砂梨(Pyrus pyrifolia)过敏原蛋白基因(Ppmal)的克隆与表达分析

Cloning and expression analysis of major allergen(Ppmal) from Sand Pear(Pyrus pyrifolia Nakai)

【目的】分离和克隆梨过敏原蛋白基因Ppmal(Gen Bank登录号为KP008110),探讨其在梨果实发育过程中的功能。【方法】以砂梨品种‘翠冠’[Pyrus pyrifolia(Burm.f.)Nakai]为试材,在盛花期后30 d对植株果柄进行涂抹赤霉素处理,利用同源克隆和RACE方法克隆目的基因全长序列,并通过实时荧光定量技术和半定量技术分析该基因在梨不同组织以及梨果实发育过程中的表达变化。【结果】Ppmal的全长是906 bp,含有480 bp的开放阅读框(ORF),编码159个氨基酸,预测的蛋白质相对分子质量为17.56 k D,等电点为5.62。生物信息学分析显示该基因没有信号肽序列,没有跨膜结构域,具亲水性。与其他物种的过敏原蛋白基因核苷酸序列同源性超过75%,与苹果和西洋梨编码的氨基酸序列同源性分别高达97.48%和96.23%,进化树分析表明该基因与苹果的进化关系最近。同时,定量结果显示经过赤霉素处理后的果实中该基因的表达量随着果实的生长发育呈现先降低后升高的趋势,并且具有组织差异表达的特点,其表达量在叶片中最大,其次是嫩叶,再次是花,枝条最低。【结论】获得梨Ppmal基因全长,对该基因在砂梨品种‘翠冠’发育过程中的表达分析显示,Ppmal受到赤霉素的调控,并在砂梨果实膨大期发生上调表达。

【Objective】The aim of this study was to isolate a major allergen gene from sand pear（Ppmal,Gen Bank accession number： KP008110） and explore its role during the development of pear.【Methods】Under the treatment of gibberellin about one month after full flowering,Ppmal was cloned from‘Cui-guan＇by homologous cloning and RACE technology,and the expression patterns of the gene in differenttissues and the development of pear were studied using real-time fluorescence quantitative PCR（q RT-PCR）.【Results】A 906 bp sequence was cloned and named as Ppmal. The open reading frame（ORF） was480 bp,encoding 159 amino acids with a molecular weight of 17.56 k D,and its p I is 5.62. Bioinformaticanalysis showed that the protein had no signal peptide sequence or transmembrane domain,and it was hy-drophilic. It shares high nucleotide sequence homology with those of other reported plants,and has97.48% and 96.23% amino acids homology with Malus domestica and Pyrus communis respectively. Phylo-genetic tree analysis indicated that Ppmal is most related to apple. At the same time,The quantitative re-sult showed that the expression of Ppmal in the fruit after treatment by GA presented a first decreased andthen increased trend with the growth and development of the fruit. What＇s more,Ppmal had the characteristic of differential distribution in various tissues,and the expression from high to low order was flowers,new shoots,leaves and shoots.【Conclusion】The full length of Ppmal was cloned and the results revealedthat this gene was controlled by GA and presented overexpression during the fruit enlargment in pear.