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摘要：昆虫在自然界中遭遇的高温和低温胁迫等不利环境非常普遍，而热激蛋白（Heat shock protein，Hsp）的表达和调控是生物体受到来自外界或者自身因素胁迫所产生的一种应激反应，使得体内新合成或含量增加的一组蛋白质。白蜡虫对极端高温和低温具有很强的耐受力，其中Hsp40对白蜡虫的这种胁迫反应具有重要意义。本实验是通过在获得中间片段的基础上，采用RACE的方法，分别克隆5′末端和3′末端的核苷酸序列，并通过片段的重叠将各个片段连接起来，从而得到白蜡虫Hsp40基因全长cDNA，对其核苷酸序列和预测编码氨基酸序列进行生物信息学分析。经序列分析表明，所有的Hsp40家族成员都含有高度保守的J-结构域（J-domain），J-结构域通常位于Hsp40的N-端，大约含有70个氨基酸，并且有一个高度保守的HPD基序。而且Hsp40能够借助J-结构域与Hsp70的功能位点结合，进而激活Hsp70的ATPase活性，促进其结合底物以及功能发挥。序列39115
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毕业论文关键词：白蜡虫；Hsp40；RACE；基因克隆与分析；序列比对
Heat Shock Protein40 Gene Cloning and Sequence Annlysis of white wax insect
Abstract：Insects encounter the adverse environment of the nature , such as high temperature and low temperature force is very common, and heat shock protein expression and regulation is a stress response when the organism being forced from the outside factors or their own factors, and it is a set of proteins which makes new synthesis and content increased in the body . White wax insect has strong tolerance in extreme high temperature and low temperature , and Hsp40 is of great significance about the stress response of a wax scale insect . This experiment is on the basis of obtaining the middle segment, using the method of RACE, Cloning, respectively, the nucleotide sequence at the end of 5 'and 3' , and connecting each fragment through overlapping the fragments ,thus getting the whole cDNA of a wax scale insect Hsp40 gene , and conducting bioinformatics analysis about the nucleotide sequence and predictive coding amino acids sequence. The sequence analysis showed that all the members of Hsp40 family contain J- domain which is highly conserved , J - domain is usually located in Hsp40 N - end of the structure, containing about 70 amino acids, and has a highly conservative HPD motif. and Hsp40 can combine with the functional sites of Hsp70 with the help of J- domain, and then activating Hsp70 ATPase activity, promoting it to combine the substrate and play its function.
Key words：Ericerus pela Chavanness； Heat shock protein40；RACE；Gene cloning and characterization；Sequence alignment
目 录
摘要 1
ABSTRUCT.1
1.引言 3
1.1热激蛋白的起源与发展3
1.2热激蛋白的分类和特点3
1.3热激蛋白的功能4
1.3.1Hsp的分子伴侣作用.4
1.3.2 Hsp与生物体耐热性4
1.3.3 Hsp与生长发育5
1.3.4 Hsp与细胞凋亡5
1.4白蜡虫热激蛋白的研究现状5
2研究目的与研究内容6
2.1研究主要目的6
2.2研究内容6
3实验材料与实验方法7
3.1供试昆虫7
3.2试剂7
3.3主要实验仪器7
3.4引物的设计与合成7
3.5 3′RACE和5′RACE7
3.6 PCR产物的纯化8
3.7 E.coli DH5α感受态细胞制备8
3.8 PCR纯化产物的连接与转化8
3.9阳性重组克隆的鉴定9
3.10测序10
4结果与分析10
5结论与讨论13
参考文献.15
致谢.18
白蜡虫Hsp40基因克隆与序列分析1 引言
1.1 Hsp的起源与发展
热激蛋白（Heat shock proteins，Hsp）是生物体受到自身或者外界因素的胁迫而产生应激反应并使得体内含量增加或者新合成的一组蛋白质，是机体进行自我保护的物质基础。Hsp广泛存在于真核和原核生物中，能被多种因素诱导，包括高温、低温、紫外线照射、重金属、杀虫剂、组织缺氧、盐度胁迫、细菌和病毒感染、寄生、种群密度、衰老、近亲繁殖、有害突变等（Sørensen et al.， 2003）。