本文選題：三角帆蚌 + 外套膜��； 參考：《上海海洋大學》2017年碩士論文

【摘要】：三角帆蚌是我國最主要的淡水育珠貝,珍珠顏色對珍珠價值有重要影響,研究表明珍珠顏色主要受供片蚌貝殼珍珠層顏色影響。酪氨酸酶參與了黑色素形成,與貝殼珍珠層顏色有關。本研究從分泌白色與紫色珍珠質三角帆蚌外套膜和珍珠囊比較轉錄組中篩選出與三角帆蚌珍珠層顏色相關的酪氨酸酶基因,以白色與紫色家系三角帆蚌為實驗材料,探究酪氨酸酶與三角帆蚌珍珠層顏色之間的關系。1.三角帆蚌Hctyr基因的克隆與表達分析克隆得到三角帆蚌酪氨酸酶基因HcTyr,全長2315bp,開放閱讀框(ORF)長1989bp,編碼662個氨基酸。HcTyr基因包含一個酪氨酸酶結構域,無信號肽,GenBank登錄號為KX447816。熒光定量結果發(fā)現(xiàn),HcTyr基因主要在三角帆蚌外套膜中表達,其他組織中幾乎不表達。白色蚌中,在外套膜前端緣膜、中央膜以及后端緣膜的表達量幾乎處于同一水平。紫色蚌中,從前端緣膜、中央膜到后端緣膜表達量逐漸上升。紫色蚌后端緣膜中基因表達量顯著高于白色蚌(p0.05)。原位雜交結果表明,HcTyr基因在外套膜的背膜上皮細胞中有明顯的陽性雜交信號。對三角帆蚌外套膜中酪氨酸酶的活性進行檢測發(fā)現(xiàn),紫色三角帆蚌外套膜中酪氨酸酶活性要顯著高于白色蚌。2.三角帆蚌HcTyp-1基因克隆與表達分析從三角帆蚌中克隆獲得一個酪氨酸酶類似蛋白HcTyp-1,基因全長3590bp,編碼了778個氨基酸。它包含了一個酪氨酸酶結構域與一個幾丁質結合域,氨基酸序列包含一個由20個氨基酸殘基構成的信號肽,GenBank登錄號為KX447817。根據(jù)熒光定量結果顯示,HcTyp-1基因主要在三角帆蚌外套膜中表達,其他組織中幾乎不表達。在白色三角帆蚌中HcTyp-1在前端緣膜以及中央膜中表達量最高,后端緣膜表達量相對較低;在紫色的三角帆蚌中HcTyp-1基因在前端緣膜表達量較高,后端緣膜表達量較低。并且白色蚌的后端緣膜以及中央膜的表達量要顯著高于紫色蚌(p0.05)。根據(jù)外套膜的原位雜交結果表明,HcTyp-1基因在三角帆蚌背膜處的上皮細胞檢測到明顯雜交信號,同時在外套膜中褶的上皮細胞中也檢測到一些雜交信號。3.三角帆蚌HcTyp-1基因SNP篩選及與珍珠層顏色性狀關聯(lián)性分析根據(jù)酪氨酸酶類似蛋白HcTyp-1基因cDNA設計引物,比較篩選獲得了8個SNP位點。研究這些多態(tài)性位點與貝殼珍珠層顏色性狀相關性發(fā)現(xiàn),C+3057T位點基因型僅在a參數(shù)上存在顯著差異(P0.05),G+2985T和T+3006C兩個位點基因型分別在L、b及a、dE上存在顯著差異(P0.05),A+2834C、C+2919T和G+2986T這3個SNPs位點基因型在L、a及dE上均存在顯著差異(P0.05)。連鎖不平衡結果顯示,HcTyp-1基因上除C+2912T、C+2983A這兩個其它6位點具有差異顯著位點之間都存在強連鎖不平衡。單倍型分析發(fā)現(xiàn),單倍型T2和T4在白色蚌中出現(xiàn)的頻率極顯著高于在紫色蚌中出現(xiàn)的頻率,T6和T8兩種單倍型在紫色群體中出現(xiàn)的頻率極顯著高于白色群體。HcTyp-1基因的部分SNPs可作為三角帆蚌抗不同內(nèi)殼色貝殼輔助育種的候選分子標記。
[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.
【學位授予單位】：上海海洋大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S917.4

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