三角帆蚌Hc-GSK3β基因鉴定及内壳色性状相关SNP筛选

IDENTIFICATION OF HC-GSK3βGENE AND SCREENING OF SNPS RELATED TO INNER SHELL COLOR OF HYRIOPSIS CUMINGII

为了探究三角帆蚌(Hyriopsis cumingii)糖原合成激酶-3β(GSK3β)基因对壳色的影响,研究采用RACE技术获得Hc-GSK3β基因cDNA全长1867 bp,其中包含1261 bp的ORF区编码420个氨基酸,ORF中含有一个S_TKc结构域,该结构域序列高度保守。组织差异表达分析发现Hc-GSK3β基因在紫色蚌鳃、斧足、内脏团和边缘膜组织中表达量高于白色蚌的表达量(P<0.05),且在斧足和边缘膜表达差异水平达到极显著(P<0.01),而在紫色蚌闭壳肌组织中表达量显著低于白色蚌(P<0.05)。原位杂交(ISH)实验结果显示在三角帆蚌外套膜的外褶、中褶、内褶、背膜区和腹膜区均有阳性信号产生,且在外褶的信号表达较强烈。该基因经重测序比较,共鉴定出6个SNP位点,其中在C+185A位点的CA基因型在紫色蚌的分布频率显著高于白色三角帆蚌(P<0.05);在紫色蚌中,T+341G位点TT基因型三角帆蚌内壳颜色参数b值显著低于TG基因型(P<0.05)。研究表明,Hc-GSK3β基因参与了三角帆蚌壳色形成,筛选的SNP标记可用于三角帆蚌壳色育种。

Melanin is an important factor for the formation of purple shells and pearls of Hyriopsis cumingii.Glycogen synthesis kinase-3β(GSK3β)is a key gene of animal melanin synthesis pathway.In order to explore the effect of Hc-GSK3βgene on shell color of H.cumingii,the full-length cDNA of Hc-GSK3βgene of 1867 bp was successfully cloned by RACE technology,including the ORF region of 1261 bp encoding 420 amino acids.The ORF contained a S_TKc domain,whose sequence is highly conserved.Tissue expression(qRT-PCR)analysis showed that the expression of Hc-GSK3βgene in purple mussel was significantly higher than that of white mussel in gill,foot,hepatopancreas and fringe mantle tissues(P<0.05),and the expression existed extremely significant difference in foot and fringe mantle tissues(P<0.01).The expression in the adductor muscle tissue of purple mussel was significantly lower than that of white mussel(P<0.05).The results of in situ hybridization(ISH)showed that positive signals appeared in the outer fold,middle fold,inner fold,dorsal mantle area and ventral mantle area of H.cumingii,and the signal expression in the outer fold was stronger.6 SNPs locus were identified by re-sequencing and comparison.The distribution frequencyof genotype CA at C+185A locus was significantly higher in purple mussels than that of white mussels(P<0.05).At the T+341G locus,the value of color parameter b of the genotype TT was significantly lower than genotype TG(P<0.05).The study showed that Hc-GSK3βgene was involved in shell color formation of H.cumingii,and SNP markers could be used for the shell color breeding of H.cumingii.