长牡蛎Wnt5b基因的鉴定及在黑色素生成中的作用

Identification of the CgWnt5b gene and its function in melanin production in the Pacific oyster Crassostrea gigas

Wnt基因家族编码一类保守的分泌型糖蛋白,广泛参与细胞分化与增殖、细胞运动、凋亡、免疫调控以及色素生成等生物学过程。本研究鉴定了一个长牡蛎Wnt家族基因——CgWnt5b,比较了黑、白壳色长牡蛎中CgWnt5b转录水平的表达情况,并对其在调控黑色素生成方面的功能进行初步研究。研究结果显示,CgWnt5b编码序列全长1116bp,编码371个氨基酸。生物信息学分析预测CgWnt5b蛋白相对分子量为42KDa,等电点为9.28。对CgWnt5b与哺乳动物Wnt基因家族19个成员进行系统发育分析,确定了CgWnt5b为哺乳动物Wnt5a和Wnt5b的同源基因。荧光实时定量PCR检测长牡蛎外套膜、鳃、消化腺、闭壳肌、性腺、唇瓣以及血淋巴组织中CgWnt5b的mRNA分布,结果显示,CgWnt5b在外套膜、闭壳肌和唇瓣组织中的相对表达水平较高,在消化腺、鳃、血淋巴和性腺组织中相对表达量较低。黑壳长牡蛎中CgWnt5b mRNA表达量显著低于白壳长牡蛎,且siRNA敲降CgWnt5b的表达后,多个黑色素生成相关酪氨酸酶基因的表达显著上调。综上,本研究在长牡蛎中鉴定了一个Wnt家族成员CgWnt5b,可能通过抑制黑色素生成关键基因TYR的表达抑制长牡蛎中黑色素的生成。

The Wnt gene family encodes a class of conserved secretory glycoproteins that are widely involved in different biological processes,including cell differentiation and proliferation,cell motility,apoptosis,immune regulation,and pigmentation.In this study,we identified a Wnt family gene,CgWnt5b,and compared the expression of CgWnt5b in black and white shelled oysters in the Pacific oyster Crassostrea gigas.A preliminary study on the function of CgWnt5b in regulating melanogenesis was also conducted.Our results reveal that the CgWnt5b coding sequence is 1116 bp in length and encodes 371 amino acids.Bioinformatics analysis predicted a relative molecular weight of 42 KDa and an isoelectric point of 9.28 for the CgWnt5b protein. Phylogenetic analysis of CgWnt5b with 19 members of the mammalian Wnt gene family revealed thatCgWnt5b is a homolog of mammalian Wnt5a and Wnt5b. Quantitative real-time PCR was performed to detect the mRNAdistribution of CgWnt5b in the mantle, gill, digestive gland, adductor muscle, gonad, labial papls, and hemolymph tissuesof the oyster. The results reveal that CgWnt5b is highly expressed in mantle, adductor muscle and labial papls tissues, andhas a relatively low expression level in digestive gland, gills, hemolymph, and gonad tissues. The expression of CgWnt5b mRNAin black-shelled oyster was significantly lower than that in white-shelled oyster, and the expression of several melanin-relatedtyrosinase genes was significantly up-regulated after knockdown of CgWnt5b by RNA interference. In summary, this studyidentified a member of the Wnt family, CgWnt5b, which may inhibit melanogenesis in C. gigas by suppressing the expressionof TYRs, the key factors controlling melanin production.