四种海藻热休克蛋白70（HSP70）基因的克隆与表达分析

IOCAS-IR > 海洋环流与波动重点实验室

	四种海藻热休克蛋白70（HSP70）基因的克隆与表达分析
其他题名	Molecular cloning and expression analysis of cytosolic HSP70 genes from four kinds of seaweeds
	付万冬
学位类型	博士
	2009-06-03
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	海带孔石莼裙带菜浒苔分子克隆热休克蛋白70 荧光定量rt-pcr Mrna表达
摘要	热休克蛋白70是热休克蛋白家族中重要的成员，参与新生蛋白的折叠、转运、重折叠变性蛋白、协助降解变性蛋白和抗逆环境胁迫等功能。海带和裙带菜是浅海潮下带典型的褐藻，孔石莼和浒苔是潮间带典型的绿藻，四种大型海藻均有重要的经济价值和生态价值。随着潮汐变化固着藻类生境理化因素变化剧烈，藻类面临着严重的环境胁迫，因此研究藻类抗逆机理有着重要的意义。本研究采用同源克隆法配合RACE-PCR，克隆了海带、孔石莼、裙带菜和浒苔HSP70基因的全序列（分别命名为LJHSP70、UPHSP70、QDHSP70和EPHSP70）。利用生物信息学方法分析了四种藻类HSP70结构特征、同源性关系和进化地位。获得的海带HSP70基因全序列长为2918 bp，5’非翻译区为248 bp，3’非翻译区为696 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.97。获得的裙带菜HSP70基因全序列长为3243 bp，5’非翻译区为248 bp，3’非翻译区为1021 bp，开放阅读框为1974 bp，编码657个氨基酸，预测的分子量为72.03 kDa，等电点为4.96。获得的孔石莼HSP70基因全序列长为2283 bp，5’非翻译区为65 bp，3’非翻译区为247 bp，开放阅读框为1971 bp，编码656个氨基酸，预测的分子量为71.13 kDa，等电点为5.04。获得的浒苔HSP70基因全序列长为2265 bp，5’非翻译区为65 bp，3’非翻译区为217 bp，开放阅读框为1983 bp，编码660个氨基酸，预测的分子量为71.39 kDa，等电点为5.03。四种海藻HSP70氨基酸序列均含有四肽重复序列GGMP，具有三个典型的HSP70签名基序。细胞质定位的HSP70 C-末端特征基序为EEID或EEVD，并且N-端氨基酸序列保守性高于C-端。海带和裙带菜HSP70蛋白同源性为98%，孔石莼和浒苔HSP70蛋白同源性为96%，四种海藻HSP70蛋白序列与陆地植物和其他藻类HSP70蛋白序列同源性为70-80%。利用荧光定量RT-PCR技术对不同胁迫条件处理的海带和孔石莼HSP70 mRNA的表达水平进行定量分析。不同热激温度（5-40 ℃）处理组中，30 ℃处理组的海带HSP70 mRNA表达量最高是10 ℃处理组的海带HSP70 mRNA表达量的3倍，而35 ℃或40 ℃处理组的海带HSP70表达量却低于25 ℃或30 ℃处理组的海带HSP70 mRNA表达量。25 ℃不同热激时间（0-12 h）处理组中，海带HSP70 mRNA表达量呈先上升后下降趋势。热激1 h后海带HSP70 mRNA表达量迅速上升，热激7 h后mRNA表达量达到最大，是对照组表达量的4倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的海带HSP70 mRNA表达量是30‰盐度处理组海带HSP70 mRNA表达量的3倍。35‰、40‰和45‰盐度处理组之间HSP70 mRNA表达量较低且无显著差异。不同热激温度（5-40℃）处理组中，20 ℃或25 ℃处理的孔石莼HSP70 mRNA表达量较低，而5 ℃、35 ℃、或40 ℃处理组的孔石莼HSP70 mRNA的表达量是25 ℃处理组孔石莼HSP70 mRNA表达量的2倍以上。30 ℃不同热激时间（0-12 h）处理组中，孔石莼HSP70 mRNA表达量也呈先上升后下降趋势。热激5 h后孔石莼HSP70 mRNA表达量达到最大，是对照组的3.5倍。不同盐度（0‰-45‰）胁迫处理组中，0‰或5‰盐度处理组的孔石莼HSP70 mRNA表达量是30‰盐度处理组孔石莼HSP70表达量的3倍。30‰、40‰和45‰盐度处理组孔石莼HSP70 mRNA表达量较低，且无显著差异。不同紫外线照射时间（0-4.0 h）和不同干燥时间（0-4.0 h）处理组中，孔石莼HSP70 mRNA表达量都在3 h后达到最高值，之后表达量维持在较高水平。为进一步研究藻类HSP70的生物学功能，将海带HSP70基因的开放阅读框区域克隆到表达载体pEASY-E2中，并转化到大肠杆菌BL21(DE3)pLysS。将阳性重组子培养于含有AMP（100 U/mL）的LB培养基，IPTG诱导表达，SDS-PAGE电泳鉴定。经5 h诱导，其表达量达到平台期，继续培养HSP70表达量并不显著增高。5 mM IPTG诱导海带HSP70蛋白表达量高于1 mM IPTG诱导蛋白表达量。
其他摘要	Heat shock protein 70 is one of important members of heat shock protein families and plays essential roles in nascent protein folding, translocation, denatured protein refolding, protein degradation, adverse stress resistance and so on. Laminaria japonica and Undaria pinnatifida, representative species of brown algae, inhabit subtidal zone; Ulva pertusa and Enteromorpha prolifera, representative species of green algae, inhabit intertidal zone. The four kinds of macroalgae have important economical and ecological values. These macroalgae confront serious environmental stresses with the turning tides and rapid alterations of other physical and chemical factors. Therefore, it is meaningful to study adverse stress resistance mechanism of macroalgae. In this study, homologous cloning coupled with the rapid amplification of cDNA ends (RACE) was used to clone full length cytosolic heat shock protein 70s of L. japonica, U. pertusa, U. pinnatifida and E. prolifera (designed as LJHSP70, UPHSP70, QDHSP70 and EPHSP70, respectively). Bioinformatics was used to analyze structural features, homologous relationship and phylogenetic position of HSP70s. The full length of LJHSP70 cDNA was 2918 bp, with a 5’ untranslated region (5UTR) of 248 bp, a 3’ untranslated region (3UTR) of 696 bp, and an open reading frame (ORF) of 1974 bp encoding a polypeptide of 657 amino acids with an estimated molecular weight (Mw) of 72.03 kDa and an estimated isoelectric point (PI) of 4.97. The full length of QDHSP70 cDNA was 3243 bp, with a 5UTR of 248 bp, a 3UTR of 1021 bp, and the ORF of 1974 bp encoding a polypeptide of 657 amino acids with an estimated Mw of 72.03 kDa and an estimated PI of 4.96. The full length of UPHSP70 cDNA was 2283 bp, with a 5UTR of 65 bp, a 3UTR of 247 bp, and the ORF of 1971 bp encoding a polypeptide of 656 amino acids with an estimated Mw of 71.13 kDa and an estimated PI of 5.04. The full length of EPHSP70 cDNA was 2265 bp, with a 5UTR of 65 bp, a 3UTR of 217 bp, and the ORF of 1983 bp encoding a polypeptide of 660 amino acids with an estimated Mw of 71.39 kDa and an estimated PI of 5.03. The HSP70s of four kinds of seaweeds had degenerate repeats of tetrapeptide GGMP and three typical HSP70 signature motifs. The C-terminus amino acid sequences of cytosolic HSP70s were EEID or EEVD and the conservation of HSP70s of N-terminus was higher than that of C-terminus. The homology between LJHSP70 and QDHSP70 was 98%, the homology between UPHSP70 and EPHSP70 was 96%, the homology between the four HSP70s and HSP70s of other algae and land plants was 70-80%. Under different stress conditions, mRNA expression levels of LJHSP70 and UPHSP70 were quantified by quantitative RT-PCR (qRT-PCR). When L. japonica samples were kept in different temperatures (5-40 ℃) for 1 h, the expression level of LJHSP70 at 30 ℃ was highest and two-fold higher than that at 10 ℃. The expression level of LJHSP70 at 35 ℃ or 40 ℃ was lower than that at 25 ℃ or 30 ℃. When L. japonica samples were kept at 25 ℃ for different times (0-12 h), the mRNA expression level of LJHSP70 had a trend of rise first then fall. The expression level of LJHSP70 reached maximum level after 7 h, which was three-fold higher than that of control group. When L. japonica samples were kept in different salt concentrations (0‰-45‰) for 2 h, the expression level of LJHSP70 at 0‰ or 5‰ salt concentration was two-fold higher than that at 30‰ for 2 h. The expression levels of LJHSP70 at 30‰, 35‰ and 40‰ were low and had no difference (P<0.05). When U. pertusa samples were kept in different temperatures (5-40 ℃) for 1 h, the expression level of UPHSP70 at 20 ℃ or 25 ℃ was low. The expression level of UPHSP70 at 5 ℃, 35 ℃, or 40 ℃ was over one fold higher than that at 25 ℃. When U. pertusa samples were kept at 30 ℃ for different times (0-12 h), the mRNA expression level of UPHSP70 also had a trend of rise first then fall. The expression level of UPHSP70 reached maximum level after 5 h, which was 2.5-fold higher than that of control group. When U. pertusa samples were kept in different salt concentrations (0‰-45‰) for 2 h, the expression level of UPHSP70 at 0‰ or 5‰ salt concentration was two-fold higher than that at 30‰ for 2 h. The expression levels of UPHSP70 at 30‰, 35‰ and 40‰ were low and had no difference (P<0.05). When U. pertusa samples were kept at UV radiation or desiccated for different times (0-4 h), the mRNA expression level of UPHSP70 reached maximum level after 3.0 h, after that maintained at high level. To further study the biological function of seaweed HSP70, the ORF of LJHSP70 gene was subcloned into expression vector of pEASY-E2. The recombinant plasmid was transformed to E.coli BL21(DE3)pLysS. The positive recombinant was cultured in LB media with 100 U/mL ampicillin, induced by IPTG and determined by SDS-PAGE. The expression level of LJHSP70 reached high level after 5 h of induction, after that the expression level increased very slowly. The expression level of LJHSP70 at 5 mM IPTG was higher than that at 1 mM.
页数	139
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1517
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	付万冬. 四种海藻热休克蛋白70（HSP70）基因的克隆与表达分析[D]. 海洋研究所. 中国科学院海洋研究所,2009.

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