【作者】 袁嘉恩；

【导师】 韩博平；

【作者基本信息】 暨南大学 ， 水生生物学， 2008， 硕士

【摘要】 热休克蛋白70(heat shock protein 70,HSP70)是热休克蛋白家族中最重要的一类成员,具有“分子伴侣”、细胞保护、抗凋亡以及肿瘤免疫治疗等独特复杂的生物学功能,因此,它的研究已成为生命科学领域研究的热点之一。根据蓝蟹(Callinectes sapidus)HSC70基因以及凡纳宾对虾(Litopenaeus vannamei)HSP70基因的cDNA全长序列,设计引物,采用RT-PCR和RACE(rapid amplification ofcDNA ends)相结合的方法,从38℃热休克的中华绒螯蟹(Eriocheir sinensis)的卵组织中克隆得到长2429bp的热休克蛋白70基因,编码649个氨基酸的多肽分子,推测的分子量为70.9kDa,理论等电点为5.3。与已知的30个其它生物的HSP70或HSC70蛋白序列的同源性为66～91%,通过构建系统发生树,发现该热休克蛋白70基因是一种组成型HSC70基因。相同方式,在38℃热休克的锯缘青蟹(Scylla serrata)的卵组织中克隆得到长2482bp的热休克蛋白70基因,编码649个氨基酸的多肽分子,分子量为71.06kDa,理论等电点为5.24。包括长1950bp的完整编码区,在poly(A)尾上游发现一加尾信号(AATAAA)。与已知的30个其它生物的HSP70和HSC70蛋白序列构建系统发生树,发现该热休克蛋白70基因也是一种组成型HSC70基因。采用半定量RT-PCR方法,对两种蟹的HSC70组织表达进行研究。中华绒螯蟹的HSC70的mRNA常温常态下在卵组织、心脏、肌肉、胃、肠组织中都有一定的表达量,经过38℃热休克以后,表达量也相应升高,部分组织表达量增加比较显著,则进一步说明该HSC70是一种组成型的热休克蛋白,其中卵组织和心脏的表达量最高。在1-4h的热应激实验中,各测试组织的表达量都呈上升趋势。锯缘青蟹的HSC70的mRNA表达情况和中华绒螯蟹相似,正常情况下在卵组织、心脏、肌肉、胃、肠组织中都有一定的表达量,经过38℃热休克以后表达量也有相应的提升,说明该HSC70也是一种组成型的热休克蛋白。更多还原

【Abstract】 Heat shock protein 70kDa (HSP70) is one of the most important proteins among members of HSPs, and mainly functions as molecular chaperone. Moreover, HSP70s play fundamental roles in cytoprotection, anti-apoptotic effects, and immune therapy of tumor, etc. Therefore, researches on HSP70s have become one of popular and prospective focuses in fields of life science.Firstly, by the study of the HSP70 gene of Callinectes sapidus and Litopenaeus vannamei, according to their sequences, I designed the proper primers and cloned a HSP70 gene from the ovum tissue after 38℃heat-stressed Eriochier sinensis by using RT-PCR and RACE. The cDNA sequence was 2429 bp in length and encoded a 649 (Mr 70.9kDa) amino-acid polypeptide with isoelectric point (pI) 5.3. Based on the phylogenic tree of HSP70s, it indicated that it belonged to the member of constitutive HSC70 family with high identities (66-91%) of amino-acid sequence with other 29 known HSP70s.In the same way, a HSP70 gene was cloned from ovum tissue of Scylla serrata. This HSP70 was 2482bp in length and encoded a 649 (Mr 71.06kDa) amino-acid polypeptide with pI 5.24. It includes a 1950bps open reading frame. There was a polyadenylation signal of AATAAA found in 3’ end of the sequence. Based on the phylogenic tree of HSP70s, this HSP70 also belonged to the constitutive HSC70 family.Semi-quantitative RT-PCR was developed to study the HSC70 transcription pattern in E. sinensis and S. serrata. The level of HSC70 mRNA is constitutively express in ovum, heart, muscle, stomach, intestines of non-stressed E. sinensis. After heat shock in 38℃water, the levels of HSC70 mRNA have up-regulated. The highest expression of HSC70 of E. sinensis was observed in ovum and heart organ. The result confirmed that this gene belonged to the constitutive HSC70 family.The RT-PCR assay demonstrated that HSC70 of S. serrata constitutively expressed in ovum, heart, muscle, stomach and intestines. After heat shock in 38℃water, the levels of HSC70 mRNA have up-regulated. The highest expression of HSC70 of S. serrata was observed in ovum. The result also suggested that this gene belonged to the constitutive HSC70 family.更多还原