【作者】 张磊；

【导师】 牟颖；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2007， 硕士

【摘要】 硒是人体必需的微量元素之一。在生物体内，它主要是通过构成各种含硒酶来发挥作用，且以硒代半胱氨酸(selenocysteine，Sec)的形式作为酶的催化基团。Sec由终止密码子UGA编码，以共翻译的形式插入到新生肽链中，又被称为第21种氨基酸。Sec在原核生物中涉及一个顺式作用元件和四个基因产物。在Escherichia coli中，Sec的表达效率只有正常氨基酸的1—3％。基因元件selA，selB和selC基因参与了Sec的掺入过程，我们考察三者单独或者一起与硒蛋白基因共表达时，Sec通读效率的变化。在本文的前期工作中，已经成功完成了含硒谷胱甘肽硫转移酶在大肠杆菌中的表达和GFP／pET-32b报告载体系统的构建及表达。在本文工作中，我们完成了另一报告载体GFP／pUC18的构建及表达，并通过两种报告系统，即绿色荧光蛋白(GFP)和β-半乳糖苷酶，来检测Sec的通读效率并加以比较以探索更好地Sec表达条件。与β-半乳糖苷酶相比，GFP测定UGA的通读效率更为方便、快捷。在测定Sec通读效率方面两个表达系统得到的结果基本相似。但是，用GFP报告系统定量测定UGA密码子与有义密码子的通读效率比值却比以β-半乳糖苷酶测定的比值小得多。这是由于GFP处于强启动子控制之下，使得荧光强度的增加与其分子数的增加不成线性关系，不利于wt-GFP的定量对比分析。我们尝试使用较弱lac启动子解决了这一问题，但同时，由于Sec-GFP的翻译效率大大降低，在lac启动子启动下，其荧光值低于荧光检测仪的灵敏度，也无法用于定量分析。鉴于GFP报告系统的缺陷，接下来我们使用β-半乳糖苷酶报告系统检测Sec的通读效率。结果表明，与Sec单独表达时相比，当与selC基因共表达时，Sec的通读效率增加了40％。而与selA，selB和selC基因共表达时，UGA的通读效率增加至2.2倍。这与GFP报告系统结果相符合。但是Sec基因只与selA或selB基因共表达时，Sec的通读效率分别下降了39％和89％。这表明selA和selB基因不但没有促进反而抑制了Sec的通读。而当与selB和selC基因共表达时，Sec的通读效率增加了11％，在此基础上增加selA基因的表达，通读效率降低了29％。因此，我们推测SelA和SelB对于Sec的通读具有反馈抑制的作用，这样可以调节Sec的过量表达。SelB可以在一定程度上抑制SelA蛋白的表达水平，同时也由于自身的过量导致翻译的终止加强，Sec通读效率的大幅度下降。可见在Sec的插入过程中，SelA、selB和selC是综合作用的，不是提高某一个基因的量就会促进Sec的表达，有时反而会产生抑制作用。只有调节好三者的相对量才能获得最适宜的Sec表达条件。通过构建一系列的表达载体，测定Sec的表达效率，我们发现，共表达selAB，selC基因时，Sec掺入效率提高了5.46倍。而目前文献报道的效率较高的Sec表达体系(共表达selA，selB和selC基因)仅可以使表达效率提高2.2倍。可见我们的方法更有利于Sec的掺入，也更能促进硒蛋白的表达。更多还原

【Abstract】 Selenium is an essential element for mammals. Many of selenoenzymes are involved in redox reactions with selenocysteine acting as an essential component of catalytic cycle, such as glutathione peroxidase, thioredoxin reductase, iodothyronine deiodinase and formate dehydrogenase. The expression efficiency of Sec is only 1 -3 % of that of normal amino acids in Escherichia coli, therefore, the selenoprotein is faintly expressed in organism.In prokaryotes, the incorporation of selenocysteine requires four gene products: on tRNA and three proteins. Interestingly, selenocysteine is encoded with UGA codon and incorporated into the polypeptide co-translationally. Since UGA codon remains its routine function in tissues expressing selenoproteins, there is a special regulatory mechanism for the incorporation of selenocysteine. In addition, recognition of UGA as a selenocysteins codon depends on secondary mRNA structures, i.e. stem loops called SECIS （Selenocysteine Inserting Sequence）, which locates 11 nucleotides downstream of UGA codon. The SECIS element from prokaryotes is inside the open reading frame of m RNA, while eukaryotic SECIS is in the 5’- or 3’-untranslated region. Furthermore, the bacterial SECIS is species-specific, while the SECIS elements in eubacteria and eukaryotes are somewhat less species-restricted.We had already acquire the expression of GST-Sec in E.coli by the special regulatory mechanism for the incorporation of selenocysteine. To research the influence of selA, selB and selC genes on the read-though efficiency of UGA codon, we use two kinds of report gene system, which are called green fluorescence protein（GFP） reporter andβ-Galactosidase reporter, to detect the read-through efficiency of selenocysteine.GFP reporter system has been constructed by ourselves, then we optimized the condition of expression and detected the read-through efficiency of selenocysteine by the reporter system successfully. The results showed that the read-through efficiency of UGA codon was increased to 1.32-fold in the presence of SECIS element compared to that without SECIS element, and the efficiency was futher increased up to 2.0-fold when GFP fusion was co-expressed with selA, selB and selC genes. These results suggest that GFP can be served as a rapid and convenient tool compared toβ-galactosidase for measuring the read-through efficiency of UGA codon in intact E. coli cells. At the same time, we detected the read-through efficiency of selenocysteine by theβ-Galactosidase reporter system ,too. The results are similar to the GFP.But because of the strong promoter of the vector GFP/pET-32b, the GFP protein will be puckered by mistake, thus the number of GFP molecules will not be in line with the fluorescence intensity of GFP protein, so it makes against to the quantitative analysis of the read-through efficiency of selenocysteine. We have tried to chang vector pUC18 which has weaker promoter to improve the status, but a new problem is that the read-through efficiency of selenocysteine is too low to be detected through the fluorescence apparatus. Perhaps another promoter may improve the matter.Because of the limitation of GFP reporter, we use theβ-Galactosidase reporter system to assess the influence of selA ,selB and selC genes on the read-through efficiency of UGA codon. When co-expressing with selC, the read-through efficiency of Sec was increased by 40% compared to the individual Sec gene. The result is reasonable. However, when co-expressing with the selA or selB gene, the read-through efficiency of UGA codon was decreased by 39% and 89%, respectively. These results suggested that selA and selB did not improve but suppress the read-through of Sec. When co-expressing with the selB and selC gene, the read-through efficiency was increased by 11%. Thus, we proposed that the overexpression of selB protein suppressed the expression level of selA in vivo and decreased the concentration of selenocystl-tRNA^Sec, which resulted in the decrease of read-through efficiency of Sec. In addition, selB may suppress the initiation of translation. At the same time, the read-through efficiency of Sec was enhanced by 5.46 times when Sec gene was co-expressed with the selAB and selC gene. However, the read-through efficiency of Sec was only increased by 2.22 times when Sec was co-expressed with pSUABC under the common conditions accepted by expressing selenoprotein. Obviously our method is better than pSUABC at the read-through efficiency of Sec or selenoprotein expression.更多还原