草鱼Rab1A基因的克隆表达、抗体制备及对微囊藻毒素-LR的响应

为了解草鱼(Ctenopharygodon idella)小分子三磷酸鸟苷(GTP)结合蛋白家族成员Rab1A的分子结构特点及其在微囊藻毒素-LR (MC-LR)胁迫中的作用,本研究根据草鱼肝脏(MC-LR诱导)转录组测序获得的unigenes序列,采用RACE技术克隆了草鱼Rab1A基因cDNA,其全长序列为806 bp,开放阅读框为609 bp,编码202个氨基酸。同源性分析结果表明,Rab1A与鲤鱼(Cyprinus carpio) Rab1A3相似性最高。系统进化分析表明草鱼Rab1A与鲤鱼、斑马鱼(Danio rerio)等鱼类聚为一大支。实时荧光定量PCR (qRT-PCR)分析发现Rab1A在草鱼各组织中广泛分布,在血液和鳃等组织中的表达较丰富。构建了pGEX-4T-1-Rab1A重组表达载体,并制备了Rab1A多克隆抗体,通过酶联免疫(ELISA)检测抗体效价为1∶512 000以上,Western blot检测显示抗体具有特异性。草鱼肝脏经微囊藻毒素-LR(MC-LR)染毒,发现25和75 μg·g^-1感染96 h后对Rab1A基因和蛋白表达的影响不明显(P>0.05),但100 μg ·kg^-1 可导致草鱼肝脏Rab1A基因和蛋白表达显著下降 (P<0.05),表明Rab1A在参与草鱼抵御MC-LR胁迫过程中起着重要的作用。本研究为进一步了解Rab1A基因的功能及其在MC-LR胁迫过程中所发挥的作用奠定了基础。

Cloning,Expression, Antibody Preparation of Rab1A in Grass Carp (Ctenopharygodon idella) and Its Response to Microcystin-LR

In order to investigate the structure and roles responding to microcystin-LR (MC-LR) Rab1A, one member of small GTPases family, the cDNA sequence of Rab1A gene in grass carp (Ctenopharygodon idella) was amplified using rapid amplification of cDNA ends (RACE). Grass carp Rab1A gene was 806 bp in length and had an ORF coding a predicted 202 amino acids protein. Sequence analyses showed that grass carp Rab1A gene was homologous to common carp (Cyprinus carpio) Rab1A3 gene. Phylogenetic analyses showed that grass carp Rab1A was assigned to a clade with common carp, zebrafish (Danio rerio) and other teleost. The grass carp Rab1A gene was ubiquitously expressed in all analyzed tissues from healthy grass carp as revealed by quantitative real-time (qRT-PCR), and was highly expressed in blood and gill. A special segment coding 1-202 AA of Rab1A was selected and cloned into pGEX-4T-1 vector to construct the prokaryotic expression vector pGEX-4T-1-Rab1A, and the polyclonal antibody of Rab1A was prepared. The antibody titer obtained by ELISA was higher than 1∶512 000. Western blot results showed that the antibody had specificity. The expression of Rab1A protein of grass carp liver was significantly down-regulated 100 μg·kg^-1 for 96 h(P<0.05), but the expression were not significantly changed with 25 and 75 μg·kg^-1 groups (P>0.05), which suggest thatRab1A gene might play an important role in defending grass carp against MC-LR stress. The above research results will provide a basis for further studies of the function of Rab1A and its roles in response to MC-LR.