瓯江彩鲤体色相关基因Sox10的分离与表达分析

Isolation and expression analysis of Sox10 gene relating body color variation in Oujiang color common carp

为探明鱼类体色变异的相关调控因子，采用RT-PCR及RACE技术获得总长度为2830bp的瓯江彩鲤（Cyprinus carpio var．color）SoxlO基因eDNA序列。该基因序列的5’和3’非编码区分别为9bp和1375bp，开放阅读框1446bp，编码481个氨基酸。氨基酸序列的系统发育分析表明，瓯江彩鲤Soxl0基因的氨基酸序列与部分物种的相似性在59％～94％。RT-PCR分析表明，该基因在瓯江彩鲤皮肤、肌肉、眼睛和鳔表达量最高，鳃和心表达量次之，肾和肝没有表达。荧光定量PCR分析显示，Soxl0基因在体色类型为白色（“粉玉”体色）的表达量显著地高于其他体色（P〈0．05），“粉花”黑色皮肤中的表达量最低，显著低其他体色类型（P〈O．05），而其他体色类型的表达差异不显著俨〉0．05）。结果发现，SoxlO基因在瓯江彩鲤的体色变异中发挥着调控作用。

The numerous and diverse body color of fish was not only excellent materials to study the molecular mechanism of body color and body color inherence, body color evolution, and it was also an ideal model to diagnosis disease. Now, five basic and stable body color patterns have existed in Oujiang color common carp （Cyprinus carpio var. color）, namely Quanhong, Dahua, Mahua, Fenyu and Fenhua, respectively. This fish can be a very good model and material for studying body color inheritance. At the melanin synthesis signaling pathway, skin color was regulated by a small group genes. SoxlO gene plays an important role in the formation of melanin, it regulates the birth, migration and differentiation of melanin. However, the effect of SoxlO gene on body color has not been reported. Four healthy lines of Oujiang color carp （Quanhong, Dahua, Fenyu, Fenhua） were collected from the provincial farm of the Zhejiang. Then the skin, muscle, eye, gill, kidney, swim bladder, heart, and liver were taken out, furthermore, the black skin and red skin were sampled separately of Dahua, the same with black skin and white skin of fenhua. Total RNA was extracted using Trizol, and reverse transcribed into the First-strand cDNA. Primers were designed by Primer5 program, and the right products were purified and sequenced. The obtained sequence was analyzed by Bioedit, Clustalw, Mega and other bioinformatics softwares. The total length ofSoxlO gene cloned in Oujiang color common carp is 2 830 bp which contains 9 bp 5＇ untranslated regions （UTR） and 1 375 bp 3＇-UTR, and 1 446 bp open reading frame （ORF） encoding 481 amino acids. The phylogenetic analyses showed that there were 59%-94% similarities in amino acid sequence with some of previously reported other species. RT-PCR analysis revealed that the gene was highly expressed in skin, muscle, eyes and swim bladder, weakly expressed in gill and heart and no expression were found in kidney and liver. Real-time PCR showed that the expression of the SoxlO gene in white skin （Whole white body color） was significantly higher than other color patterns （P〈0.05）, and the significantly lowest expression in black skin of Fenhua color pattern （P〈0.05）, but there were no significant difference in expression level among the other color patterns （P〉 0.05）. The results primarily indicated that the SoxlO gene is important in regulating body color variation in Oujiang color common carp. The information that obtained provides an important basis for conducting future studies on body color, and regulatory mechanisms in melanin synthesis signaling pathway, and provides an important theoretical basis for further research in body color selection during selective breeding in fish.