光棘球海胆的主要卵黄蛋白cDNA序列分析

Analysis of cDNA encoding major yolk protein(MYP) in sea urchin Strongylocentrotus nudus

提取成熟光棘球海胆(Strongylocentrotus nudus)性腺中的RNA做模板,根据NCBI数据库中已知海胆(编号:AB097218、AB192414、AY090112)主要卵黄蛋白MYP cDNA保守序列设计引物,用LA PCR方式分段扩增并测序得到了光棘球海胆MYP cDNA的全序列。扩增的cDNA全长4 061 bp,包含4 047 bp的开放阅读框,共编码1 349个氨基酸。用CluxtalX1.83对光棘球海胆与其他几种已知海胆MYP cDNA及推导的氨基酸序列进行比对,用Mega3.01计算遗传距离及构建进化树,结果表明,光棘球海胆与其他7种海胆的MYP具有高度的同源性。从氨基酸水平上看,光棘球海胆与红海胆(Pseudocentrotus depressus)亲缘关系最近,遗传距离为0.069±0.01;与同科的马粪海胆(Hemicentrotus pulcher-rimus)、紫球海胆(S.purpuratus)、中间球海胆(S.intermedius)的遗传距离分别是0.095±0.012、0.098±0.011和0.101±0.012;与白棘三列海胆(Tripneustes gratilla)、拟球海胆(Paracentrotus lividus)及绿海胆(Lytechinus variega-tus)亲缘关系相对较远,遗传距离分别是0.216±0.017、0.218±0.017和0.535±0.028。获得光棘球海胆MYP

Major yolk protein （MYP） is found in both sexes of sea urchins where it is presumed to play a physiological role in gametogenesis, embryogenesis, or both. Some studies demonstrate that MYP is a transferrin-like, iron binding protein and one of MYP＇s transferrin-like properties is to shuttle iron to developing germ cells. Although the precise functions of MYP are not clear, MYP is necessary for the reproduction of sea urchins. To learn the cDNA sequence encoding MYP of sea urchin is very important for the advanced research on the function and phylogenesis of MYP gene. The overall sequence of cDNA encoding MYP of Strongylocentrotus nudus was determined by amplifying and sequencing. The templet RNA was extracted from gonad of mature sea urchin, and the primers were designed based on the known cDNA of other sea urchins （AB097218,AB192414,AY090112）on NCBI database. The strategy of amplification is Long and Accurate （LA） PCR. Two overlapped fragments covering the full-length cDNA were amplified and sequenced. The full-length cDNA sequence was obtained by joining the two fragments together. The full-length cDNA amplified is 4 067 bp and the nucleotide sequence contains an open reading frame （ORF） of 4 047 bp encoding 1 349 amino acids. The obtained nucleotide sequence and also the deduced amino acids were compared with nucleotide and amino acids sequence of other species of sea urchins accumulated on the NCBI database （AY090112, AB097218, AB192414 AF318300, AY274929, AY026514, AY090113） using CluxtalX1.83. Genetic distances were computed, and phylogenetic trees were constructed using Mega3.01. The results indicate that the nucleotide and deduced amino acids sequences of MYP cDNA in Strongylocentrotus nudus show high homology with other seven species of sea urchins. The relationship between Strongylocentrotus nudus and Pseudocentrotus depressus is close at the level of amino acids, and the genetic distance is 0.069±0.01. The genetic distances between Strongylocentrotus nudus and other three species of sea urchins （Hemicentrotus pulcherrimus , Strongylocentrotus purpuratus , Strongylocentrotus intermedius） in the same family are 0.095±0.012, 0.098±0.011 and 0.101±0.012, respectively. The relationships between Strongylocentrotus nudus and Tripneustes gratilla, Paracentrotus lividus, Lytechinus variegates are correspondingly far,and the genetic distances are 0.216±0.017, 0.218±0.017 and 0. 535±0.028, respectively. The MYP cDNA sequence obtained of Strongylocentrotus nudus provides the bases for advanced research on the function and phylogenesis of MYP gene.