Gao Xue, Li Zheng-Guo, Fan Jin, Yang Ying-Wu
Key Lab of Functional Gene and Regulation Technologies under Chongqing Municipal Education Commission, Genetic Engineering Research Center, College of Bio-Engineering, Chongqing University, Chongqing 400044, China.
Zhi Wu Sheng Li Yu Fen Zi Sheng Wu Xue Xue Bao. 2007 Feb;33(1):71-6.
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.
本研究以‘奉节’脐橙果实的有斑果皮和无斑果皮为实验材料，通过抑制性消减杂交（SSH）构建并筛选与果皮斑相关的基因。结果表明，抑制性消减杂交非常有效。构建了差异表达基因的cDNA文库。该文库包含约200个克隆，平均插入片段大小约为300 bp。随机挑选部分阳性克隆进行测序。50个克隆中有6个未发现同源序列，3个在GenBank中功能未知。根据同源性分析，选择了4个同源基因（钙结合蛋白、半胱氨酸蛋白酶、NAC结构域蛋白和扩张蛋白），通过半定量RT-PCR分析检测它们在有斑和无斑脐橙果实中的表达。4个基因在有斑果皮中的表达均高于无斑果皮。这表明SSH cDNA文库中的这些基因可能与果皮斑有关，可作为未来研究柑橘果实斑形成分子生物学机制的研究对象。
