蚤状溞(Daphnia pulex)热休克蛋白Hsp90基因的克隆与表达分析

利用RACE技术从蚤状溞(Daphnia pulex)中克隆到Hsp90基因cDNA全长为2568bp,开放阅读框为2155bp,编码718个氨基酸残基,Hsp90蛋白中存在GxxGxG、LxxLL模块(亮氨酸拉链)和C末端的MEEVD序列。同源性比对结果显示蚤状溞Hsp90基因与日本对虾和刀额新对虾的同源性最高为85%,与其它甲壳纲物种的同源性保持在79%及以上。进化分析发现,蚤状溞Hsp90基因与剑水蚤、日本沼虾、红螯相手蟹等甲壳纲的亲缘关系最近。用Real Time PCR技术,检测了Hsp90mRNA在蚤状溞不同生殖状态下的表达水平:Hsp90 mRNA在两性溞(带冬卵)中的表达量明显高于孤雌溞(带夏卵)(P0.05),且在冬卵中的表达量最低。推测Hsp90可能参与了蚤状溞的生殖转化调控。Hsp90 mRNA在雄溞中的表达量是孤雌溞的2.4倍,说明Hsp90可能参与了精子的形成过程。

CLONING AND EXPRESSION ANALYSIS OF A Hsp90 GENE IN DAPHNIA PULEX

The full-length cDNA of a Hsp90 gene (DpHsp90) was cloned from cladoceran Daphnia pulexusing rapid amplification of complementary DNA ends (RACE) method. The DpHsp90 cDNA is 2568bp in length; and it has a 2155-bp open reading frame that encodes a 718-amino-acid polypeptide containing GxxGxG, LxxLL module (leucine zipper) and C-terminal MEEVD sequence. In addition, DpHsp90 shared homology of up to 85% with Marsupenaeus japonicusand Metapenaeus ensis, and 79% with other crustacean species. Phylogenetic analysis revealed that DpHsp90 protein has a close genetic relationship with crustacea such as Tigriopus japonicus,Macrobrachium nipponense, Chiromantes haematocheirand so on. Results of qPCR (Real-time Quantitative PCR) show that the DpHsp90 expression was significantly higher (P<0.05) in ephippial female (with winter eggs) than in parthenogenetic female (with summer eggs), and was the lowest in the resting egg. Therefore, Hsp90 was closely related to the reproduction conversion of Daphnia pulex. Meanwhile, Hsp90 mRNA expression in male was about 2.4 times higher than the parthenogenetic ones, indicating that Hsp90 may have been involved in the formation of sperm.