【作者】 董晓慧；

【导师】 尹新明；

【作者基本信息】 河南农业大学 ， 农业昆虫与害虫防治， 2004， 硕士

【摘要】 利用RT-PCR技术构建了赤拟谷盗（Tribolium castaneum）热激蛋白70基因的竞争定量PCR系统.竞争性对照模板的构建方法是：利用计算机辅助优选设计引物，在合适的退火温度下，得到单一的特异性强的赤拟谷盗的热激蛋白70靶标基因的条带，而后降低退火温度，从几条非靶标的条带中选择一条，后经胶回收纯化、连接、转化，最后得到纯的质粒。此质粒就是在赤拟谷盗的竞争定量PCR系统中的竞争性对照模板，它与赤拟谷盗的热激蛋白70靶标基因的两端都含有PCR引物DNA序列，但是它们的PCR产物的分子量不同，并且能通过琼脂糖凝胶电泳区分开来。在竞争PCR中，利用系列浓度稀释后的竞争性对照模板与靶标DNA在同一试管中反应，由于它们互相竞争PCR引物的结果，靶标PCR产物和竞争性对照模板PCR产物的量呈线性相关。利用光密度分析仪得到竞争性对照模板PCR产物和靶标PCR产物的相对量，并可以得到它们之间的比值。由它们之间的比值，我们绘制了竞争性对照模板的初始浓度与竞争性对照模板PCR产物和靶标PCR产物比值之间的标准曲线，并得到它们之间的线性方程为Y=1.032X-1.618，其中相关系数r2=0.975。 本研究利用降低退火温度的方法得到了对赤拟谷盗热激蛋白70基因进行竞争PCR定量的内部竞争物，通过内部竞争物和目的基因共同竞争底物和引物，得到它们的PCR产物的比值，绘制它的标准曲线，建立了它的竞争定量PCR系统，为它的热激蛋白70基因定量提供了方便，并为研究热激蛋白在转录水平上的表达规律奠定了基础。同时，为在昆虫中构建竞争定量PCR系统的理论提供了依据。更多还原

【Abstract】 Using the RT-PCR skills we build the system of competitor quantitativePCR of the heat shock protein70 gene of Tribolium castaneum. The way to get thecompetitive contraposition template is: according to the gene sequence published onGeneBank, using computer softwares to select primers. In the appropriate temperature, wecan get the simple ribbon of the heat shock protein70 gene of Tribolium castaneum. Thenlower the temperature, we can get several ribbons and select one except for the one of theheat shock70 gene of Tribolium castaneum, finally, we get the pure plasmid of the oneselected. And the plasmid is the competitive contraposotion template of the system ofcompetitor quantitative PCR of the heat shock protein70 gene of Tribolium castaneum. Theplasmid and the the heat shock protein70 gene of Tribolium castaneum we get all have theprimer sequence, but the molecular weight of their PCR offspring are different ,so we candivide them easily .In the competitive PCR, we put a series diluent plasmid and the samequantum Heat shock70 gene of Tribolium castaneum to the same tube. Because they needthe same primer and the primer is limited, their quantum of PCR have interdependency.Using a kind of machine we can get the ratio of their PCR offspring, so we can draw thestandard curve and get the equation betweem the original concentration and the ratio oftheir PCR offspring, and the equation is Y=1.032X-1.618，and r2=0.975。 From the research we get the inside competitor and build the system of competitorquantitative PCR of the heat shock protein70 gene of Tribolium castaneum. So it is veryconvenient to study the quantum of the expression of heat shock70 gene of Triboliumcastaneum, and it is the foundation to study the expression regularity of heat shock70 gene. 51<WP=58>At the same time, it gives light to the theory of building the system of competitorquantitative PCR on insects.更多还原