砂梨脂氧合酶cDNA片段克隆与RNAi载体构建

Cloning of Lipoxygenase cDNA Fragment of Pear (Pyrus pyrifolia) and Construction of Its Corresponding RNAi Vector

以砂梨‘若光’的成熟果实为材料,根据脂氧合酶氨基酸保守区设计1对简并引物,采用RT-PCR克隆到1段长827 bp的序列,经Blast比对和DNAstar软件聚类分析,结果表明由该序列推导出的氨基酸序列含有脂氧合酶氨基酸保守结构域,与马铃薯脂氧合酶基因的氨基酸序列相似性达到77.5%,判断其为砂梨脂氧合酶基因片段,命名为LOX1,并将序列登录到GenBank,登录号为EF215448。根据RNAi载体的构建原则,选择LOX1一开放阅读框设计携带酶切位点的特异引物,通过PCR扩增正、反向基因片段,再与YYT间隔区串连,插入植物表达载体pYF7713中的相应位置,成功地构建了干扰LOX基因表达的RNAi的植物双元表达载体pYL028,为深入研究该基因在砂梨果实成熟过程中的功能及耐贮藏基因工程育种奠定了基础。

The total RNA was isolated from ripen fruit of pear （Pyrus pyrifolia Nakai ‘ Wakahikari＇）. A specific fragment of the expected size was amplified by Reverse Transcription-Polymerase Chain Reaction with degenerate oligonuclectide primers designed on the basis of conserved domain of other plant lipoxygenase. The cDNA fragment was 827 bp. The amino acid sequence revealed that the cDNA fragment had lipoxygenase conserved domain and it was 77.5% homologus with the lipoxygenase of potato,through blasting and clustering analysis. We presumed that the fragment cDNA came from lipoxygenase gene of P. pyrifolia designated as LOX1. The cDNA sequence of this clone was deposited at GenBank （EF215448）. The specific primers with enemzy site were designed from an open read frame （ORF） of the cDNA sequence according to the principles of construction of its corresponding RNAi vector. Those two fragments were amplified from PCR,one is forward orientation, another is reverse orientation, ligated with YYT interval region. The fusion fragment was introduced into pYF7713,it was successful to construct the recombinant plasmid pYL028,which could express dsRNA of LOX gene. It established a base for transformation of storable pear and studies of this gene function in fruit ripening of P. pyrifolia.