近缘新对虾PCNA基因的克隆及表达分析

Cloning and expression analysis of PCNA in Metapenaeus affinis

增殖细胞核抗原(Proliferating cell nuclear antigen, PCNA)作为DNA聚合酶δ的辅助蛋白,在DNA复制过程中发挥重要作用。近缘新对虾(Metapenaeus affinis)卵巢发育过程中存在细胞增殖活动旺盛的阶段,但目前关于其卵巢发育的分子机制研究较少。利用RACE (Rapid amplification of cDNA ends)技术获得近缘新对虾PCNA (MaPCNA)基因的cDNA序列全长,并通过实时荧光定量PCR (qRT-PCR)对其进行卵巢发育相关的表达分析。MaPCNA全长为1 144 bp,包含140 bp的5’非编码区,221 bp的3’非编码区,开放阅读框(ORF)为783 bp,编码260个氨基酸。MaPCNA蛋白的相对分子质量为28.82 kD,理论等电点为4.5。多重比对分析表明,PCNA氨基酸序列在甲壳动物中较为保守。组织表达结果显示,MaPCNA基因在检测的组织中均有表达,其中在卵巢中的表达量最为显著(P<0.05)。在不同发育阶段的卵巢中,MaPCNA基因的表达出现变化(P<0.05),从Ⅰ期开始逐渐上升,到Ⅲ期表达量最高,之后显著降低并趋于平稳。MaPCNA基因在幼体发育不同时期的表达呈现出一定的规律性趋势,在受精卵时期的表达量最高,之后呈下降趋势,从无节幼体Ⅵ期开始表达平稳。研究结果提示PCNA基因可能在近缘新对虾的卵巢发育过程中发挥重要作用。

As a coprotein of DNA polymeraseδ,proliferating cell nuclear antigen(PCNA)plays an important role in the pro-cess of DNA replication.There is a stage of vigorous cell proliferation during the ovarian development in Metapenaeus affinis,but little attention has been paid to its molecular mechanism.In this study,we applied rapid amplification of cDNA ends(RACE)technique to obtain the full length cDNA sequences of PCNA in M.affinis(MaPCNA),and analyzed the expression of MaPCNA related to ovarian development by real-time fluorescent quantitative PCR(qRT-PCR).The total length of MaPCNA was 1144 bp,including 140 bp of 5'untranslated region,221 bp of 3'untranslated region and 783 bp ORF encoding 260 amino acids.The molecular mass of MaPCNA protein was 28.82 kD and the theoretical isoelectric point was 4.5.The protein homo-logy analysis shows that MaPCNA had high homology with other crustaceans.The result of tissue expression shows that Ma-PCNA was expressed in all tested tissues,with the highest expression in ovary(P<0.05).The expression of MaPCNA in ovary at different developmental stages showed significant changes(P<0.05),increasing from stage I to stage III gradually,decreasing sig-nificantly and then tending to be stable.The expression of MaPCNA showed a regular change trend at different larval develop-mental stages.The expression level of MaPCNA was the highest in oosperm,then began to decline,and became stable from the Nauplius VI.The results suggest that PCNA may play an important role in the ovarian development of M.affinis.