【作者】 王瑞娴；

【导师】 沈卫德；

【作者基本信息】 苏州大学 ， 特种经济动物饲养， 2011， 硕士

【摘要】 家蚕(Bombyx mori)是重要的经济昆虫和模式生物。热激蛋白(heat shock protein,HSP)是在生物界广泛存在的蛋白质,不仅在应激条件下高表达,在正常情况下的组织中也广泛存在。蜕皮激素属于类固醇激素,能促进核酸和蛋白质的合成,影响糖类、脂类的代谢,影响中枢神经系统并参与免疫调节。芸香苷是食物中常见的类黄酮物质,属黄酮醇类,有广泛的生物学活性,能抑制细胞增殖和诱导细胞凋亡。我们从NCBI上获得了家蚕10种热激蛋白基因和蛋白序列,用生物信息学的方法对BmHSP进行分析,并采用双跟踪标定实时荧光定量RT-PCR (dual-spike-in qPCR)方法,对家蚕BmHSP的基因转录水平进行了定量研究,主要研究结果如下:1、热激蛋白的生信息学分析用生物信息学方法对热激蛋白进行分析,结果表明:Bmhsp20.4、Bmhsp20.8和Bmhsp19.9的亲缘关系最近,小分子热激蛋白的同源性较高, Bmhsp21.4在进化中处于独立的分支。对小分子热激蛋白二级结构域的分析比较认为,家蚕的7种小分子热激蛋白序列有较高的保守性,而且都相似的α螺旋区域。对启动子区域的转录因子结合位点预测发现,结合位点存在差异,又有相似性,推测与基因的功能相关。2、蜕皮激素对BmHSP的诱导表达定量分析。测定了家蚕幼虫经蜕皮激素诱导后,热激蛋白在中肠、脂肪体和马氏管中的表达水平。结果表明:Bmhsp70在各个组织中检测不到表达。在中肠中, Bmhsp21.4的表达水平在刺激后显著升高,表明该基因在中肠中起重要作用;Bmhsp25.4在刺激后的脂肪体中持续高表达,表明该基因在脂肪体中有重要作用。不同的基因在不同的组织中,表达水平表现出时序的差异。3、芸香苷诱导家蚕HSP基因的表达变化用实时荧光定量RT-PCR的方法测定了芸香苷刺激家蚕后热激蛋白在中肠、脂肪体和马氏管中的表达水平。结果表明:Bmhsp70在各个组织中检测不到表达;不同的组织中基因的转录水平差异很大,并且不同的基因呈现出不同的表达模式,且基因表达时序的不同与启动子结合位点的差异可能有一定的相关性,Bmhsp19.9、Bmhsp20.1、Bmhsp25.4均有较高的转录水平和相似的转录的模式,推测他们在家蚕对芸香苷的代谢中是有着非常重要的作用。本文对家蚕10种热激蛋白基因和蛋白序列进行了生物信息学分析,并通过定量的方法检测了家蚕热激蛋白在蜕皮激素和芸香苷的诱导的表达水平,发现在不同的刺激下,不同组织、不同时间内,不同的基因有不同的表达模式,且基因表达时序的差异可能与转录因子的结合有关。为热激蛋白在生物体内的作用提供参考,为后续的研究提供基础和依据。更多还原

【Abstract】 Silkworm is an important economic insects and model organisms. Heat shock protein (heat shock protein, HSP) exists in biology widely, not only in the high expression under stress, but also widespread under normal circumstances. Ecdysone can promote the synthesis of nucleic acids and proteins, affecting carbohydrate lipid metabolism, affecting the central nervous system and immune regulation. Rutin is a common flavonol of flavonoids can inhibit cell proliferation and induce apoptosis. In this paper, we analysised BmHSPs with the method of bioinformatics, investigated the transcription levels of BmHSP genes in Bombyx mori with dual-spike-in qPCR method. The main results are as follows:1 bioinformatics analysis of heat shock proteinsUsing bioinformatics methods analyzed heat shock proteins, the results showed that: Bmhsp20.4, Bmhsp20.8 and Bmhsp19.9 have the closest relationship, small heat shock proteins were highly homologous, and Bmhsp21.4 was an independent branch in evolution. The secondary structure analysis of small heat shock proteins revealed that the sequences of 7 silkworm small heat shock proteins were quite conservative containing the similarα-helix region. Predicting the binding sites of transcription fators on promoters of BmHSPs revealed that the binding sites were different but had similarities, suggesting that maybe they were associated with fuctions,2 the expressions of BmHSPs in tisusses inducted by ecdysoneExpressions were determined in the midgut, fat body and malpighian tubules after induction by ecdysone. The results showed that: we could not detect the expression of Bmhsp70. In midgut, expression of Bmhsp21.4 increased at 2h after induction implied its importance. In fat body, transcription of Bmhsp25.4 kept in a high level. The expressions of genes were different in tusseus and timing,3 the expressions of BmHSPs in tisusses inducted by rutinWe detected the expression of each gene in tissues with qRT-PCR. The results showed that: the expression of Bmhsp70 was not detected; transcriptions were very different of genes, and different genes showed different expression patterns.We inferred the promoter binding sites may have some correlation with gene expressions.In this study we analysised ten heat shock protein genes with bioinformatics method, illustrate the expression levels of BmHSPs in different organizations inducted by ecdysone and rutin, we found that different gene has different expression modols after different induction in tissues and time, and the differences in gene expression may be related to the binding positions of transcription factors.The results can provide data for studying the function of BmHSPs in vivo and for subsequent research.更多还原