【作者】 陈大睿； 沈中元； 唐旭东； 徐莉； 朱峰； 陶恒平； 管蕊；

【Author】 Chen Da-rui 1 Shen Zhong-yuan 1,2,3* Tang Xu-dong 1,2,3 Xu Li 1,2,3 Zhu Feng 2 Tao Heng-ping 2 Guan Rui 2 (1 Jiangsu University of Science and echnology,Zhenjiang Jiangsu 212018,China;2 The Key Laboratory of Silkworm and Mulberry Genetic Improvement,Ministry of Agriculture,Sericultural Research Institute,Chinese Academy of Agricultural Sciences,Zhenjiang Jiangsu 212018,China)

【机构】 江苏科技大学生物与应用化学学院； 中国农业科学院蚕业研究所； 农业部蚕桑遗传改良重点开放实验室；

【摘要】 丙硫咪唑能够特异性结合在微孢子虫的β微管蛋白基因上,抑制β微管蛋白与α微管蛋白组装微管或使已组装的微管解聚,使纺锥体不能形成,从而抑制细胞的有丝分裂。为了从分子生物学角度验证丙硫咪唑对家蚕微孢子虫治疗作用的效果,阐明其药理机制,本文选用家蚕品种P50为实验对象,采取2种不同处理方式(1、接种家蚕微孢子虫(Nosema bombycis)2、接种N.bombycis后再添食丙硫咪唑),在6个不同时段(丙硫咪唑处理后6h、18h、30h、42h、66h、90h)分别取中肠组织,以家蚕β-actin基因为内参基因进行不同样品之间mRNA量校正,分析家蚕微孢子虫β-tubulin基因相对表达量差异。提取中肠组织总RNA后,检测其质量和浓度,然后去基因组DNA,再反转录成cDNA,再用PCR检测模板质量,最后用实时荧光定量PCR检测和分析结果,建立SYBR Green实时荧光定量PCR基因相对定量表达技术平台。结果表明:PCR内参基因和目的基因只有特异性目的条带,溶解曲线呈单峰,无基因组DNA污染,扩增效率在80%-120%之间,用2-△△Ct法计算得到在中肠中随着时间增长,添食丙硫咪唑的家蚕β-tubulin基因表达量相对于只添食家蚕微孢子虫的值越来越小,在90h达到最低为0.004,抑制作用越来越明显。更多还原

【Abstract】 Albendazole could be combined with β-tubulin gene to inhibit β-tubulin and α-tubulin to form microtubule or makes the packed microtubule depolymerize to destroy spindle,preventing the cell mitosis of microsporidia in silkworm.In order to validate the inhibiton of albendazole effects on Nosema bombycis from molecular biology level,this study chose Bombyx mori P50,adopted 2 different kinds of treatment ways(inoculated N.bombycis,inoculated N.bombycis followed by feeding albendazole),and obtains midgut,through 6 phases(6h,18h,42h,66h,90h after feeding albendazole) in order to calibrate mRNA in various of samples and analyze the relative expression of β-tubulin gene in N.bombycis.According to RNA extracted from midgut in various of phases after different treatments,the quality and concentration were measured respectively.After removing genome DNA and RT-PCR,the quality of templates was measured.Subsequently,measured and analyzed the results with real time PCR,as a result,relative quantitation technology platform of gene expression with SYBR Green real time PCR was established.The results indicated the control gene and target gene had only one specific strip after PCR respectively.The dissociation curve had only one peak,which means there were no contamination of genome DNA.The efficiency of amplification was between 80%-120%,according to 2 -△△Ct,with the progress of time,the relative expression of β-tubulin gene in midgut which were fed on albendazole was decreased little by little and reached the lowest point 0.004 at 90h,compared with that are fed on N.bombycis only.The inhibition effect of N.bombycis β-tubulin gene expression is more and more obvious.更多还原