砂梨扩展蛋白基因cDNA克隆及全序列分析

Cloning and sequencing of expansion gene from Pyrus pyrifolia

设计植物EXP扩展蛋白简并引物,以砂梨果实cDNA为模板,克隆得到EXP基因cDNA片段,该片段长465 bp。根据该片段序列,分别设计2条5’和3’末端扩增的特异引物,利用RACE技术,获得了该片段的5’端和3’端序列。用DNAMAN5.22软件对3个序列进行拼接和分析,获得了该基因的cDNA全长,命名为Pyp-EXP。该cDNA全长为1 395 bp,5’端起始密码子ATG始于72 bp,3’端终止子TAG止于830 bp,Ploy+(A)从1 363～1 395 bp。该基因已在GenBank基因数据库注册,注册号为EF602031。Pyp-EXP核苷酸序列有一个759 bp完整的开放阅读框,编码区与西洋梨、苹果、湖北海棠、桃的同源性分别为96%,96%,94%和86%;该cDNA推导编码252个氨基酸,含有1个组氨酸(His_Phe_Asp,HFD)功能域,与西洋梨、苹果、湖北海棠、桃中相应序列同源性分别为98%,97%,95%和93%。该基因的克隆为研究扩展蛋白的时空表达及其在果实发育和成熟过程中的作用奠定了基础。

Degenerate oligonueleotide primers were designed on the basis of conserved domain of other expansions, and a partial fragment of 465 bp, was cloned from eDNA library of ripening fruit of Pyrus pyrifolia Nakai. According to the eDNA fragment sequence, the 5＇ end and 3＇ end fragment sequences were obtained RACE technology respectively. The full length of eDNA of expansion gene, designated as P）p-EXP, was 1 395 bp by DNAMAN5.22 from above three sequences. The 5＇ end included a putative translation start codon （ATG） at position 72 bp, the 3 ＇end included an end codon （TAG） at position 830 bp and a Ploy＋（A） tail at position fi：om 1 363 bp to 1 395 bp （the accession number in GenBank database is EF602031）. The Pyp-EXP gene had an open reading frame （ORF） of 759 nucleotides. Its blasing shows that it is 96%, 96%, 94% and 86% identical with the sequences of EXP genes in Pyrus communis, Malus xdomestica, Prunus persica, Malus hupehensis. Its code for a polypeptide of 252 amino acids, and it contained histidine function region. Its biasing shows that it is 98%, 97%, 95% and 93% identical with the sequences of EXP proteins in Pyrus communis, Malusxdomestica, Prunus persica, Malus hupehensis. Coning of Pyp -EXP will contribute to study the space and time expression and the function during fruit development and ripening of EXP genes.