过量铜及甲基紫精对MSR的表达调控研究
毕业论文关键词：甲硫氨酸亚砜还原酶；ROS；Cu；甲基紫精
Expression Regulation of MSR by Excess Copper and Methyl Viologen
Abstract： In order to study the effects of Cu and methyl viologen (MV) on the expression of methionine sulfoxide reductase (MSRs), we found that excessive Cu and MV treatment by real time quantitative PCR could affect rice Expression of OsMSRA4.1 in MSR Gene Family. In order to further understand its mechanism, the promoter sequence of OsMSRA4.1 and OsMSRB3 gene in the MSR gene family was cloned, and the promoters (ProA4 and ProB3) were introduced into the vector pGWB3 by GateWay. The ProA4-pGWB3 and ProB3-pGWB3 recombinant vectors were successfully constructed. Two recombinant vectors were invaded into the callus of rice by Agrobacterium transformation. Among them, ProB3-pGWB3 gene transfer has been differentiated from positive transgenic rice. However, the GUS staining of transgenic ProB3-pGWB3 rice does not show GUS activity, whose cause needs further analysis.
Key words: Methionine sulfoxide reductase；ROS；Cu；Methyl Viologen
目 录
摘要1
关键词1
Abstract1
Key words1
引言1
1 材料与方法2
1.1 实验材料 2
1.2 实验方法 3
2 结果与分析5
2.1 Cu和MV处理下OsMSRA4.1基因相对表达量的测定5
2.2 启动子序列克隆6
2.3 启动子-pGWB3重组载体的构建6
2.4 ProA4及ProB3的序列分析7
2.5 含启动子-pGWB3重组载体的农杆菌构建8
2．6 含启动子-pGWB3基因农杆菌侵染愈伤并诱导分化9
2.7 转启动子-pGWB3基因水稻幼苗的鉴定9
2.8 Cu及MV处理对GUS活性的影响 10
3 讨论10
3.1 转启动子-pGWB3基因水稻苗的构建 10
3.2 分析转ProB3-pGWB3基因水稻幼苗无GUS活性的原因 11
3.3 启动子上游短引物的设计 11
3.4 创新之处 11
致谢11
参考文献11
过量铜及甲基紫精对MSR的表达调控研究
引言甲硫氨酸（methionine，Met）是多肽和蛋白质中重要的含硫氨基酸残基。由于硫很容易与活性氧（reactive oxygen species，ROS）反应，因此Met易被臭氧、过氧化氢及羟自由基等活性氧氧化成为甲硫氨酸亚砜(MetSO)，这会使其构成的蛋白质生物学活性降低或丧失。但是，甲硫氨酸亚讽还原酶可以将生物体内的MetSO还原为Met，使其所在蛋白质重新恢复活性,并且可以间接清除ROS[1]。
大多数的生物都拥有A型（MARAs）和B型（MARBs）两种类型的MARs蛋白，两者分别特异性的作用于S构型和R构型的MetSO，但两种类型的MARs在序列上或结构上并没有相似性[2,3]，两种类型的MARs将MetSO文持在一个较低的水平。
尽管MSRAs和MSRBs之间缺乏相似性，但两种通常共享一套包含三个步骤的反应机制：a.半胱氨酸的每还原一摩尔的甲硫氨酸亚砜就形成一摩尔的亚磺酸中间体。b.在半胱氨酸另一个巯基的作用下两者形成分子内二硫键。c.最后通过还原剂还原二硫键，重新形成半胱氨酸（图1-A）[4]。而对于1-Cys MSRBs，由于它只有一个具有氧化还原活性的半胱氨酸，无法形成分子内二硫键，因而只能与还原剂作用形成分子间二硫键。参与这一特殊反应的还原剂有硫氧还蛋白，谷氧还蛋白（Grx）以及谷胱甘肽（GSH）（图1-B）[5]。