Cloning of lipoxygenase cDNA fragment of pear (Pyrus pyrifolia) and construction of its corresponding RNAi vector

Hu-Zhong-Dong; Qiao-Yu-Shan; Wang-San-Hong; Yao-Quan-Hong; Zhang-Zhen

Xibei Zhiwu Xuebao 27(7): 1285-1290

2007


Accession: 015292374

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Abstract
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 inter val 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.