坛紫菜hsp70基因克隆与表达

HSP70家族是一类最保守和最重要的热应激蛋白家族,在藻类中编码HSP70家族的基因也是普遍存在的,从原始单细胞藻到多细胞的大型藻,hsp70基因在藻类适应环境的过程中起了重要的作用.为了进一步探讨hsp70基因的生物学作用及其在系统进化中的保守性,研究采用简并PCR,扩增获得了坛紫菜(Porphyra haitanensis)hsp70基因片段,随后利用基因组步移获取了坛紫菜hsp70基因全序列.该基因全长2449个碱基,其中1866bp为编码区域,共编码621个氨基酸,与同属紫菜(P.purpurea)hsp70的氨基酸一致性达90%以上.利用Real-time PCR技术研究热激及恢复过程中坛紫菜hsp70的表达情况,结果表明:坛紫菜hsp70对热激应答极为迅速,35℃热激15 min,hsp70 mRNA的表达就提升了15倍;在恢复培养3 h后坛紫菜hsp70的表达进入第二个高峰,表达量提升了48倍,且在随后的过程中始终高于对照值.在整个热激处理中,坛紫菜HSP70蛋白除了作为胁迫的应激蛋白外,始终作为重要因素参与了防御与损伤修复的过程.

Cloning and Expression of hsp70 for Porphyra haitanensis

The genes encoding HSP70s belong to one of the most important and highly conserved gene family,which are ubiquitous in algae,both unicellular and multicellular organisms and play an important role in adapting to the environment.In order to study the biological functions and the conservation of hsp70,the whole hsp70 sequence is extracted from the red seaweed Porphyra haitanensis through degenerate PCR and genomic DNA walking.The whole sequence includes 2 236 bp,among which there are 1 866 bp encoding 621 amino acids.The comparison of the amino acids sequences predicted from the gene sequences with that of another Porphyra(P.purpurea) show that the homology is more than 90%.The relative expression and the relevance of hsp70 gene from P.haitanensis under heat shock are detected by the real-time quantitative PCR technique.The results indicate that the level of mRNA expression of hsp70 increases dramatically in response to the heat shock.The expression of mRNA of hsp70 is 15 times higher than the normal after being heat-shocked under 35 ℃ for 15 min,and reaches the second expression peak as high as 48 times of the control when it is recovered from heat for 3 hours.The gene expression stays higher than the control during the recovery stage.It indicates that the HSP70 not only acts as the stress responding protein but also plays an important role in the stress resistance and damage remedying procedures.