马氏珠母贝育珠和生长性状与相关基因的关联分析 - 图文(2)

差；全组织荧光定量显示，四个基因在各组织均表达，血淋巴中表达量低。TβR I和EGFR在鳃和足中表达量最高，而GHITM和FGF18在性腺中表达量显著高于其他组织（P < 0.05）；

关键词：马氏珠母贝，育珠性状，生长性状，功能基因，关联分析

II

Correlation Analysis of Pearl Production and Growth Traits with the Related Genes from Pinctada martensii

Abstract

In this study, we evaluated the differences in pearl production and growth traits of the fifth-generation selected lines (F5), the control stocks and the common cultured stocks, calculated the correlation between pearl production and growth traits of the three stocks and related genes, then cloned and analyzed the four growth genes. The results are summarized as follows:

1. Cultured for 210 days after nucleus-inserting operation,compared pearl production traits of the three groups: (1) Compared to the control stocks and the common cultured stocks, survival rate of F5 increased by 8.79% and 7.61%, respectively. (2) Compared to the control stocks and the common cultured stocks, nuclear retention rate of F5 increased by 47.94% and 46.06%, respectively, significant difference (P < 0.05). (3) Compared to the control stocks and the common cultured stocks, weight of pearl of F5 increased by 22.02% and 18.75%, respectively, the difference was not significant; Compared to the control stocks and the cultured stocks, pearl thickness of F5 increased by 30.24% and 24.28%, respectively, significant difference (P < 0.05);

2. Cultured for 210 days after nucleus-inserting operation. Correlation analysis showed that the correlation between NACREIN, MSI60, PIF177 and pearl traits (pearl weight and pearl thickness) were positive, with correlation coefficients ranging from 0.408 to 0.979. The correlation coefficients between N19 and pearl traits were negative, correlation coefficients of N19 and pearl thickness and weight of pearl were -0.214 and -0.270, respectively.

3. Four growth related genes, transforming growth factor receptor type I (TβR I), epidermal growth factor receptor (EGFR), growth hormone-induced transmembrane protein (GHITM) and fibroblast growth factors (FGF18) were selected in the experiment. Correlation coefficients between each of the four genes and growth traits of P. martensii were calculated. The results showed a positive correlation between eight growth traits of P. martensii and the four genes, with correlation coefficients ranging from 0.387 to 0.998. The correlation coefficient of TβR I and shell length was significant (r = 0.998, P <0.05). 4. Full-length cDNA of TβR I was 2210 bp, ORF of 1569 bp, encoding 522 amino acids, precursor protein includes a signal peptide, extracellular domain, transmembrane domain and the intracellular domain. Extracellular domain contained an activin type I receptor, intracellular domain includes GS domain (189-215 aa) and serine/threonine kinase domain

III

(217-508 aa). Full-length cDNA of EGFR was 4934 bp, ORF of 4269 bp, encoding 1422 amino acids. The protein consisted of the extracellular domain of the N-terminal, a transmembrane domain and the intracellular domain of the C-terminal. Extracellular domain was a ligand binding site, transmembrane domain includes 22 amino acid residucing, the receptor anchored in the cell membrane, intracellular domain contains a tyrosine kinase domain (872-1128 aa). Full-length cDNA of GHITM was 1468 bp, ORF of 1017 bp, encoding 338 amino acids, there was a region same with Bax1-I family. Full-length cDNA of FGF18 was 2534 bp, ORF of 714 bp, encoding 237 amino acids, protein precursors had an N-terminal signal peptide structure. The mature protein consisted of 213 amino acid residues, co-existence of conserved sequences at the 45-178 aa with FGF family. Amino acid sequence homology compared found TβR I, GHITM and EGFR was conserved, homology in the mollusks and vertebrates was high. FGF18 had relatively poor conservation. RT-PCR detected the gene expression in the tissues of P. martensii. Four genes were expressed in various tissues and expression in hemocytes was the lowest. TβR I and EGFR expression were highest in gill and foot, while GHITM and FGF18 expression in gonad was significantly higher than in the other tissues (P < 0.05).

Key Words: Pinctada martensii, pearl production traits, growth traits, functional genes, correlation analysi

IV

目 录

摘 要 .................................................................................................................................... I Abstract .............................................................................................................................. III 1前言 .................................................................................................................................... 1 1.1马氏珠母贝的研究现状 .............................................................................................. 1 1.1.1马氏珠母贝的概述 ............................................................................................... 1 1.1.2马氏珠母贝珍珠质矿化的研究 ........................................................................... 1 1.1.3马氏珠母贝生长的研究 ....................................................................................... 3 1.2研究的目的和意义 ...................................................................................................... 5 2育珠性状与珍珠质矿化相关基因表达量的相关分析 .................................................... 6 2.1实验材料 ...................................................................................................................... 6 2.2实验方法 ...................................................................................................................... 6 2.2.1马氏珠母贝三个群体育珠性状的统计分析 ....................................................... 6 2.2.2马氏珠母贝三个群体珍珠质矿化相关基因的荧光定量 ................................... 6 2.2.3 育珠性状与珍珠质矿化相关基因的相关分析 .................................................. 9 2.3实验结果 ...................................................................................................................... 9 2.3.1马氏珠母贝三个群体育珠性能的比较 ............................................................... 9 2.3.2 珍珠质矿化相关基因表达的定量结果 ............................................................ 10 2.3.3 珍珠层厚度与珍珠质矿化相关基因的相关性 ................................................ 10 2.4 讨论 ........................................................................................................................... 11 3生长性状与生长相关基因表达量的相关分析 .............................................................. 13 3.1实验材料 .................................................................................................................... 13 3.2实验方法 .................................................................................................................... 13 3.2.1马氏珠母贝三个群体生长性状的跟踪测量 ..................................................... 13 3.2.2马氏珠母贝三个群体生长相关基因的荧光定量 ............................................. 13 3.2.3 生长性状与相关基因表达量的相关分析 ........................................................ 14 3.3实验结果 .................................................................................................................... 14 3.3.1马氏珠母贝三个群体生长的比较 ..................................................................... 14 3.3.2 生长相关基因的荧光定量结果 ........................................................................ 15 3.3.3 生长性状与生长相关基因表达量的相关性 .................................................... 16 3.4 讨论 ........................................................................................................................... 16 4马氏珠母贝生长相关基因的克隆和组织表达研究 ...................................................... 18 4.1实验材料 .................................................................................................................... 18 4.1.1实验动物和菌种 ................................................................................................. 18 4.1.2主要仪器设备 ..................................................................................................... 18 4.1.3主要试剂 ............................................................................................................. 19 4.1.4主要的溶液和培养基及配制方法 ..................................................................... 20 4.2实验方法 .................................................................................................................... 20 4.2.1引物的设计 ......................................................................................................... 20 4.2.3 RNA的抽提 ........................................................................................................ 21

4.2.4 cDNA 末端的快速扩增(RACE)获取基因的全长 ............................................ 21 4.2.5 序列的拼接及生物信息学分析 ........................................................................ 24 4.2.6荧光定量PCR ..................................................................................................... 24 4.3实验结果 .................................................................................................................... 25 4.3.1 生长相关基因的克隆结果 ................................................................................ 25 4.3.2 生长相关基因的序列分析 ................................................................................ 26 4.3.3 序列的同源性及进化分析 ................................................................................ 36 4.3.4 生长相关基因在各组织中的表达 .................................................................... 43 4.4 讨论 ........................................................................................................................... 46 4.4.1 生长相关基因的序列特征 ................................................................................ 46 4.4.2 生长相关基因的组织表达分析 ........................................................................ 48 5结论 .................................................................................................................................. 50 参考文献 ............................................................................................................................. 51 致 谢 ............................................................................................................................. 59 导师简介 ............................................................................................................................. 60

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