三角帆蚌酪氨酸酶相关基因克隆及对珍珠层颜色影响研究

发布时间：2018-06-13 04:20

本文选题：三角帆蚌 + 外套膜　；参考：《上海海洋大学》2017年硕士论文

【摘要】：三角帆蚌是我国最主要的淡水育珠贝,珍珠颜色对珍珠价值有重要影响,研究表明珍珠颜色主要受供片蚌贝壳珍珠层颜色影响。酪氨酸酶参与了黑色素形成,与贝壳珍珠层颜色有关。本研究从分泌白色与紫色珍珠质三角帆蚌外套膜和珍珠囊比较转录组中筛选出与三角帆蚌珍珠层颜色相关的酪氨酸酶基因,以白色与紫色家系三角帆蚌为实验材料,探究酪氨酸酶与三角帆蚌珍珠层颜色之间的关系。1.三角帆蚌Hctyr基因的克隆与表达分析克隆得到三角帆蚌酪氨酸酶基因HcTyr,全长2315bp,开放阅读框(ORF)长1989bp,编码662个氨基酸。HcTyr基因包含一个酪氨酸酶结构域,无信号肽,GenBank登录号为KX447816。荧光定量结果发现,HcTyr基因主要在三角帆蚌外套膜中表达,其他组织中几乎不表达。白色蚌中,在外套膜前端缘膜、中央膜以及后端缘膜的表达量几乎处于同一水平。紫色蚌中,从前端缘膜、中央膜到后端缘膜表达量逐渐上升。紫色蚌后端缘膜中基因表达量显著高于白色蚌(p0.05)。原位杂交结果表明,HcTyr基因在外套膜的背膜上皮细胞中有明显的阳性杂交信号。对三角帆蚌外套膜中酪氨酸酶的活性进行检测发现,紫色三角帆蚌外套膜中酪氨酸酶活性要显著高于白色蚌。2.三角帆蚌HcTyp-1基因克隆与表达分析从三角帆蚌中克隆获得一个酪氨酸酶类似蛋白HcTyp-1,基因全长3590bp,编码了778个氨基酸。它包含了一个酪氨酸酶结构域与一个几丁质结合域,氨基酸序列包含一个由20个氨基酸残基构成的信号肽,GenBank登录号为KX447817。根据荧光定量结果显示,HcTyp-1基因主要在三角帆蚌外套膜中表达,其他组织中几乎不表达。在白色三角帆蚌中HcTyp-1在前端缘膜以及中央膜中表达量最高,后端缘膜表达量相对较低;在紫色的三角帆蚌中HcTyp-1基因在前端缘膜表达量较高,后端缘膜表达量较低。并且白色蚌的后端缘膜以及中央膜的表达量要显著高于紫色蚌(p0.05)。根据外套膜的原位杂交结果表明,HcTyp-1基因在三角帆蚌背膜处的上皮细胞检测到明显杂交信号,同时在外套膜中褶的上皮细胞中也检测到一些杂交信号。3.三角帆蚌HcTyp-1基因SNP筛选及与珍珠层颜色性状关联性分析根据酪氨酸酶类似蛋白HcTyp-1基因cDNA设计引物,比较筛选获得了8个SNP位点。研究这些多态性位点与贝壳珍珠层颜色性状相关性发现,C+3057T位点基因型仅在a参数上存在显著差异(P0.05),G+2985T和T+3006C两个位点基因型分别在L、b及a、dE上存在显著差异(P0.05),A+2834C、C+2919T和G+2986T这3个SNPs位点基因型在L、a及dE上均存在显著差异(P0.05)。连锁不平衡结果显示,HcTyp-1基因上除C+2912T、C+2983A这两个其它6位点具有差异显著位点之间都存在强连锁不平衡。单倍型分析发现,单倍型T2和T4在白色蚌中出现的频率极显著高于在紫色蚌中出现的频率,T6和T8两种单倍型在紫色群体中出现的频率极显著高于白色群体。HcTyp-1基因的部分SNPs可作为三角帆蚌抗不同内壳色贝壳辅助育种的候选分子标记。
[Abstract]:Hyriopsis cumingii is the most important freshwater pearly shellfish in China. The color of Pearl has an important influence on the value of pearl. The study shows that the color of pearl is mainly influenced by the color of the shell of the shell of mussel. Tyrosinase is involved in the formation of melanin and the color of the shell pearl. The tyrosinase gene related to the pearl layer color of Hyriopsis cumingii was screened in the PSA comparative transcriptional group. The relationship between the color of the tyrosinase and the pearl layer of Hyriopsis cumingii was studied with the white and purple clam. The cloning and expression of the Hctyr gene of Hyriopsis cumingii.1. was cloned and cloned to get the tyrosinase gene H of Hyriopsis cumingii CTyr, full length 2315bp, open reading frame (ORF) long 1989bp, encoding 662 amino acid.HcTyr gene contains a tyrosinase domain, no signal peptide, GenBank login number is KX447816. fluorescence quantitative results found, HcTyr gene is mainly expressed in the mantle of Hyriopsis cumingii, almost non expression in his tissue. White mussel, in the front-end edge of mantle membrane The expression of the membrane, the central membrane and the posterior marginal membrane was almost at the same level. In the purple mussel, the expression of the membrane in the central membrane and the posterior marginal membrane increased gradually. The gene expression in the posterior marginal membrane of the purple mussel was significantly higher than that of the white mussel (P0.05). The results of in situ hybridization showed that the HcTyr gene was obviously positive in the outer membrane epithelial cells of the mantle. The activity of tyrosinase in the mantle of Hyriopsis cumingii was detected. It was found that the tyrosinase activity in the mantle of the cuminga cumingii was significantly higher than that of the white clam.2., HcTyp-1 gene cloning and expression analysis. A tyrosinase similar protein HcTyp-1 was cloned from clam cumingii, and the full length of 3590bp was encoded. It contains 778 amino acids. It contains a tyrosinase domain and a chitin binding domain. The amino acid sequence contains a signal peptide composed of 20 amino acid residues. The GenBank login number is KX447817. based on the fluorescence quantitative results, and the HcTyp-1 gene is mainly expressed in the mantle of Hyriopsis cumingii and almost non expression in other tissues. In the Hyriopsis cumingii, the expression of HcTyp-1 in the front-end membrane and the central membrane is the highest, and the expression of the back edge membrane is relatively low. In the purple cumingii, the expression of HcTyp-1 gene in the front-end membrane is higher and the expression of the posterior marginal membrane is low, and the expression of the posterior marginal membrane and the central membrane of the white mussel is significantly higher than that of the purple mussel (p0.0 5). According to the results of in situ hybridization of the mantle, the HcTyp-1 gene was detected in the epithelial cells of the dorsal membrane of Hyriopsis cumingii. At the same time, some hybrid signals were detected in the pleated epithelial cells of the mantle,.3., HcTyp-1 gene SNP and the correlation analysis of the color traits of the nacre based on the tyrosinase class. 8 SNP loci were obtained by designing primers like protein HcTyp-1 gene cDNA. The correlation of these polymorphic loci with color traits of shell nacre showed that the genotype of C+3057T locus was only significant difference in a parameters (P0.05), and there were significant differences between G+2985T and T+3006C in L, B and a, respectively. There were significant differences between the 3 SNPs loci genotypes of 2834C, C+2919T and G+2986T in L, a and dE (P0.05). The linkage disequilibrium results showed that there were strong linkage disequilibrium between the HcTyp-1 genes except C+2912T, C+2983A, and the two other 6 loci. Haplotype T2 and the frequency of the occurrence in white mussels was found by haplotype analysis. The rate was significantly higher than that in the purple clam. The frequency of the two haplotypes of the two haplotypes of T6 and T8 in the purple population was significantly higher than that of the white group.HcTyp-1 gene, which could be used as a candidate marker for the resistance of Hyriopsis cumingii to the different shell color shell assisted breeding.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S917.4

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