 amino acids with theoretical molecular weight of 51.6 kD, and the genetic distance between phylogenetic tree analysis and adult disk growth factor of Drosophila melanogaster was close. The recombinant plasmid of p EASY-E1-MDCII. MDCII with correct gene sequence was constructed and expressed in different developmental stages of Musca domestica. In the third instar larva, the expression level was higher in salivary gland and fat body, while in Candida albicans, the expression level was higher in the third instar larva, and in Candida albicans. The expression of MDCII gene in Staphylococcus aureus and Escherichia coli were obviously up-regulated. MDCII gene belonged to adult disk growth factor in chitinase, which participated in the growth and development of housefly and played a certain role in immune defense.
【作者单位】： 贵州医科大学;贵州医科大学附属医院肿瘤生物治疗中心;
【基金】：国家自然科学基金项目(81560337,81160204)
【分类号】：Q78;Q966
，
本文编号：2214045
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