黄瓜果实弯曲相关基因Cs14-3-3的克隆及表达分析

Cloning and Expression Analysis of Fruit Bending Related Gene Cs14-3-3 in Cucumber

以黄瓜(Cucumis sativus L.)果实易发生弯曲的品种‘长春密刺’为试验材料,采用RT-PCR技术从果皮中分离并克隆了14-3-3蛋白基因,并将此基因命名为Cs14-3-3。Cs14-3-3基因cDNA全长792bp,编码263个氨基酸,分子量29589.287D,等电点为4.585。生物信息学分析表明此基因含有14-3-3蛋白基因的典型结构域,与其他物种14-3-3蛋白基因核苷酸序列同源性达到75%以上,编码的氨基酸序列同源性达到85%以上,属于14-3-3蛋白基因家族。同时,采用实时荧光定量PCR方法对顺直果实和弯曲果实腹部及脊部在果实开花的2、4、6、8、10、12d的基因表达量进行了研究。结果显示,在果实发育各个时期,Cs14-3-3的表达量均为在弯曲果实腹部>顺直果实>弯曲果实脊部的表达量;在各个部位,Cs14-3-3在果实开花2d的表达量明显高于开花后其他时期。试验结果表明,Cs14-3-3基因为黄瓜果实弯曲相关基因,在黄瓜果实开花早期发育过程中起重要作用。

By RT-PCR technology, we separated and cloned the 14-3-3 protein gene from the peel of the cucumber （ Cucumis sativus L. ） bending variety ＇Changchun Mici＇ , named Cs14-3-3. The full length cDNA of Cs14-3-3 is 792 bp, encoding a protein of 263 amino acids. Its molecular mass is 29 589.287 D, and pI is 4.585. Bioinformatic analysis showed that the protein of Cs14-3-3 possesses the basic structure of 14-3-3 proteins, and shares high homology with the known 14-3-3 proteins, which is belonging to the 14-3-3 protein family. At the same time, by using real-time fluorescence quantitative PCR （RT-qPCR）, we studied the expression of mRNA in straight fruit and the abdomen and the ridge of the bending fruit in the periods of 2, 4, 6, 8, 10, 12 days after flowering. The result showed that in each period, the expressionlevel of Cs14-3-3 from high to low order is bending fruit abdomen, straight fruit, bending fruit ridge, and in each part, the expression level of Cs14-3-3 in 2 days after flowering obviously higher than that in the other periods. In conclusion,＂ it was preliminarily revealed that Cs14-3-3 gene is related to fruit bending in cucumber, and plays an important role in the period of early fruit development after flowering.