【作者】 唐婷；

【导师】 任国栋； 柳峰松；

【作者基本信息】 河北大学 ， 动物学， 2009， 硕士

【摘要】 谢氏宽漠王Mantichorula semenowi Reitter隶属于鞘翅目Coleoptera拟步甲科Tenebrionidae漠甲亚科Pimeliinae漠甲族Pimeliini宽漠王属Mantichorula Reitter,是分布在我国内蒙古阿拉善东部、宁夏中北部和蒙古国南缘的一个特有物种,其成虫能在沙表74℃的极端高温下活动并利用小生境繁衍生息,受环境胁迫形成了特殊的生理生态适应特征。了解谢氏宽漠王的热休克蛋白(heat shock protein,HSP)基因调控,研究其在恶劣环境下的生物抗逆机制在理论和应用上都有重要的意义。本论文以谢氏宽漠王为研究材料。研究内容主要分为3个部分：第1部分：通过RT-PCR及RACE技术从谢氏宽漠王体内克隆获得与昆虫抗高温机制相关的2种不同hsp70基因的cDNA序列,并对其核苷酸和编码的蛋白序列进行了生物信息学分析；第2部分：通过同源克隆得到谢氏宽漠王β-actin基因,检测了β-actin基因作为分子内标的可靠性,并建立了1种以在昆虫体内稳定表达的管家基因β-actin为分子内标的半定量RT-PCR方法。以此方法对热激条件下谢氏宽漠王体内Mshsp70、Mshsc70基因的mRNA表达水平进行相对定量研究；第3部分：构建了谢氏宽漠王Mshsp70基因的原核表达载体,并在大肠杆菌DE3体内进行诱导表达,并以纯化后的蛋白免疫家兔制备抗体。研究结果如下：(1)谢氏宽漠王Mshsp70基因cDNA序列2137 bp,编码区长1950 bp,5′端非翻译区为19 bp,3′端非翻译区为168 bp。可编码649个氨基酸残基,理论分子量为71.34 KD,同源性分析表明该基因与异色瓢虫Harmonia axyridis的核苷酸序列相似性高达85%,推导的氨基酸序列与赤拟谷盗Tribolium castaneum蛋白序列相似性达92%。(2)谢氏宽漠王Mshsc70基因cDNA序列1908 bp,编码区长1808 bp,3’UTR为100 bp。编码601个氨基酸残基。理论分子量为57.85 KD,同源性分析表明该基因的核苷酸序列和蛋白序列与赤拟谷盗的序列相似性分别为80%,85%。(3)谢氏宽漠王β-actin基因cDNA序列全长1372 bp,包含一个长度为1131 bp的开放阅读框,编码376个氨基酸。5′和3′末端非翻译区域(UTR)分别为66 bp和175bp；该数据与其他动物β-actin基因核苷酸序列具有96%～99%高度同源性。将该序列提交GenBank,获得序列号为EU825991。β-actin表达量检测结果显示热激后不同恢复时间其表达量无明显变化,且与未经热激处理的对照相比无显著差异,可以作为研究受外界环境胁迫作用下昆虫体内不同基因表达水平高低的可靠内部参照标准。(4)通过半定量RT-PCR分析：谢氏宽漠王在未经热处理的正常条件下,Mshsp70和Mshsc70均有表达,但是室温恢复过程中,2个基因具有截然不同的表达趋势。经42℃热激1 h后立即诱导Mshsp70表达至最高峰；在恢复到室温的1～4 h内Mshsp70表达量逐渐降低,但仍然高于未热激对照组。而Mshsc70在42℃热激1 h后表达受到抑制,但在恢复2～4 h内有少量的表达。(5)利用高效原核表达载体pET-DsbA,成功构建了谢氏宽漠王Mshsp70基因的原核表达载体,并在大肠杆菌DE3中37℃培养,IPTG终浓度为1 mM诱导3 h获得成功表达。SDS-PAGE蛋白电泳显示,融合蛋白分子量约为60 KD,目的条带与预期分子量一致。利用His亲和层析纯化得到融合蛋白,以纯化后的蛋白免疫家兔制备HSP70抗体,经过Western Blot检测到与预期大小一致的特异性条带。更多还原

【Abstract】 Mantichorula semenowi Reitter,1889 (Coleoptera:Tenebrionidae) is an endemic species distributed in the floating dune of Southeast Alxa, China and South Mongolia which adjoins China. The species evolved special physiological and ecological characteristics of adaptation under environmental stress. Its imago can survive on the surface of dune with extreme temperature as high as 70℃and use the microhabitat to produce offspring. The present results provide new insights into mechanisms used by M. semenowi to adapt to stressful environments.This thesis contains 3 parts.1.The cloning and expression analysis of Mshsp70 and Mshsc70 from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods.2.The cloning and expression analysis ofβ-actin from M. semenowi. The cDNA was cloned using RT-PCR and RACE methods, and detected reliability ofβ-actin genes as internal control within insects after heat shock. To establish a stable expression in vivo geneticβ-actin steward for the internal control semi-quantitative RT-PCR method. By this method of heat shock the gene mRNA expression level of Mshsp70 and Mshsc70 relative quantitative were researched in M. semenowi.3.The Mshsp70 prokaryotic expression vector was constructed and in DE3 induction expression, to purify the protein making antibody in rabbit.The results are offered as follows:(1) The full-length cDNA of Mshsp70 is 2137 bp, containing a 3’-untraslate region of 168 bp, which encords a deduced 649 amino acid peptide with a predicted molecular mass of 71.34 KD, the result of sequence alignments analysis showed that this amino acids shares 80% identity with Harmonia axyridis and protein shares 85% identity with Tribolium castaneum.(2) The cDNA of Mshsc70 is 1908 bp, containing a 3’-untraslate region of 100 bp, which encords a deduced 601 amino acid peptide with a predicted molecular mass of 57.85 KD, the result of sequence alignments analysis showed that this nucleotide and protein shares 80% and 85% identity with Tribolium castaneum respectively.(3) The full-length cDNA ofβ-actin is 1372 bp, containing a 5’UTR of 66 bp and 3’UTR of 175 bp. The open reading frame ofβ-actin is 1131 bp, which encords a deduced 376 amino acid peptide. The result of sequence alignments analysis showed that this nucleotide share 96-99% identity with other animals. The sequence of theβ-actin was submitted to GenBank and assigned the accession number EU825991. Profiles ofβ-actin expression in different recovering time after heat shock were expressed similarly and not significantly different with that of the unstimulated control, suggesting an ubiguitous expression pattern can be used as internal control in gene mRNA expression level.(4) Semi-quantitative RT-PCR analysis showed that 1 h heat shock treatment at 42℃caused rapid increase of Mshsp70 mRNA, which reached maximum levels at the end of the heat shock treatment, and decreased gradually after being moved to room temperature. Expression of Mshsc70, however, was inhibited after heat-shock treatment:during recovering 2-4 h, the expression levels were only little times that of the control.(5) The Mshsp70 prokaryotic expression vector was constructed by prokaryotic vector pET-DsbA, and the recombined plasmid was transfected into DE3. After induction by cultivating 37℃, eventually concentration IPTG 1mM, the specific expression of the DsbA-fused protein was detected by SDS-PAGE. The result of SDS-PAGE showed that the molecular weight of the recombined protein is approximately 59.4 KD, which is consistent with its theory molecular weight. Using His affinity chromatography purified get fusion protein, to preparation HSP70 antibody, after western blot detected and expected the same size of specific bands.更多还原