【作者】 王德前；

【导师】 徐宁迎；

【作者基本信息】 浙江大学 ， 动物遗传育种与繁殖， 2013， 博士

【摘要】 热休克蛋白作为分子伴侣,参与维持细胞蛋白质空间构象,促进变性蛋白质修复,增加细胞抵抗力。本试验选取绍兴鸭HSP60、HSP70、HSP90基因进行cDNA序列克隆,用荧光定量方法检测HSP基因在不同组织中、不同热应激状态下的表达情况,运用RNAi方法研究HSP基因在肝细胞中对细胞凋亡相关基因的调控作用,运用iTRAQ分析热应激对肝脏差异蛋白表达的影响。主要结果如下：1) HSP60cDNA序列全长2027bp (GenBank accession number:JQ669386),其中包括58bp的5’UTR、262bp的3’UTR和1707bp的编码区(CDS),翻译编码569个氨基酸。HSP60氨基酸序列与红色原鸡同源性为93.1%,与火鸡同源性为92.6%、与斑马雀同源性为90.9%；绍兴鸭HSP70基因cDNA序列全长1532bp,包括68bp的5’UTR、339bp的3’UTR和1125bp的编码区(CDS),翻译编码375个氨基酸。HSP70氨基酸序列与火鸡同源性为99.5%、与红色原鸡同源性为99.4%,与珍珠鸡和鹌鹁同源性为99.2%；绍兴鸭HSP90基因cDNA序列全长为1350bp,包含251bp5-UTR、214bp3-UTR与1086bp CDS,编码362个氨基酸。HSP90氨基酸序列与斑胸草雀、红色原鸡、火鸡同源性均达到100%、与日本鹌鹑同源性为99.7%。2) HSP60、HSP70和HSP90基因在整个绍兴鸭孵化期均低水平mRNA表达;正常情况下,绍兴鸭HSP60mRNA在肝脏表达量最高,HSP70和HSP90mRNA在心脏中表达量最高；持续性热应激时,HSP60mRNA在心脏、肝脏、肾脏、脾脏与胰腺5种组织中的表达量增加,HSP70mRNA在心脏、肝脏、肾脏、脾脏4种组织中的表达量增加,HSP90mRNA在心脏、肝脏中表达量增加；急性热应激时,HSP60、HSP70mRNA在心脏、肝脏2种组织中表达量升高,HSP90mRNA在心脏、肝脏、肾脏3种组织中表达水平升高；心脏HSP60mRNA热应激恢复2小时后表达量降至正常水平,心脏和肝脏HSP70mRNA表达热应激恢复2小时降至正常水平,心脏和肝脏HSP90mRNA热应激恢复3小时后降至正常水平。3)用RNAi方法对鸭肝细胞HSP基因表达进行干涉,发现HSP60mRNA表达量降低了75.69%,同时CCO基因表达量升高704.7%、SAPK基因升高4325%、PKB基因降低95.9%；HSP70表达量降低了80.0%,CCO基因表达量升高1133.2%、TRAIL基因表达量升高146.2%、SAPK基因表达量升高82.4%、PKB基因表达量降低98.4%；HSP90表达量降低了80.0%,CCO基因表达量升高892.6%、SAPK基因表达量升高3502.1%、PKB基因降低97.8%。4) iTRAQ鉴定出98个热应激表达差异蛋白,其中57个蛋白表达上调,41个蛋白表达下调。GO注释和Pathway富集分析表明这些蛋白主要是与糖酵解和糖异生、三羧酸循环和丙酮酸代谢、半胱氨酸和蛋氨酸代谢、脂肪酸代谢、不饱和脂肪酸生物合成、细胞粘连等生物过程或功能相关,蛋白质相互作用网络表明热休克蛋白60(HSP60)、热休克蛋白90(HSP90)是最核心的蛋白。更多还原

【Abstract】 Heat shock proteins (HSP) were ubiquitously expressed in living organisms when subjected to a variety of stimuli such as extreme temperatures, oxygen radicals, heavy metals, nutrient deprivation, bacterial and viral infections and malignant transformation. The HSPs are well known as molecular chaperones which help in normal folding for various polypeptides, assist mis-folded proteins to regain their native states, regulate protein degradation and help in translocation of proteins to different cellular compartmentsIn the present study, HSP cDNA were cloned and characterized from Anas platyrhyncho. HSP60cDNA was obtained by a combination of homology and rapid amplification of cDNA ends (RACE). HSP70cDNA and HSP90cDNA were determined by RT-PCR. Expression profiles of HSP genes in different tissues by various treatments were determined by fluorescent real-time quantitative RT-PCR.The RNAi technique was used to knockdown HSP gene in hepatocyte to explore its regulation to the apoptosis related genes. And iTRAQ was used to analysis the difference of proteins in liver due to heat treatment. All results were presented as fellows:1. The full-length of HSP60is2027bp in length, with an open reading frame of1707bp encoding a putative protein of569amino acids. Comparison of amino acid sequence of HSP60revealed that Shaoxing duck shared93.1%,92.6%,90.9%amino acid identity to those of red jungle fowl, turkey and zebra finche. The full-length of HSP70is1532bp in length, with an open reading frame of1125bp encoding a putative protein of375amino acids. Comparison of amino acid sequence of HSP70revealed that Shaoxing duck shared99.5%,99.4%,99.2%amino acid identity to those of turkey, red jungle fowl, Guinea fowl and quail.The full-length of HSP90is1350bp in length, with an open reading frame of1086bp encoding a putative protein of362amino acids. Comparison of amino acid sequence of HSP90revealed that Shaoxing duck shared100%,99.4%,99.2%amino acid identity to those of Zebra finches, red jungle fowl and turkey, shared99.7%amino acid identity to that of Japanese quail.2. Fluorescent real-time quantitative RT-PCR was applied to determine HSP60expression after exposure to different thermal shocks. HSP transcripts were at low expression level throughout the embryo development. HSP transcripts were expressed in all tested tissues of untreated laying duck, with a maximum HSP60level in the liver and a maximum HSP70and HSP90level in heart. Under the long term treatment with both30℃and35℃, HSP60transcripts in heart, liver, kidney, spleen, pancreas and HSP70transcripts in heart, liver, kidney, spleen and HSP90transcripts in heart, liver were significantly up-regulated. Under acute challenge with40℃, HSP60mRNA and HSP70mRNA expression in heart and liver were increased, HSP90mRNA expression in heart, liver and kidney were increased. A time-dependent expression pattern of HSP60was found in the recovery period after heat shock reaction, HSP60expression levels in liver recoveryed to pre-challenge level after2h post challenge, HSP70expression levels in heart and liver recoveryed to pre-challenge level after2h post challenge, HSP90expression levels in heart and liver recoveryed to pre-challenge level after3h post challenge.3.The knockdown of HSP60gene inhibited its mRNA expression by75.69%, meanwhile, the mRNA expression of CCO, SAPK genes were significantly increased by704.7%and4325%, and PKB gene was decreased by95.9%in the hepatocytes.The knockdown of HSP70gene inhibited its mRNA expression by80.0%, the mRNA expression of CCO, TRAIL, SAPK genes were significantly increased by1133.2%,146.2%,82.4%, and PKB gene was decreased by98.4%.The knockdown of HSP90gene inhibited its mRNA expression by80.0%, the mRNA expression of CCO, SAPK genes were significantly increased by892.6%and3502.1%, PKB gene was decreased by97.8%in the hepatocytes.4. Isobaric tags for relative and absolute quantization (iTRAQ) were used to analysis protein expression in liver under heat treatment compared with that of the contro1.98differentially expressed proteins were successfully identified, with57up-regulated and41down-regulated. GO annotation and pathway enrichment analysis showed that these proteins are mainly associated with glycolysis and gluconeogenesis, pyruvate metabolism, cysteine and methionine metabolism, fatty acid metabolism, unsaturated fatty acid biosynthesis, cell adhesion and other biological processes or function.Interaction network analysis of expressed proteins showed that HSP60, HSP90are the most central proteins involved.更多还原