美洲鲥hsp70的分子特征及其对运输应激的应答

Molecular Characterization of Hsp70 and Its Response to Transport Stress in American Shad(Alosa sapidissima)

运用同源克隆和c DNA末端快速扩增(RACE)技术获取了美洲鲥(Alosa sapidissima)热应激蛋白70(hsp70)的全长c DNA序列,其总长度为2 545 bp,开放阅读框(ORF)为1 914 bp,推测编码637个氨基酸,其氨基酸的二级结构含有HSP70家族的3个特征序列。氨基酸同源性分析显示,美洲鲥hsp70与墨西哥脂鲤(Astyanax mexicanus)等鱼类的相似性达84%以上,与无脊椎动物果蝇(Drosophila auraria)及大肠杆菌(Escherichia coli)的相似性分别为73%和38%。荧光定量PCR检测显示,该基因在美洲鲥鳃、肌肉、头肾中高表达,在脑和心中相对高表达,在肝、脾、肠、肾中微量表达。采用荧光定量PCR方法研究运输应激对美洲鲥未经繁殖的亲鱼hsp70 m RNA水平的影响,在运输实验中鳃、肝hsp70 m RNA水平呈先升高后降低的变化趋势,头肾hsp70 m RNA水平呈现递增趋势。该结果表明克隆到的基因符合诱导型hsp70的特点,而且美洲鲥鳃、肝、头肾组织的hsp70 m RNA对运输应激表现出明显的应答作用。

American shad（Alosa sapidissima） belongs to Clupeomorpha,Clupeiformes,Clupeidae,Alosa.As eurythermal,euryhaline anadromous and migratory fish,American shad and Chinese shad are not only very similar in appearance and habits,but also have high nutritional value and economic value.American shad is extremely sensitive to changes in the external environment,vulnerable to the impact of environmental factors change（such as sound,light,water physical and chemical factors,etc.） and the influence of manual operation such as fishing,transportation,etc.Heat stress proteins（HSPs）,also known as heat shock proteins,are widely present in different species.It is a family of non-specific cell protective proteins associated with resilience,and similar to a number of house-keeping genes products.The heat stress proteins,especially the heat stress protein 70（hsp70） family,are highly conserved in evolution.It also has a variety of biological functions.As a molecular chaperone,it assists proteins folding,assembly,transportation,and regulation,as well as repair of damaged proteins,and disintegration of denatured proteins under stress.It can also protect mitochondria from cytokines damage,and play an important role in anti-apoptosis,anti-oxidation and immune response.Hsp70 c DNA was cloned,analyzed,and the expression level of hsp70 m RNA was detected in a variety of fish species,such as bluntnose black bream（Megalobrama amblycephala）,grass carp（Ctenopharyngodon idellus）,silver carp（Hypophthalmichthys molitrix）,nile tilapia（Tilapia nilotica）,roughskin sculpin（Trachidermus fasciatus）,bronze gudgeon（Coreius guichenoti）,siberian sturgeon（Acipenser baerii）,sword tali（Xiphophorus hellerii）,bastard halibut（Paralichthys olivaceus） etc.Studies show that the expression of hsp70 gene is regulated at the transcriptional level.This study aimed to investigate the expression of hsp70 gene in American shad broodstocks tissues and its differential expression before and after the transport stress,in order to rovide a theoretical basis for resistance mechanism study of this species.In this experiment,by means of artificial transportation stress,we took 6 tails as the transport 0 h sampling,then collected 24 tails randomly into 12 bags,each containing 2 tails：6 bags for 2 h transport stress sampling,while the other 6 bags for 4 h sampling.The results were expressed by Mean ± SE,and the differences between groups were analyzed by One-way ANOVA.The full-length c DNA of hsp70 of American shad was cloned by reverse transcription polymerase chain reaction（RT-PCR） and rapid amplification of c DNA ends（RACE） methods.The results revealed that the length of hsp70 c DNA was 2 545 bp,containing an open reading frame（ORF） of 1 914 bp specified a peptide of 637 amino acids.The secondary structures contained three signature sequences of HSP70 family（Fig.2）.The homology analysis indicated that hsp70 of American shad shared more than 84% identity with the hsp70 s of other fishes such as Astyanax mexicanus,it also shared as high as 73% and 38% identity with Drosophila auraria and Escherichia coli（Table 2）.The tissue-specific expressions of hsp70 m RNAs were detected.A high expression was observed in the gill,muscle and head kidney,while relatively high expression was encountered in brain and heart,and a weak expression was found in the liver,spleen,intestine and kidney（Fig.4）.Real-time quantitative PCR showed that hsp70 m RNA content in gill and liver increased significantly after transport for 2 hours and then dropped after transport for 4 hours（Fig.5 and Fig.6）,while hsp70 m RNA showed an increasing trend in head kidney（Fig.7）.These results suggest that our cloned gene is consistent with the characteristics of hsp70,and that the hsp70 m RNA in gill,liver,and head kidney showed obvious response to transport stress.