甜菜夜蛾P450基因上游调控序列分析
毕业论文关键字：细胞色素P450；组成型表达；顺式作用元件；调控机制
Upstream regulatory sequence analysis of P450 gene of Beet armyworm
Abstract：P450 mediated pesticide metabolism in the body by an insect detoxification enhancement is a common and important mechanism of insect resistance. Including the increased amount of P450 enzymes is the main reason to the insecticide detoxification metabolism ability enhancement. In recent years, the research shows that resistance related to the increased amount of P450 enzymes may be cis or trans factors in the process of gene transcription, or cis and trans factors jointly control results. Therefore in this study, we through the fluorescence quantitative PCR technology in the detection of beet armyworm CYP450 gene expression quantity of resistant strains in the fields. The results showed that CYP321A8, CYP321A16, CYP332A1 and CYP6AE10 50 ~ 100 times in the resistant strain of high expression. We infer that the four CYP450 gene may be associated with resistance to a variety of pesticides in beet armyworm. Use of beet armyworm genome as a template, We got the four CYP450 gene cloning 5 'flanking sequences of 1600 ~ 2200 bp sequence. After a software and web site prediction, We got the 4 different genes possible transcription start site and transcription factor binding sites. By the results of prediction in this experiment, will help us to beet armyworm CYP450 gene resistant strains in the field of high expression provides a molecular basis. Then the resistance mechanism of beet armyworm detoxifying enzymes provides theory basis.
Key words:CYP450; constitutive expression; cis-acting element; regulatory mechanism
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1材料与方法2
1.1材料 2
1.2方法 2
1.2.1RNA提取2
1.2.2RT-PCR3
1.2.3定量PCR检测mRNA表达量3
1.2.4DNA提取3
1.2.5基因组步移3
1．3转录因子结合位点预测 5
1.4统计分析 5
2结果与分析5
2.1CYP450在田间抗性品系中过表达5
2.2CYP450基因上游序列的克隆5
2.3启动子位点分析表6
2.4转录因子结合位点6
3讨论 7
致谢9
参考文献10
甜菜夜蛾P450基因上游调控序列分析
前言：自从人类第一次发现害虫对化学药剂产生抗药性以来，已有近千种害虫对各种化学药剂产生了抗性。使用化学农药导致的昆虫抗药性问题，已经成为控制害虫（农业害虫和卫生害虫）的一大障碍。由害虫抗药性引发的用药剂量和用药次数的不断增加导致了一系列问题的发生，如食品安全、环境污染、防虫投入成本增加、虫煤疾病的流行等给人类生产生活造成了巨大的损失。由细胞色素P450介导的昆虫抗药性直接影响到杀虫剂对昆虫选择性以及昆虫对杀虫剂的耐受性[1]。对某药剂产生抗性的昆虫通过P450酶代谢和降解杀虫剂的有效成分，使此杀虫剂在昆虫体内失活，这是P450介导抗性的化学本质[2]。P450酶量的增加是昆虫抗药性增强的主要原因,而P450基因的过量表达是P450酶量增加的主要原因，因此掌握P450基因表达与调控机制，从源头揭示P450基因介导抗性的奥秘，可以使害虫的抗性问题得到有效的解决方案。