缩骨鲫(Carassius auratus sogu var.)MC4R克隆、组织分布以及与生长相关SNPs的筛选

黑色素皮质素受体-4(Melanocortin reporter-4,MC4R)是G蛋白偶联受体超家族成员,在能量调控中发挥重要作用。本实验采用同源克隆以及染色体步移对缩骨鲫(Carassius auratus sogu var.)MC4R基因进行克隆,对其核苷酸序列和推测的氨基酸序列进行生物信息学分析,通过Real-time PCR对其m RNA组织分布进行研究,并对缩骨鲫MC4R编码区潜在的与生长相关的SNPs进行筛选。本实验克隆MC4R基因组基因长度为2854bp,只包含一个外显子,长度为981bp,共编码326个氨基酸,MC4R共有七个跨膜结构,是较为稳定的疏水蛋白。缩骨鲫MC4R与鲫鱼的MC4R氨基酸序列的同源度最高达到99.3%,与MC3R和MC5R同源度相对其他MCRs成员较高。缩骨鲫MC4R m RNA在脑中表达量最为显著,眼中次之,肌肉,卵巢和肾脏中表达较少,在鳃、肠、心脏和肝胰腺中微量表达。在MC4R的CDs区内检测到6个与生长潜在相关的SNPs:两个错义突变(A199G和A881C),4个同义突变(A123G,C231T,A444G和C480T)。本实验可为缩骨鲫生长和发育的分子机制研究以及遗传育种提供基础资料。

CLONE,EXPRESSION,AND SCREENING OF GROWTH ASSOCIATED SNPS OF CARASSIUS AURATUS SOGU VAR.

Melanocortin reporter-4 (MC4R) belongs to G-protein-coupled receptors family; it plays an important role in energy regulation. By homologous cloning and chromosome walking, MC4R gene of Carassius auratus sogu var. was cloned, and the expression of MC4R gene were studied based on real-time PCR. Meanwhile, SNPs (single nucleotide polymorphisms) of the MC4R in CDs region were detected by Sanger sequences. The result show that the length of MC4R genome gene was 2854bp that contain one 981-bp exon, the CDs code 326 amino acid. The MC4R contained 7 transmembrane structure and was a stable hydrophobin. Through sequence alignment of MC4Rs from different species and other members of MCRs, we found that the C. auratus is the closest species to C. auratus sogu var. The homology ratio is 99.3%, the MC4R was far from other MCRs members. The real-time PCR result shows that MC4R mRNA was most expressed in brain, and then in eyes; and less expressed in muscle, ovary, and kidney. Weak MC4R mRNA was expressed in gill, intestine, heart and hepatopancreas. In the codon sequence, six SNPs were detected:two missense mutations, and four silent mutations. The results shall be insightful to understand the growth of C. auratus sogu var., and offer fundamental data for molecular markers of C. auratus sogu var.