圆斑星鲽piwil2基因的克隆与表达分析

Cloning and Expression of the piwil2 Gene in the Spotted Halibut (Verasper variegatus)

中文关键词: 圆斑星鲽  piwil2  基因克隆  表达分析

英文关键词: Verasper variegates  piwil2  Gene cloning  Expression analysis

中文摘要:

      本研究采用RACE末端扩增方法，得到全长为3872 bp的圆斑星鲽(Verasper variegatus) piwil2基因序列，命名为Vvpiwil2，开放阅读框(ORF)长为3192 bp，编码1063个氨基酸，5-UTR和3-UTR的长度分别140 bp和540 bp。基于ExPASy、SMART、Signal4.1和NCBI的保守结构域(CDD)数据库在线分析对蛋白序列结构进行预测，推断Vvpiwil2编码的氨基酸分子量为118.6 kDa，理论等电点为9.02，无跨膜结构及信号肽，有3个结构域：ArgoL1结构域、PAZ结构域及PIWI结构域。利用实时荧光定量PCR技术对圆斑星鲽不同发育时期的胚胎、仔稚鱼以及雌雄成鱼的不同组织表达模式进行分析。结果显示，Vvpiwil2基因从胚胎发育早期至高囊胚期均大量表达，之后呈下降趋势，直至孵化阶段。由于胚胎从卵裂至囊胚时期的发育过程主要受细胞质成分引导，直至原肠早期，mRNA开始大量转录合成，实现由母源型向合子型的过渡，推断Vvpiwil2是母源性基因。孵化后仔稚鱼68 d时，Vvpiwil2基因表达量显著高于其他时期，表明Vvpiwil2的功能可能与圆斑星鲽性腺分化过程相关；Vvpiwil2基因在雌雄成鱼性腺中的表达量显著高于其他组织，且卵巢中的表达量显著高于精巢，推测Vvpiwil2基因在卵巢功能的维持中发挥重要作用。本研究结果为解析圆斑星鲽性别决定机制提供了新的靶标基因，为建立全雌化苗种繁育技术打下坚实的理论基础。

英文摘要:

      The spotted halibut (Verasper variegatus) is a rare and valuable marine fish species that inhabits the coast of northern China. Mature females are much larger than males because of their faster growth rate. It will create substantial economic benefits to establish an all-female breeding technique for V. variegatus. A better understanding of sex-related genes will contribute to the improvement of a single-sex breeding technique. In this study, we successfully isolated the piwil2 gene of V. variegatus, which was named Vvpiwil2. The total length was 3872 bp, including a 3192 bp open reading frame (ORF), encoding 1063 amino acids; the 5′UTR was 140 bp and the 3′UTR was 540 bp. Based on ExPASy, SMART, Signal4.1, and the NCBI Conservative Domain Database (CDD) biological analysis, the ORF encoded a putative protein, with a predicted molecular weight of 118.6 kDa and an isoelectric point of 9.02. No transmembrane structure or signal peptide site was detected. There were three domains: the ArgoL1, PAZ, and PIWI domains. Real-time fluorescence quantitative PCR technique was used to analyze the expression patterns of the Vvpiwil2 gene at different stages of embryo and larvae. The results showed that the Vvpiwil2 gene was abundantly expressed from early development to the high blastocyst stage, and then declined until the hatching stage. The developmental stage of the embryo from cleavage to the blastocyst stage was mainly guided by the cytoplasmic component. The mRNAs began to be transcribed and synthesized in a large amount at the early gastrula stage, then the transition from maternal to zygote occurred. Therefore, the results indicated that the Vvpiwil2 gene was a maternal gene. After hatching, the expression of the Vvpiwil2 gene at 68 days post hatching was significantly higher than during other stages, which demonstrated that the Vvpiwil2 gene was associated with gonadal differentiation. The expression level of the Vvpiwil2 gene in gonads was significantly higher than in other tissues, and the expression level in the ovary was significantly higher than in the testis, revealing that the Vvpiwil2 gene might play an important role in the maintenance of ovarian functions. The results of this study provide a potential sex determinant gene for V. variegatus and lay a solid theoretical foundation for the establishment of an all-female breeding technique.