摘要
【目的】克隆甘蔗14-3-3基因并预测分析其编码蛋白的结构,为研究甘蔗基因功能和代谢调控机制提供参考。【方法】以水稻14-3-3基因为模板,BLAST甘蔗EST数据库,依据序列拼接结果及RT-PCR技术获得编码甘蔗14-3-3蛋白的全长基因,并用生物信息学方法对该蛋白的二级、高级结构和功能活性位点进行预测。【结果】克隆得到长784bp的甘蔗14-3-3基因,最大开放阅读框为771 bp,编码256个氨基酸;该蛋白的分子量与理论等电点分别为28.88 kDa和4.79。蛋白聚类分析结果表明,甘蔗14-3-3蛋白与水稻、高粱、玉米14-3-3蛋白的同源性均在90%以上。Cn3D V.4.1预测位于第3和第5两个二聚体上的Lys-50、Arg-57、Arg-131和Try-132组成一个凹穴,是结合靶蛋白的作用面;第60、65、188、218位的4个丝氨酸是磷酸化的活性位点。实时定量PCR检测结果表明,14-3-3基因在甘蔗不同组织中均有表达,在茎和分生组织中14-3-3基因高丰度表达,可能与糖代谢和细胞分裂的调控相关;而在根中可能参与矿物元素的吸收和代谢。【结论】甘蔗14-3-3基因可作为信号转导调控蛋白的候选基因之一。
[Objective]Cloning the 14-3-3 gene of sugarcane,analyzing and predicting its coding protein structure were condueted to provide references for gene function and metabolic regulatory mechanism studies of sugarcane.[Method]In the present study,a cDNA encoding 14-3-3 protein from Saccharum spp.hybrids was cloned and sequenced by RT-PCR technique based on comparing EST sequences of 14-3-3 protein found in sugarcane.The secondmy structure,advanced structure and functional activity sites of 14-3-3 protein were predicted by bioinformatics.[Result]The cDNA length was 784 bp,28.88 kDa of molecular weight and 4.79 of isoelectric point.It contained 771 bp of open reading frame encoding a polypeptide of 256 amino acids.Cluster analysis revealed that the sugarcane polypeptide shared more than 90％ identity with rice,broomcorn and maize.The binding motif for the target protein consisted of a basic pocket composed of Lys-50,Arg-57 and Arg-131,as well as Tyr-132,within the third and fifth helices.The activity sites for the phosphoserine were also shown in the 60th,65th,188th and 218th serines.Sugarcane 14-3-3 gene transcripts were detected by real time-PCR in different tissues.The results revealed that 14-3-3 gene could express in different tissues of sugarcane.It expressed highly in stalk and meristem,which may be related with glucose metabolism and cell division.In roots,sugarcane 14-3-3 maybe participate into absorption and metabolic of mineral elements.[Conclusion]Sugarcane 14-3-3 gene could act as one of the candidate genes for signal transduction regulating protein.
出处
《南方农业学报》
CAS
CSCD
北大核心
2013年第11期1757-1764,共8页
Journal of Southern Agriculture
基金
中国热带农业科学院中央级公益性科研院所基本科研业务专项项目(1630062012009)
