三角帆蚌HcCUBDC基因cDNA的全长克隆与表达分析

Full-length c DNA cloning and expression analysis of Hc CUBDC gene from Hyriopsis cumingii

为寻找珍珠层颜色形成相关基因,以转录组文库中标注的序列为模板,利用cDNA末端快速扩增(rapid amplification of cDNA ends,RACE)方法,获得了三角帆蚌HcCUBDC基因的全长cDNA序列,共5 158 bp,开放阅读框(ORF)1 920 bp,编码639个氨基酸,5'端非编码区576 bp,3'端非编码区2 662 bp,Gen Bank登录号为KP067952。生物信息学分析表明,三角帆蚌HcCUBDC蛋白含有一个由19个氨基酸组成的信号肽和4个CUB结构域,但未能比对上已知的蛋白。经荧光定量PCR检测,HcCUBDC基因在紫色和白色蚌前端缘膜、后端缘膜、中央膜、鳃、斧足、肝胰腺、肠和肾8个组织中均有表达,并且都是肝胰腺中表达量最低。在紫色蚌中,后端缘膜表达量极显著高于前端缘膜;在白色蚌中,前端和后端缘膜之间表达差异不显著。HcCUBDC基因在紫色蚌后端缘膜中的表达量极显著高于白色蚌,在紫色和白色蚌前端缘膜中的表达量差异不显著。外套膜原位杂交结果显示,HcCUBDC基因主要在缘膜外上皮细胞中表达。研究表明,HcCUBDC基因在三角帆蚌贝壳珍珠层颜色形成中发挥作用,可为进一步深入研究该基因在珍珠颜色形成过程中的功能及其调控机理提供基础资料。

This study aims to find genes related to the formation of nacre color. According to the sequence annotated from the transcriptome,the full-length cDNA sequence of HcCUBDC gene was cloned from Hyriopsis cumingii by using rapid amplification of cDNA ends（ RACE） approaches. The entire HcCUBDC cDNA was 5 158 bp,including a 1 902 bp open reading frame（ ORF） encoding a polypeptide of 639 amino acids,a 576 bp 5＇ UTR,and a 2 622 bp 3＇ UTR（ Gen Bank accession number is KP067952）. Bioinformatics analysis showed that the HcCUBDC gene had a signal peptide of 19 amino acids,and four CUB domains,but shared no identity with known proteins. Real-time Q-PCR revealed that HcCUBDC gene was ubiquitously expressed in all tissues,including anterior mantle pallial（ a M P）,posterior mantle pallial（ p M P）,mantle center（ M）,gill（ G）,foot（ F）,hepatopancreas（ H）,intestine（ I） and kidney（ K）,with the lowest level of transcripts in H. In the purple mussels,the expression level of p M P was significantly higher compared with a M P. In the white mussels,there was no difference between a M P and p M P. HcCUBDC gene expression level of the p M P in purple mussels was significantly higher than that of white mussels,and there was no difference about the mRNA levels of a M P between the purple and white mussels. In situ hybridization showed that HcCUBDC mRNA was mainly expressed in outer epithelial cells at the mantle pallial. The results showed that HcCUBDC gene played important roles in the inner- shell color formation of H. cumingii,and may provide useful information for further studies on function and regulation mechanism of HcCUBDC gene in the color formation of the pearls.