三角帆蚌细胞周期蛋白基因筛选及其表达分析

Screening and expression of cyclins gene in Hyriopsis cumingii

细胞周期蛋白(cyclin)对细胞生长繁殖起到关键的调节作用。该研究采用高通量测序技术,构建了三角帆蚌(Hyriopsis cumingii)转录组文库,进行了cyclins基因筛选与生物信息分析,通过qRT-PCR技术,探讨其在不同组织(闭壳肌、内脏团、外套膜、斧足、心脏、血液、鳃、性腺)与性别中的表达特征。转录组测序共得到257457条unigene(N50为1796 bp),注释率为86.7%;通过分析,cyclin A、cyclin D2、cyclin E被筛选出,且cyclin A、cyclin D2与cyclin E蛋白的氨基酸序列均与美洲牡蛎(Crassostrea virginica)、太平洋牡蛎(Crassostrea gigas)具有较高相似性。实时荧光定量分析结果表明,cyclin A、cyclin D2、cyclin E在三角帆蚌各组织中均有表达,且存在显著的组织差异:cyclin A、cyclin E基因在性腺中表达量显著高于其他组织(P<0.05),在斧足中表达量最低;cyclin D2基因在鳃中表达量最高(P<0.05),在血液中表达量最低。在同一组织的不同性别之间:cyclin A基因在性腺、闭壳肌、心脏、血液中表达存在显著性别差异(P<0.05);cyclin D2在外套膜、心脏、性腺中的表达具有显著(P<0.05)的性别差异;cyclin E在性腺、斧足、鳃、心脏中的表达具有显著(P<0.05)的性别差异;cyclin A、cyclin E基因在雌性心脏和性腺中表达量显著(P<0.05)高于雄性,而cyclin D2反之(P<0.05),cyclin D2基因在雌性外套膜表达量显著高于雄性(P<0.05),暗示cyclin A、cyclin E与性腺发育关系密切,cyclin D2可能与鳃损伤修复有关。本研究初步探究了三角帆蚌cyclin A、cyclin D2、cyclin E基因的功能,为建立三角帆蚌细胞系提供了分子基础。

Cyclins plays an important role in cell cycle entry and progression,they can modulate the catalytic activities of cyclin-dependent kinases(CDKs),cyclins can binding the catalytic subunits of CDKs to regulate cell cycle.To select cyclin genes in Hyriopsis cumingii,explore the function of cyclins,total RNA from mixed sample of mantle,gonad,visceral mass and hemolymph were isolated and sequenced by Illumina HiSeq 2000.Based on the sequencing results,select the cyclin A,cyclin D2,cyclin E genes,through cloning their conserved sequence to verify cyclins genes in H.cumingii.Amino acid sequence similarity analysis showed that cyclin A,cyclin D2 and cyclin E of H.cumingii have high sequence identity with Crassostrea virginica and Crassostrea gigas.At the transcriptional level,quantitative real-time PCR(qRT-PCR)was used to detect expression of cyclin genes in different tissues.The result revealed that all cyclin genes were expressed in tested tissues(adductor,foot,visceral mass,gill,heart,mantle,gonad,blood)with variable levels,expression levels of cyclin A and cyclin E genes in gonad were significantly(P<0.05)higher than those in other tissues,expression level of cyclin D2 gene in gills was significantly(P<0.05)higher than those in other tissues.Between different genders in the same tissue,there were significant(P<0.05)gender differences in the relative expression of cyclin A gene in gonad,adductor,heart and blood.In gonads and heart,there were significant(P<0.05)gender differences in cyclin D2 and cyclin E expression.Cyclin A and cyclin E genes were significantly(P<0.05)higher expressed in female hearts and gonads than those in males,indicated that cyclin A and cyclin E genes play an important role in gonad development.The high expression of cyclin D2 in gill suggested that it might be associated with gill damage repair.In this study,cyclin A,cyclin D2 and cyclin E genes and their functions were preliminarily studied,which laid a molecular basis for further regulation of the cell growth in vitro.