为了解文蛤体内类胡萝卜素代谢相关基因清道夫受体B类I(Mm-SRBI)与红壳色形成的关联性，采用直接测序法对基因编码区进行SNP筛查和关联分析，并用免疫荧光、蛋白免疫印迹分析了Mm-SRBI蛋白在红、白壳色文蛤外套膜中的表达差异。结果显示，在Mm-SRBI基因的编码区筛选到15个SNP位点，位于保守序列上的4个突变位点c.647C/T、c.723A/G、c.818C/T、c.1037A/G与红、白壳色存在显著关联，其中c.723A/G位点为非同义突变(异亮氨酸突变为缬氨酸)，且4个位点在遗传上存在强连锁性；蛋白免疫印迹与免疫荧光结果表明，Mm-SRBI在红壳中的蛋白表达量显著高于白壳文蛤，主要表达部位为外套膜边缘膜的纤毛处。研究表明，Mm-SRBI基因的高表达与变异可能导致文蛤对类胡萝卜素的代谢产生差异，从而在生长发育过程中导致红壳色的形成。

The hard clam (Meretrix meretrix) is one of the most commercially important cultured shellfish in China. The red shell clam is richer in carotenoids, which makes its shell color red and has higher nutritional value compared with the white shell clam. In order to understand the mutation of scavenger receptor class B type I (SRBI) and its association with the formation of red shell color in M. meretrix, we analyzed the SNPs of Mm-SRBI gene CDS by using direct sequencing among 181 red shell clams and 207 white shell clams. Immunofluorescence (IF) and Western blot (WB) were used to analyze the expression characteristics of Mm-SRBI protein in the mantle of two shell color strains. The results showed that a total of 15 single nucleotide polymorphisms (SNPs) were detected, and there were only two types of mutation in Mm-SRBI: transition and transversion, and the ratio of the two was about 3∶1. Among the 15 loci, 4 loci were significantly associated with shell color. The statistical results indicated that in red shell strain the range of H_o was 0.309 4−0.442 0, H_e 0.468 8−0.494 6, and N_e 1.863 4−1.973 4, while in the white shell strain H_o was 0.231 9−0.328 5, H_e 0.324 4−0.500 0, and N_e 1.639 9−1.995 3. The PIC value evidenced that these loci were all moderately polymorphic (0.5>PIC>0.25). Additionally, all of them were in conserved sequences, in which c.723 A/G site was non-synonymous mutation that led to amino acid changes Ile723Val, and 4 loci were genetically strongly linked (D′>0.75). The results of IF and WB revealed that Mm-SRBI protein was higher expressed in the mantle of red-shelled clams compared to that of the white-shelled clams and was mainly expressed at the outer epithelium of the mantle. Moreover, the quantitative analysis of gray values demonstrated that Mm-SRBI protein expression in red-shell clams was approximately 4.5 times higher than that in white-shell clams. In summary, our results suggested that the mutations of Mm-SRBI and the high expression of its protein may lead to differences in the metabolism of carotenoids in M. meretrix, which may further lead to the formation of red shell color. It also provided a helpful basis to explore the molecular mechanisms of carotenoids metabolism underlying shell coloration and could be potentially applied to marker-assisted selection breeding programs for M. meretrix.

Q786；S917.4

国家自然科学基金(31772846)；国家重点研发计划(2018YFD0901404)；国家现代贝类产业技术体系专项(CARS-49)；浙江省生物工程一流学科(A类)自设课题(ZS2019001)项目