奉节脐橙果皮褐变差减文库的构建及初步分析

以奉节脐橙果实为材料,采用抑制差减杂交技术,分别以褐变与未褐变柑橘果皮作为检测方和驱动方,成功构建了果皮褐变的差减cDNA文库,对部分克隆进行了序列测定并与GenBank进行了同源性比较。选择其中的4个基因:钙结合蛋白同源基因、半胱氨酸蛋白酶同源基因、NAC蛋白质家族同源基因和膨胀素同源基因进行半定量RT-PCR分析,结果表明它们在褐变果皮中的表达量均高于未褐变果皮,说明这些基因的增强表达可能与脐橙果皮褐变有密切关系。

Construction and Preliminary Analysis of Subtractive Library for Peel Pitting of 'Fengjie' Navel Orange Fruit

In this study, the pitted peel and non-pitted peel of 'Fengjie' navel orange fruits were used as experimental materials to construct and screen the peel pitting related genes by suppression subtractive hybridization (SSH). The results showed that suppression subtractive hybridization was very effective. A cDNA library of differentially expressed genes was constructed. The library included about 200 clones with an average insert size of around 300 bp. Part of the positive clones were picked up randomly and sequenced. Six of the 50 clones had no homologous sequences being found and three had unknown functions in GenBank. According to the analysis of the homology, four homologous (Ca2+ binding protein, cysteine proteinase, NAC-domain protein and expansin) genes were chosen to examine their expressions through semi-quantitative RT-PCR analysis in pitted and non-pitted navel orange fruits. The expression of four genes were all higher in pitted peel than that in non-pitted peel. It suggests that these genes in the SSH cDNA library may be involved with peel pitting and can be subject of future investigation to explore the molecular biological mechanism of the pitting of citrus fruit.