【作者】 吴琪；

【导师】 张国范；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2010， 博士

【摘要】 牡蛎为世界性分布种类,是重要的经济贝类。结合素蛋白(bindin)是巨蛎属牡蛎精子顶体的主要成分,它与精子和卵子的受精识别作用有关。海藻糖凝集素结构域(Fucose binding lectin repeat, F-lectin repeat)是bindin蛋白的功能结构域,其中包括三个重要的保守的功能识别位点(H37,R64,R70)和四个保守的半胱氨酸位点(C27,C36,C88,C105)。本研究利用3’RACE、5’RACE的方法得到熊本牡蛎bindin蛋白cDNA全长1134bp,包括774 bp开放阅读框,共编码257个氨基酸,其中包括由24个氨基酸组成的信号肽序列。并扩增得到熊本牡蛎bindin蛋白DNA全长5353 bp,其中包括四个外显子和三个内含子。得到11个F-lectin repeat单倍型序列,单倍型多样性指数为0.846。熊本牡蛎的十一个单倍型之间dN/dS值在O.158-1.9275之间,与太平洋牡蛎比较后dN/dS值在0.9317-1.7175之间,均未检测出正选择压力。与长牡蛎的F-lectin repeat五个单倍型序列比较后发现5个受正向选择压力的位点,其中三个位点在外显子1上(40,66and 67; mean dN/dS= 7.59),两个在外显子2上(121 and 123; mean dN/dS= 7.41),从3D结构模型图上可以看到这五个位点都位于三个保守的氨基酸位点周围。葡萄牙牡蛎cDNA全长1049 bp,包括774 bp开放阅读框,共编码257个氨基酸序列,包括24个氨基酸组成的信号肽序列。扩增得到葡萄牙牡蛎bindin蛋白DNA全长8508 bp,其中包括四个外显子和三个内含子。对葡萄牙牡蛎F-lectin repeat内部的内含子测序,共发现3个F-lectin repeat单倍型,并且与这三个单倍型相伴的内含子(intron-4)根据序列长度也可分为三种型(typeⅠ, typeⅡ, typeⅢ)。将来自熊本牡蛎的11个F-lectin repeat单倍型序列,葡萄牙牡蛎的3个单倍型序列,长牡蛎的5个单倍型序列进行比较,计算F-lectin repeat的dN/dS值,结果表明在这三种牡蛎之间并没有检测出正向选择压力。经过比较三个种的功能结构域共发现五个受正向选择压力的位点,其中三个在F-lectin repeat外显子1上(40,66 and 67；dN/dS=6.848),两个在外显子2上(123 and 125;dN/dS=6.346),这五个位点都位于三个保守的识别基序(H37,R64,R70)周围,推测这五个位点可能与种特异的识别有关。结合素蛋白与其在卵子上的受体在受精识别过程中是协同进化的,这种配子识别蛋白之间的进化最终引起种的分化和新种形成。研究配子识别系统可以帮助我们更好的理解种形成和适应的复杂过程。更多还原

【Abstract】 Oysters are marine bivalve molluscs widely distributed in world oceans. Bindin is a major protein packaged as insoluble granules in sperm acrosomes. It is responsible for species-specific recognition and binding of sperm to a glycoprotein receptor on the surface of eggs. Fucose binding lectin repeat (F-lectin repeat) is the main function domain of bindin including three conservative recognition motif (H37, R64, R70) and four Cysteines (C27, C36, C88, C105). We cloned a novel bindin gene from the Kumamoto oyster Crassostrea sikamea by 3’and 5’RACE. The full-length cDNA of C. sikamea bindin was 1134 bp with a 774 bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA we obtained was 5353 bp containing 4 exons and 3 introns. Eleven haplotypes of fucose lectin (F-lectin) repeat were detected. Haplotype diversity was 0.846. The ratios of nonsynonymous (dN) and synonymous (dS) substitutions per nucleotide site between haplotyes were from 0.158 to 1.9275. And the dN/dS values were from 0.9317 to 1.7175 between two species. No positive selection has been found. Five positive selected positions (p>95%) were identified among sixteen haplotypes from C. sikamea and C. gigas. Three sites were in the F-lectin repeat exon-1 (position 40,66 and 67; dN/dS= 7.59), and two were in the F-lectin repeat exon-2 (position 121 and 123; dN/dS= 7.41). They all located on the F-lectin binding face around the three recognition motif residues (H37, R64 and R70) in the 3D model. The full-length bindin cDNA of C. angulata was 1049 bp with a 774 bp open reading frame encoding 257 amino acids. The deduced amino acid sequence contained a putative signal peptide of 24 amino acids. The length of the bindin genomic DNA was 8508 bp containing four exons and three introns. Three haplotypes of F-lectin repeat were detected from seven sequences of F-lectin repeat of six male oysters. Intron-4 in the middle of F-lectin repeat is highly variable in both size and sequence. We classified intron-4 into three types (typeⅠ, typeⅡ, typeⅢ) according to their size and the F-lectin repeat they were located in. We compared the number of nonsynonymous substitutions (dN) and synonymous substitutions (dS) per nucleotide site among 19 F-lectin haplotypes of the three species. No positive selection has been found between three species. Five positive selected positions (p>95%) are identified among nineteen haplotypes of three species. Three were in the F-lectin repeat exon-1 (position 40,66 and 67; dN/dS= 6.848), and two were in the F-lectin repeat exon-2 (position 123 and 125; dN/dS= 6.346). They are located on the F-lectin binding face around the three recognition motif residues. We assume that these five clustered amino acids are related with species specific recognition. Bindin and its cognate receptor must evolve together for recognition during fertilization. The rapid divergence of GRPs within species ultimately leads to incompatibility of sperm and egg between species. The gamete recognition systems could help us understand the complex speciation and adaptation.更多还原