马氏珠母贝TTLL基因的序列特征、SNP筛选及其与生长性状的关联分析  

Sequence characteristics, SNP screening and association with growth traits of TTLL gene in pearl oyster Pinctada fucata martensii

作　　者：尹诗欣 李志屿 杨晶淼 廖永山 杨创业[1,3] 邓岳文 王庆恒[1,3] YIN Shixin;LI Zhiyu;YANG Jingmiao;LIAO Yongshan;YANG Chuangye;DENG Yuewen;WANG Qingheng(College of Fisheries,Guangdong Ocean University,Zhanjiang 524088,China;Pearl Research Institute,Guangdong Ocean University,Zhanjiang524088,China;Guangdong Technology Research Center for Pearl Aquacultureand Process,Zhanjiang 524088,China)

机构地区：[1]广东海洋大学水产学院,广东湛江524088 [2]广东海洋大学珍珠研究所,广东湛江524088 [3]广东省珍珠养殖与加工工程技术研究中心,广东湛江524088

出　　处：《大连海洋大学学报》2022年第6期949-959,共11页Journal of Dalian Ocean University

基　　金：广东省教育厅科研项目(2020ZDZX1045);北海市科技计划项目(2020165010);广东省农业农村厅科研项目(粤财农[2020]4号)。

摘　　要：为了探究微管蛋白酪氨酸连接酶样(tubulin tyrosine ligase-like, TTLL)在马氏珠母贝Pinctada fucata martensii中的作用,对TTLL基因的序列特征、表达模式及其SNP位点进行了相关分析。结果表明:采用RACE技术克隆获得马氏珠母贝TTLL基因cDNA全长为1 962 bp,其中,开放阅读框(ORF)为1 611 bp,编码536个氨基酸,5′UTR长134 bp, 3′UTR长191 bp;TTLL蛋白靠近N端处具有一个TTL结构域;多序列比对及系统进化树分析显示,马氏珠母贝与其他物种的TTLL氨基酸序列具有较高的同源性,并与长牡蛎Crassostrea gigas等双壳贝类聚为一支;qRT-PCR分析显示,TTLL基因在马氏珠母贝外套膜、闭壳肌、性腺、肝胰腺、鳃和足中均有表达,在性腺中表达量最高(P<0.05),在生长性状较好的大规格群体和杂交家系中高表达(P<0.05),在卵和受精卵中的表达量也显著高于其他发育时期(P<0.05);马氏珠母贝TTLL基因共有277个SNP位点,其中,109个SNP位点符合哈迪-温伯格平衡,6个位点为中度多态,103个位点为低度多态;对基因外显子SNPs单倍型分析发现,4个SNP位点形成了2个单倍块、5种单倍型,单倍型GG为优异单倍型,其生长性状显著高于单倍型AA;对基因突变位点分析发现,7个SNP位点发生了突变,包括4个错义突变和3个同义突变,其中,位点g.7572350 T>G和g.7572368 A>G与壳宽显著性关联(P<0.05),位点g.7572405 C>T与壳长、壳高显著性关联(P<0.05),而其他突变位点对生长性状无显著性关联(P>0.05)。研究表明,TTLL基因参与马氏珠母贝的生长发育过程,且其优异单倍型GG及突变位点g.7572350 T>G、g.7572368 A>G和g.7572405 C>T均有利于马氏珠母贝的生长。In order to explore the function of tubulin tyrosine ligase-like(TTLL) in pearl oyster Pinctada fucata martensii, the sequence characteristics, expression patterns and SNP screening of the TTLL gene were analyzed in the pearl oyster. The results showed that the full-length sequence of TTLL cDNA of the pearl oyster cloned by RACE technology was 1 962 bp, including 1 611 bp of open reading frame(ORF) encoding 536 amino acids, 134 bp of 5′UTR and 191 bp of 3′UTR. It is predicted that there was a TTL domain in the N-terminal of TTLL protein sequence in the pearl oyster. Multi-sequence alignments and the construction of phylogenetic tree analysis of its amino acid sequences showed that the TTLL had high homology with TTLL of other species, and was clustered with other bivalves like Pacific oyster Crassostrea gigas. qRT-PCR results revealed that TTLL was ubiquitously expressed in mantle, adductor muscle, gonads, hepatopancreas, gills and feet of the pearl oyster, with the maximal expression level in gonads(P<0.05). The TTLL was highly expressed in large-scale populations and hybrid families with better growth traits(P<0.05). There was significantly higher expression level of TTLL gene in eggs and fertilized eggs than that in other developmental stages(P<0.05). SNPs analysis of the TTLL gene revealed that 277 SNP were obtained, in which 109 SNP sites were in accordance with Hardy-Weinberg equilibrium, 6 SNP sites were moderately polymorphic and 103 SNP sites low-level polymorphisms. Haplotype analysis of TTLL gene of the pearl oyster exon region SNPs showed that 4 SNP sites formed 2 haploblocks and 5 haplotypes, in which the haplotype GG was an excellent haplotype, with significantly better growth characteristics than that in the haplotype AA. Analysis of TTLL gene mutation sites revealed that 7 SNP sites were mutated, 4 missense mutations and 3 synonymous mutations, in which locus g.7572350 T>G and locus g.7572368 A>G were significantly correlated with shell width(P<0.05), g.7572405 C>T was significantly correlated

关 键 词：马氏珠母贝 微管蛋白酪氨酸连接酶样(TTLL) 序列特征 表达模式 SNPS 生长性状 

分 类 号：S917.4[农业科学—水产科学]