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Volume 9 Issue 11
Nov.  2021
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Mutation in SRBI gene and its association with the red shell color in Meretrix meretrix

  • 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 Ho was 0.309 4−0.442 0, He 0.468 8−0.494 6, and Ne 1.863 4−1.973 4, while in the white shell strain Ho was 0.231 9−0.328 5, He 0.324 4−0.500 0, and Ne 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.
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  • [1] Cook L M. Reflections on molluscan shell polymorphisms[J]. Biological Journal of the Linnean Society, 2017, 121(4): 717-730. doi: 10.1093/biolinnean/blx033
    [2] 惠伯棣. 类胡萝卜素化学及生物化学[M]. 北京: 中国轻工业出版社, 2005.Hui B D. Carotenoid chemistry and biochemistry[M]. Beijing: China Light Industry Press, 2005 (in Chinese).
    [3] de Carvalho C C C R, Caramujo M J. Carotenoids in aquatic ecosystems and aquaculture: a colorful business with implications for human health[J]. Frontiers in Marine Science, 2017, 4: 93.
    [4] Bergamonti L, Bersani D, Mantovan S, et al. Micro-Raman investigation of pigments and carbonate phases in corals and molluscan shells[J]. European Journal of Mineralogy, 2014, 25(5): 845-853. doi: 10.1127/0935-1221/2013/0025-2318
    [5] Stemmer K, Nehrke G. The distribution of polyenes in the shell of Arctica islandica from North Atlantic localities: a confocal Raman microscopy study[J]. Journal of Molluscan Studies, 2014, 80(4): 365-370. doi: 10.1093/mollus/eyu033
    [6] Hedegaard C, Bardeau J F, Chateigner D. Molluscan shell pigments: an in situ resonance raman study[J]. Journal of Molluscan Studies, 2006, 72(2): 157-162. doi: 10.1093/mollus/eyi062
    [7] 詹艳玲. 文蛤(Meretrix meretrix)壳色的鉴定及其相关基因和microRNA研究[D]. 宁波: 浙江万里学院, 2015.Zhan Y L. Identification of shell color and the related gene and microRNA research of Meretrix meretrix[D]. Ningbo: Zhejiang Wanli University, 2015 (in Chinese).
    [8] Shete V, Quadro L. Mammalian metabolism of β-carotene: gaps in knowledge[J]. Nutrients, 2013, 5(12): 4849-4868. doi: 10.3390/nu5124849
    [9] Quadro L, Giordano E, Costabile B K, et al. Interplay between β-carotene and lipoprotein metabolism at the maternal-fetal barrier[J]. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2020, 1865(11): 158591. doi: 10.1016/j.bbalip.2019.158591
    [10] 赵羽卒, 张奎, 唐梅, 等. 家蚕B类清道夫受体BmSCRB8基因的克隆及表达[J]. 生物工程学报, 2016, 32(10): 1408-1421.Zhao Y Z, Zhang K, Tang M, et al. Cloning and expression of scavenger receptor class B BmSCRB8 in silkworm Bombyx mori[J]. Chinese Journal of Biotechnology, 2016, 32(10): 1408-1421 (in Chinese).
    [11] Dong Z P, Chai C L, Dai F Y, et al. Expression pattern and tissue localization of the class B scavenger receptor BmSCRBQ4 in Bombyx mori[J]. Insect Science, 2015, 22(6): 739-747. doi: 10.1111/1744-7917.12158
    [12] Sakudoh T, Kuwazaki S, Iizuka T, et al. CD36 homolog divergence is responsible for the selectivity of carotenoid species migration to the silk gland of the silkworm Bombyx mori[J]. Journal of Lipid Research, 2013, 54(2): 482-495. doi: 10.1194/jlr.M032771
    [13] Altmann S W, Davis Jr H R, Yao X R, et al. The identification of intestinal scavenger receptor class B, type I (SR-BI) by expression cloning and its role in cholesterol absorption[J]. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2002, 1580(1): 77-93. doi: 10.1016/S1388-1981(01)00190-1
    [14] van Bennekum A, Werder M, Thuahnai S T., et al Class B scavenger receptor-mediated intestinal absorption of dietary β-carotene and cholesterol[J]. Biochemistry, 2005, 44(11): 4517-4525. doi: 10.1021/bi0484320
    [15] 李西雷, 李卿青, 任名栋, 等. 三角帆蚌hcSRCR1基因的克隆及在不同壳色选育系中的表达模式[J]. 水产学报, 2018, 42(11): 1719-1728.Li X L, Li Q Q, Ren M D, et al. Cloning and tissue expression of a novel hcSRCR1 gene in differential inner-shell color pearl mussel Hyriopsis cumingii[J]. Journal of Fisheries of China, 2018, 42(11): 1719-1728 (in Chinese).
    [16] Lei C, Hao R J, Zheng Z, et al. Molecular cloning and characterisation of scavenger receptor class B in pearl oyster Pinctada fuctada martensii[J]. Electronic Journal of Biotechnology, 2017, 30: 12-17. doi: 10.1016/j.ejbt.2017.08.003
    [17] 任晓亮. 虾夷扇贝(Patinopecten yessoensis)闭壳肌积累类胡萝卜素相关基因的筛查[D]. 青岛: 中国海洋大学, 2011.Ren X L. Identification of genes relating to carotenoids accumulation in adductor muscles of Yesso scallops (Patinopecten yessoensis)[D]. Qingdao: Ocean University of China, 2011 (in Chinese).
    [18] Liu H L, Zheng H P, Zhang H K, et al. A de novo transcriptome of the noble scallop, Chlamys nobilis, focusing on mining transcripts for carotenoid-based coloration[J]. BMC Genomics, 2015, 16(1): 44. doi: 10.1186/s12864-015-1241-x
    [19] 庄启谦. 中国动物志: 软体动物门 双壳纲 帘蛤科[M]. 北京: 科学出版社, 2001.Zhuang Q Q. Fauna Sinica: Molluscs Lamellibranchia Veneridae[M]. Beijing: Science Press, 2001 (in Chinese).
    [20] 林志华, 董迎辉. 文蛤“万里红”[J]. 中国水产, 2015(10): 50-52. doi: 10.3969/j.issn.1002-6681.2015.10.026Lin Z H, Dong Y H. "Wanlihong" hard clam (Meretrix meretrix)[J]. China Fisheries, 2015(10): 50-52 (in Chinese). doi: 10.3969/j.issn.1002-6681.2015.10.026
    [21] 崔宝月, 董迎辉, 赵家熙, 等. 文蛤SRBI基因克隆及其在不同壳色群体中的差异表达[J]. 水生生物学报, 2018, 42(3): 488-493. doi: 10.7541/2018.061Cui B Y, Dong Y H, Zhao J X, et al. Cloning and expression analysis of SRBI gene in different shell-color strains of Meretrix meretrix[J]. Acta Hydrobiologica Sinica, 2018, 42(3): 488-493 (in Chinese). doi: 10.7541/2018.061
    [22] Botstein D, White R L, Skolnick M, et al. Construction of a genetic linkage map in man using restriction fragment length polymorphisms[J]. American Journal of Human Genetics, 1980, 32(3): 314-331.
    [23] During A, Hussain M M, Morel D W, et al. Carotenoid uptake and secretion by CaCo-2 cells: β-carotene isomer selectivity and carotenoid interactions[J]. Journal of Lipid Research, 2002, 43(7): 1086-1095. doi: 10.1194/jlr.M200068-JLR200
    [24] During A, Harrison E H. Mechanisms of provitamin A (carotenoid) and vitamin A (retinol) transport into and out of intestinal Caco-2 cells[J]. Journal of Lipid Research, 2007, 48(10): 2283-2294. doi: 10.1194/jlr.M700263-JLR200
    [25] Harrison E H. Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids[J]. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2012, 1821(1): 70-77. doi: 10.1016/j.bbalip.2011.06.002
    [26] During A, Dawson H D, Harrison E H. Carotenoid transport is decreased and expression of the lipid transporters SR-BI, NPC1L1, and ABCA1 is downregulated in Caco-2 cells treated with ezetimibe[J]. The Journal of Nutrition, 2005, 135(10): 2305-2312. doi: 10.1093/jn/135.10.2305
    [27] Shyam R, Vachali P, Gorusupudi A, et al. All three human scavenger receptor class B proteins can bind and transport all three macular xanthophyll carotenoids[J]. Archives of Biochemistry and Biophysics, 2017, 634: 21-28. doi: 10.1016/j.abb.2017.09.013
    [28] Wang J L, Li Q, Zhong X X, et al. An integrated genetic map based on EST-SNPs and QTL analysis of shell color traits in Pacific oyster Crassostrea gigas[J]. Aquaculture, 2018, 492: 226-236. doi: 10.1016/j.aquaculture.2018.04.018
    [29] Rasal K D, Chakrapani V, Pandey A K, et al. Status and future perspectives of single nucleotide polymorphisms (SNPs) markers in farmed fishes: way ahead using next generation sequencing[J]. Gene Reports, 2017, 6: 81-86. doi: 10.1016/j.genrep.2016.12.004
    [30] Zanoni P, Khetarpal S A, Larach D B, et al. Rare variant in scavenger receptor BI raises HDL cholesterol and increases risk of coronary heart disease[J]. Science, 2016, 351(6278): 1166-1171. doi: 10.1126/science.aad3517
    [31] Goodarzynejad H, Boroumand M, Behmanesh M, et al. The rs5888 single nucleotide polymorphism in scavenger receptor class B type 1 (SCARB1) gene and the risk of premature coronary artery disease: a case-control study[J]. Lipids in Health and Disease, 2016, 15: 7. doi: 10.1186/s12944-016-0176-9
    [32] Zeng T T, Tang D J, Ye Y X, et al. Influence of SCARB1 gene SNPs on serum lipid levels and susceptibility to coronary heart disease and cerebral infarction in a Chinese population[J]. Gene, 2017, 626: 319-325. doi: 10.1016/j.gene.2017.05.020
    [33] Wang H B. Relationship between SR-BI genetic polymorphism and coronary heart disease and blood lipid level[J]. International Journal of Clinical and Experimental Medicine, 2016, 9(10): 19886-19892.
    [34] Borel P, Lietz G, Goncalves A, et al. CD36 and SR-BI are involved in cellular uptake of provitamin A carotenoids by CaCo-2 and HEK Cells, and some of their genetic variants are associated with plasma concentrations of these micronutrients in humans[J]. The Journal of Nutrition, 2013, 143(4): 448-456. doi: 10.3945/jn.112.172734
    [35] Toomey M B, Lopes R J, Araújo P M, et al. High-density lipoprotein receptor SCARB1 is required for carotenoid coloration in birds[J]. Proceedings of the National Academy of Sciences of the United States of America, 2017, 114(20): 5219-5224. doi: 10.1073/pnas.1700751114
    [36] Wei W, Zheng C L, Zhu M, et al. Missense mutations near the N-glycosylation site of the A2 domain lead to various intracellular trafficking defects in coagulation factor VIII[J]. Scientific Reports, 2017, 7: 45033. doi: 10.1038/srep45033
    [37] Liu Y, Ren S F, Xie L Q, et al. Mutation of N-linked glycosylation at Asn548 in CD133 decreases its ability to promote hepatoma cell growth[J]. Oncotarget, 2015, 6(24): 20650-20660. doi: 10.18632/oncotarget.4115
    [38] Zwart M P, Schenk M F, Hwang S, et al. Unraveling the causes of adaptive benefits of synonymous mutations in TEM-1 β-lactamase[J]. Heredity, 2018, 121(5): 406-421. doi: 10.1038/s41437-018-0104-z
    [39] Lugos M D, Davou G I, Choji T P P, et al. Using immunohistochemistry without linkers to determine the optimum concentrations of primary antibodies for immunofluorescence staining of formalin-fixed paraffin-embedded tissue sections[J]. Applied Immunohistochemistry & Molecular Morphology, 2020, 28(3): 249-257.
    [40] Tsuchida K, Sakudoh T. Recent progress in molecular genetic studies on the carotenoid transport system using cocoon-color mutants of the silkworm[J]. Archives of Biochemistry and Biophysics, 2015, 572: 151-157. doi: 10.1016/j.abb.2014.12.029
    [41] Hansen H G, Niels-Christiansen L L, Immerdal L, et al. Scavenger receptor class B type I (SR-BI) in pig enterocytes: Trafficking from the brush border to lipid droplets during fat absorption[J]. Gut, 2003, 52(10): 1424-1431. doi: 10.1136/gut.52.10.1424
    [42] Miquel J F, Moreno M, Amigo L, et al. Expression and regulation of scavenger receptor class B type I (SR-BI) in gall bladder epithelium[J]. Gut, 2003, 52(7): 1017-1024. doi: 10.1136/gut.52.7.1017
    [43] Cardoso R M, Creemers E, Absalah S, et al. Hyperalphalipoproteinemic scavenger receptor BI knockout mice exhibit a disrupted epidermal lipid barrier[J]. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids, 2020, 1865(3): 158592. doi: 10.1016/j.bbalip.2019.158592
    [44] Acton S, Rigotti A, Landschulz K T, et al. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor[J]. Science, 1996, 271(5248): 518-520. doi: 10.1126/science.271.5248.518
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Mutation in SRBI gene and its association with the red shell color in Meretrix meretrix

    Corresponding author: DONG Yinghui, dongyinghui118@126.com
    Corresponding author: LIN Zhihua, zhihua9988@126.com
  • 1. Key Laboratory of Aquatic Germplasm Resources of Zhejiang, College of Biology and Environment Sciences, Zhejiang Wanli University, Ningbo    315100, China
  • 2. Ninghai Marine Biological Seed Industry Research Institute, Zhejiang Wanli University, Ningbo    315100, China

Abstract: 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 Ho was 0.309 4−0.442 0, He 0.468 8−0.494 6, and Ne 1.863 4−1.973 4, while in the white shell strain Ho was 0.231 9−0.328 5, He 0.324 4−0.500 0, and Ne 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.

  • 贝类壳色变异是由内部遗传到与生物化学、生活基质或营养有关等多种因素组合而产生的[1]。类胡萝卜素是一种最常见的生物色素,能显现出黄色到红色范围内的颜色[2]。贝类通过摄取富含类胡萝卜素的食物,从中积累类胡萝卜素[3]。目前已经确定多种贝类的贝壳中存在类胡萝卜素[4-7],贝类需要通过体内受体吸收脂蛋白中含有的类胡萝卜素[8]。清道夫受体B类I (scavenger receptor class B, member 1,SRBI)作为一种多配体受体,能够结合多种脂蛋白,从而促进动物体内类胡萝卜素的吸收及转运,是类胡萝卜素代谢途径中的关键基因[9]。迄今已在家蚕(Bombyx mori)[10-12]、鼠[13-14]等模式动物中克隆得到SRB家族基因,并证明其与体内类胡萝卜素代谢有关。在贝类中,已克隆得到三角帆蚌(Hyriopsis comingii)[15]、马氏珠母贝(Pinctada fucata martensii)[16]SRB基因,并推测不同组织中类胡萝卜素的含量与该基因表达高低有关。任晓亮[17]对虾夷扇贝(Patinopecten yessoensis)类胡萝卜素代谢相关基因进行了筛查和分析,发现不同闭壳肌颜色群体中的SRB基因表达量存在显著差异;Liu等[18]干扰华贵栉孔扇贝(Chlamys nobilis) SRB-like-3基因表达后,发现血液中类胡萝卜素含量显著降低,为SRB基因与扇贝中类胡萝卜素累积有关提供了证据。

    文蛤(Meretrix meretrix)是我国四大养殖贝类之一,具有较高的经济价值和营养价值[19]。文蛤壳色变异丰富且可稳定遗传给后代,本课题组已培育出枣红壳色的文蛤“万里红”品种[20],并发现文蛤红、白壳色的差异主要是由于贝壳中类胡萝卜素含量不同引起的[7]。崔宝月等[21]克隆得到Mm-SRBI全长,并检测发现红壳文蛤外套膜中Mm-SRBI基因表达量显著高于白壳文蛤。然而,目前对Mm-SRBI基因在红壳文蛤中的变异及其蛋白表达差异与红壳色形成的关联性等研究尚未见报道。

    本研究以红、白壳色文蛤为对象,采用直接测序法筛选分析Mm-SRBI基因中与壳色关联的单核苷酸多态性(single nucleotide polymorphism, SNP)位点,为分子标记辅助选育提供基础;用免疫荧光(immunofluorescence, IF)和蛋白免疫印迹(Western blotting, WB)技术对Mm-SRBI蛋白在文蛤外套膜中的表达特性进行分析,探究Mm-SRBI表达蛋白差异与壳色变异间的关系,为深入解析文蛤体内类胡萝卜素代谢过程提供理论基础。

    • 红壳文蛤为“万里红”新品种,白壳文蛤为同属江苏群体的白壳品系,二者均取自宁波海洋与渔业科技创新养殖基地。选取红壳和白壳文蛤各210粒,取外套膜组织于液氮中速冻,保存于−80 °C超低温冰箱中,用于后续SNP筛选实验;另取红壳、白壳文蛤各5粒,取新鲜外套膜组织于4%多聚甲醛中固定过夜,用于免疫荧光实验。

    • 根据本实验室克隆得到的Mm-SRBI基因(GenBank登录号:KY434116)序列,使用Primer Premier 5软件设计其编码序列(coding sequence, CDS)区的特异引物(表1)。用Trizol法提取红壳、白壳文蛤外套膜组织RNA,依照TaKaRa公司PrimeScriptTM RT试剂盒说明书反转录合成cDNA,PCR扩增后用1.0%琼脂糖凝胶电泳检测PCR产物,将扩增结果为单一条带的PCR产物送测(杭州擎科生物)。利用ContigExpress软件对测序结果进行拼接,基于序列比对来查找SNP位点,使用Mutation Surveyor软件查看测序图谱来进行人工校对以及统计突变位点,用SPSS 22软件对与壳色性状关联的SNP位点基因型进行相关性分析。利用POPgen 32软件计算两种壳色文蛤群体Mm-SRBI基因的SNP位点信息,利用在线分析网站(http://analysis.bio-x.cn/myAnalysis.php)分析突变位点间的连锁不平衡(LD)。

      引物名称
      primer
      引物信息(5′→3′)
      sequence (5′→3′)
      SRBI-F1 CTTTGGAATACTAAAACAGCGTGG
      SRBI-R1 TAGCCCATTCAGTGCTCCAG
      SRBI-F2 CCTTTGATGACACGCCCTT
      SRBI-R2 ATTGTCACCATGACTTGTTACCTG

      Table 1.  Primer sets used for Mm-SRBI CDS sequence amplification

    • 取4%多聚甲醛固定过的红壳、白壳文蛤外套膜边缘组织,经梯度乙醇脱水,二甲苯/正丁醇透明,石蜡包埋切片(厚度为4 μm)。将烤片后的组织切片,经二甲苯脱蜡,梯度乙醇复水,PBS冲洗,Tris-EDTA抗原修复,用5% BSA封闭液室温封闭1 h,甩干滴加Mm-SRBI一抗(1∶200),切片平放于4 °C孵育湿盒中过夜;PBST冲洗3×10 min,PBS冲洗2×10 min,在暗室中滴加FITC荧光标记的鼠抗兔IgG二抗(1∶150,含DAPI),室温避光孵育1 h;将玻片置于PBS中洗涤3×10 min;在Nikon 80i荧光显微镜下观察拍照。

    • 取红壳、白壳文蛤新鲜外套膜组织,加入适量RIPA裂解液,匀浆;利用BCA法测蛋白质浓度;配置合适浓度的SDS-PAGE凝胶,等量上样。蛋白胶经切割后转至PVDF膜上,用5%脱脂奶粉封闭液室温孵育1 h,加入封闭液稀释的一抗(1∶100)孵育袋中4 °C过夜;TBST漂洗3×10 min,加入经生物素HRP标记的鼠抗兔二抗(1∶2000),室温孵育1 h;TBST漂洗3×10 min;暗室中配置增强化学发光法(enhanced chemiluminescent, ECL)发光底物混合液,滴加至PVDF膜上,Bio-Rad凝胶成像仪拍照;利用Image J软件对红壳、白壳文蛤外套膜中SRBI表达蛋白的WB结果进行灰度值计算;用SPSS 22软件对红、白壳色文蛤的蛋白印迹灰度值进行t检验,分析其显著性差异。

    2.   结果
    • Mm-SRBI基因编码区进行扩增,成功测序了181粒红壳文蛤、207粒白壳文蛤,序列比对共发现15个SNP位点。SNP位点的位置命名以Mm-SRBI基因的mRNA全长序列第1位碱基距离目的SNP位点的碱基数设定,对15个SNP位点突变的个体基因型、基因型频率及等位基因频率与壳色关联性进行分析,结果显示在红、白壳色群体中,c.647C/T、c.723A/G、c.818C/T、c.1037A/G共4个位点与壳色存在显著关联(P<0.05)(表2)。对位点突变导致的氨基酸多态性进行分析,其中c.723A/G位点为非同义突变所在位点编码的异亮氨酸(Ile)突变为缬氨酸(Val),其余位点为同义突变,所在位点编码的氨基酸未发生变化。

      位点
      locus
      基因型
      genotype
      数量(基因型频率/%)
      number (genotype frequency)
      等位基因
      allele
      等位基因频率/%
      allele frequency
      χ2 (P值)
      χ2 (P -value)
      红壳
      red shell
      白壳
      white shell
      红壳
      red shell
      白壳
      white shell
      c.647 C/T CC 77(25.97) 68(32.85) C 58.01 47.58 6.201
      CT 56(30.94) 61(29.47) T 41.99 52.42 (0.045)*
      TT 48(26.52) 78(37.68)
      c.723A/G AA 75(41.44) 128(61.84) A 63.54 73.43 20.626
      AG 80(44.20) 48(23.19) G 36.46 26.57 (0.000033)*
      GG 26(14.36) 31(14.98)
      c.818C/T CC 67(37.02) 57(27.54) C 56.91 43.96 12.966
      CT 72(39.78) 68(32.85) T 43.09 56.04 (0.002)*
      TT 42(23.20) 82(39.61)
      c.1037A/G AA 64(35.36) 46(22.22) A 55.80 37.68 12.598
      AG 74(40.88) 64(30.92) G 44.20 62.32 (0.002)*
      GG 43(23.76) 97(46.86)
      注:*为χ2检验结果显示P<0.05
      Notes: * represents P<0.05 of χ2 test results

      Table 2.  Genotype and gene frequency of SNPs sites in Mm-SRBI gene of two shell color M. meretrix

      对15个SNP位点突变类型进行统计分析,发现Mm-SRBI基因在突变类型上只存在转换和颠换两种变异类型,其中转换的频率大于颠换的频率,二者比例约为3∶1(表3)。

      变异类型
      variation type
      基因型
      genotype
      位点
      sites
      总数
      total number
      转换
      transition
      GA 723、944、1037、1043、
      1190、1316
      6
      CT 503、647、653、
      818、974
      5
      颠换
      transversion
      TA 1049 1
      AC 992 1
      GT 1061 1
      CG 1091 1

      Table 3.  Variation type and number of genotypes in two shell color M. meretrix SRBI gene

    • Mm-SRBI基因CDS区SNP位点进行分析,得到与壳色显著关联的4个SNP位点在红壳、白壳群体中的遗传参数(表4)。依照Botstein等[22]划分原则,位点的PIC值在0.25~0.5为中度多态,PIC<0.25为低度多态。统计发现,与壳色相关联的4个突变位点都属于中度多态性位点(0.5>PIC>0.25)。在红壳文蛤群体中,观测杂合度为0.309 4~0.442 0,期望杂合度为0.468 8~0.494 6,有效等位基因数为1.863 4~1.973 4;白壳文蛤群体中,观测杂合度为0.231 9~0.328 5,期望杂合度为0.324 4~0.500 0,有效等位基因数为1.639 9~1.995 3。

      群体   
      strain   
      位点   
      locus   
      观测杂合度
      Ho
      期望杂合度
      He
      有效等位基因数
      Ne
      多态性信息含量
      PIC
      红壳文蛤
      red shell M. meretrix
      c.647C/T 0.3094 0.4885 1.9499 0.3685
      c.723A/G 0.4420 0.4688 1.8634 0.3560
      c.818C/T 0.3978 0.4918 1.9626 0.3702
      c.1037A/G 0.4088 0.4946 1.9734 0.3716
      白壳文蛤
      white shell M. meretrix
      c.647C/T 0.2947 0.5000 1.9953 0.3744
      c.723A/G 0.2319 0.4939 1.6399 0.3141
      c.818C/T 0.3285 0.3244 1.9712 0.3713
      c.1037A/G 0.3092 0.4708 1.8855 0.3594

      Table 4.  Polymorphic parameters of SNP loci of Mm-SRBI gene in two shell color M. meretrix strains

      Mm-SRBI基因突变位点间的连锁不平衡分析显示,在红壳、白壳文蛤群体内与壳色关联的突变位点之间都存在强连锁不平衡(D′>0.75),表明这些位点存在一定程度上的连锁遗传现象 (表5)。

      位点
      loci
      c.647C/Tc.723A/Gc.818C/Tc.1037A/G
      c.647C/T 0.973 (1) 0.918 (1) 0.940 (1)
      c.723A/G 0.750 (0.328) 1 (1) 1 (1)
      c.818C/T 0.814 (0.864) 0.766 (0.284) 1 (1)
      c.1037A/G 0.807 (0.666) 0.725 (0.219) 0.945 (0.771)
      注:对角线上方为D′,对角线下方为R2,括号外为红壳文蛤群体位点信息,括号内为白壳文蛤群体位点信息
      Notes: the figure above the diagonal represents D′, the figure below the diagonal represents R2; outside the parentheses is the linkage disequilibrium analysis of red shell color M. meretrix strain, inside the parentheses is white shell color M. meretrix strain

      Table 5.  Linkage disequilibrium analysis of four SNP loci of Mm-SRBI gene in two shell color M. meretrix strains

    • 免疫荧光结果显示,Mm-SRBI 蛋白在文蛤外套膜各部位均有表达(FITC绿色荧光信号),但在红壳中的表达量明显高于白壳文蛤(图1),在外套膜组织中,其边缘膜处的荧光信号显著高于其他部位。DAPI染色显示,边缘膜组织纤毛处含有大量排列整齐的单层柱状细胞,其余分布少量的肌纤维。合并图显示,绿色荧光与蓝色荧光未重合,表明Mm-SRBI蛋白为核外蛋白,在外套膜组织中,边缘膜为分泌Mm-SRBI蛋白的主要部位。

      Figure 1.  Distribution of SRBI in mantle of two shell color M. meretrix

    • WB结果显示,在预测Mm-SRBI蛋白分子量大小为56.82 ku处出现目的条带,而Mm-SRBI在红壳文蛤外套膜组织中的表达量明显高于白壳文蛤(图2-a)。WB结果的灰度值分析显示,红壳文蛤灰度值平均为5.9×105,白壳文蛤为1.3×105,二者存在极显著差异(P<0.01)(图2-b)。

      Figure 2.  Analysis of Mm-SRBI protein and the result of Western blotting in two shell color M. meretrix

    3.   讨论
    • 类胡萝卜素所呈现出的特征颜色在非光合组织和生物体中起着色素的作用。早期研究表明,在肠道上皮组织中SRBI转运蛋白负责α-胡萝卜素、β-胡萝卜素、叶黄素、β-隐黄素等类胡萝卜素的吸收[23-24],并将类胡萝卜素转运到相应的组织细胞中[25]。SRBI蛋白的差异表达影响着生物体内类胡萝卜素的含量,在类胡萝卜素水平较低的组织中,SRBI蛋白表达下调[26]。另外,有学者揭示了灵长类动物体内循环中的类胡萝卜素富集于视网膜中的机制,推测SRBI蛋白的高表达是导致该区域呈现黄斑的原因[27]。在贝类中,已有研究证明SRB基因与类胡萝卜素含量有关[15-18],而在本研究中,红壳文蛤外套膜内高表达的Mm-SRBI基因可能与高水平的类胡萝卜素相关,并影响着贝壳着色过程。

      编码区突变在调控和编码蛋白质中起着重要作用,从而影响表型性状。在分子水平上,单核苷酸多态性(SNP)标记作为第三代分子标记,因其位点丰富、遗传稳定、易于分析等优点,被广泛用于遗传作图和性状连锁分析[28-29]。在高等动物中已有不少针对SRBI基因多态性与个体性状的关联性分析,如在小鼠和人中,SRBI基因多态性会造成体内脂蛋白和甘油酸酯的水平异常,从而显现出不同群体脂类代谢的差异[30-33],不同基因型个体也与血液中的类胡萝卜素含量相关联[34];在鸟类中,SRBI基因突变会破坏类胡萝卜素的转运功能,导致羽毛着色发生变化[35]。对本实验结果的15个SNP位点进行信息注释,发现在突变类型上只存在转换和颠换两种类型,且CT之间的转换频率大于AG,可能是因为胞嘧啶(C)常发生甲基化,自发脱氨形成胸腺嘧啶(T)。经序列比对发现,与壳色显著关联的4个SNP位点都位于Mm-SRBI基因保守序列上,并且非同义突变位点c.723A/G位于糖基化位点上。糖基化是重要的蛋白修饰之一,糖基化位点突变会使蛋白的功能、理化性质发生改变,如凝血因子Ⅷ基因上的N-糖基化位点突变,导致蛋白的转运功能发生变化[36];膜糖蛋白CD133上的糖基化位点突变降低了其与受体的相互作用[37]。因此推测,Mm-SRBI基因上c.723A/G位点多态性可能使表达蛋白活性或与脂蛋白结合能力发生变化,从而导致血液和外周组织类胡萝卜素代谢水平的差异;其余3个位点虽为同义突变,未导致编码的氨基酸发生变化,但也有研究证明同义突变可影响mRNA的剪接、稳定性和结构以及tRNA的翻译效率,从而影响蛋白质合成的功能[38],但要证明这些位点突变与红、白文蛤类胡萝卜素代谢差异相关还有待进一步研究。

      免疫荧光和蛋白免疫印迹技术将蛋白进行可视化,对目标蛋白进行检测和评价,已然成为蛋白表达特性的有效研究方法[39]。在分子水平上,已验证Mm-SRBI基因在红壳文蛤外套膜组织中的表达量高于白壳文蛤,且存在显著差异[21]。在蛋白水平上,对家蚕[40]、猪[41]、人[42]等SRBI蛋白的定位分析中发现,该蛋白表达位置位于细胞膜上。本实验结果表明,Mm-SRBI蛋白在文蛤外套膜中均有表达,但主要表达在文蛤外套膜边缘膜纤毛上皮细胞膜上,且红壳文蛤的蛋白表达量显著高于白壳文蛤。WB结果显示在预期Mm-SRBI蛋白分子量大小处出现条带,灰度值比较发现,Mm-SRBI蛋白在红壳外套膜中的表达量约为白壳的4.4倍,与IF结果基本一致。在小鼠中已有研究发现,SRBI蛋白的缺失会导致小鼠体内相关脂类的表达下调[43],功能上SRBI蛋白介导高密度脂蛋白对胆固醇酯的选择性摄取,并对细胞内类胡萝卜素的代谢物进行转运,将类胡萝卜素及代谢产物运输至相应的组织中[44]。因此,推测红壳、白壳文蛤中Mm-SRBI蛋白表达存在差异可能与其类胡萝卜素代谢水平相关联。红壳文蛤相较于白壳文蛤对类胡萝卜素代谢更为旺盛,外套膜作为文蛤贝壳的生壳组织,高表达的SRBI蛋白促进其对类胡萝卜素吸收及转运,进而形成红、白文蛤在壳色外观上的差异。

      综上所述,本实验在文蛤体内类胡萝卜素代谢相关的关键基因Mm-SRBI中筛查到4个与壳色相关联的SNP位点,并对Mm-SRBI蛋白在外套膜边缘组织中进行细胞定位和表达分析,研究结果可为今后红壳色文蛤分子辅助育种提供候选标记,为深入解析文蛤类胡萝卜素代谢途径及与红壳色形成的机制研究提供理论基础。

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