中国对虾V-ATPase c亚基基因的克隆及其在高pH胁迫下的表达分析

采用RACE技术克隆了中国对虾(Fenneropenaeus chinensis)V-ATPase c亚基基因,命名为FcVHA-c基因,检测该基因在高pH胁迫下的基因应答,并采用RNAi技术验证其功能。基因分析表明, FcVHA-c基因cDNA全长为2128 bp,开放阅读框483 bp,编码160个氨基酸,预测蛋白分子量为16 kD,理论等电点7.82,具有4个跨膜结构域。同源性和系统进化分析表明, FcVHA-c具有较高的保守性,其中与南美白对虾(Penaeus vannamei)同源性最高,为99%,与南美白对虾首先聚为一支。组织表达分析显示, FcVHA-c基因在中国对虾各个组织中均有表达,在鳃中表达量显著高于其他组织(P<0.05)。pH 8.8胁迫下,该基因的表达量在12 h达到峰值,为对照组的1.206倍,在48 h达到最低值,为对照组的0.166倍;pH 9.2胁迫下,该基因表达量在1 h达到峰值,为对照组的1.577倍,在12 h达到最低值,为对照组的0.104倍。结果表明,高pH对该基因具有一定的抑制作用。干扰结果显示,高pH胁迫下干扰组较对照组的死亡率显著增高(P<0.05),表明该基因表达量越高,越有利于中国对虾的存活。本研究结果表明, FcVHA-c基因参与了高pH胁迫下中国对虾的离子调控,高pH胁迫抑制FcVHA-c基因的调控能力。

Expression analysis of V-ATPase subunit c under high pH stress in Fenneropenaeus chinensis

Vacuolar-type H⁺-ATPase (V-ATPase) is a kind of hydrogen ion transport enzyme, in which subunit c plays an important role in maintaining the normal acid-base environment in cells. To investigate the function of the V-ATPase subunit c in Fenneropenaeus chinensis under high pH stress, the full-length cDNA of V-ATPase subunit c in F. chinensis, named FcVHA-c, was first cloned by rapid amplification of cDNA ends (RACE). Then, the response of FcVHA-c under high pH stress was detected. Subsequently, RNA interference (RNAi) technology was used to verify gene function. The full length of FcVHA-c cDNA sequences consisted of 2128 base pairs (bp), including a 483 bp open reading frame (ORF), which encodes a 160-amino acid peptide with four transmembrane domains. The predicted protein molecular weight of the FcVHA-c protein was 16 kD, and the theoretical isoelectric point (pI) was 7.82. Homology and phylogenetic analysis showed that the deduced FcVHA-c amino acid sequence was highly conservative, had the highest amino acid sequence identity (99%) with Penaeus vannamei, and clustered into one group first with P. vannamei as well. Results of RT-qPCR showed that FcVHA-c was expressed in all tissues of F. chinensis, and the expression level in the gills was significantly higher than that in other tissues (P<0.05). Under the stress of pH 8.8, the expression level of FcVHA-c reached a peak at 12 h, which was 1.206 times that of the control group, and reached the lowest value at 48 h, which was 0.166 times that of the control group. Under pH 9.2 stress, the expression level of FcVHA-c peaked at 1 h. It was 1.577 times that of the control group and reached its lowest value at 12 h, which was 0.104 times that of the control group. The results showed that high pH had a certain inhibitory effect on FcVHA-c. Silencing the FcVHA-c by RNAi, under the high pH stress, resulted in mortality that was significantly higher than that in the control group (P<0.05). The results suggested that the FcVHA-c may play an important role in ion regulation under the condition of high pH stress, whereas excessive high pH-stress inhibits the ability to regulate FcVHA-c.