枸杞Lb14-3-3基因家族cDNA克隆及生物信息学分析

在开花植物中,14-3-3蛋白对植物生长发育具有重要的调控作用。本研究在前期枸杞花药发育差异蛋白质组研究基础上,利用RT-PCR技术,从宁夏枸杞‘宁杞1号’花药中克隆了5条长度分别为768 bp、747 bp、777 bp、753 bp和777 bp的cDNA序列,分别命名为Lb14-3-3a、Lb14-3-3b、Lb14-3-3c、Lb14-3-3d和Lb14-3-3e,对Lb14-3-3a、Lb14-3-3b、Lb14-3-3c、Lb14-3-3d和Lb14-3-3e,5个基因编码蛋白质的结构预测发现,其蛋白质的分子量大小在60.91~64.14 kD之间,编码蛋白质的长度在248~259个aa之间,理论等电点在4.95~4.99之间,均为酸性氨基酸,且都为不稳定的亲水性蛋白。α-螺旋(Alpha helix)是这5个蛋白质二级结构的主要成分,三级结构主要是由α-螺旋构成的对称结构,具有高度的相似性。该家族蛋白既没有信号肽也无明显的跨膜区,亚细胞定位显示Lb14-3-3a定位在线粒体上,Lb14-3-3b和Lb14-3-3e定位在叶绿体上,Lb14-3-3c和Lb14-3-3d定位在细胞质中,Lb14-3-3a蛋白发生磷酸化修饰的可能性最高,Lb14-3-3b磷酸化的可能性最低。多序列比对分析显示,该家族蛋白之间的相似性为77.51%,具有高度的保守性。进化分析显示Lb14-3-3a与其它Lb14-3-3蛋白家族的亲缘关系较远,Lb14-3-3c和Lb14-3-3e,Lb14-3-3d和Lb14-3-3b处于同一分支,亲缘关系较近,Lb14-3-3家族蛋白与马铃薯、番茄、烟草等亲缘关系较近。研究结果为阐明该蛋白家族基因在枸杞花药发育过程中潜在的调控机理提供科学依据。

cDNA Cloning and Bioinformatics Analysis of Lb14-3-3 Gene Family in Wolfberry

In flowering plants, 14-3-3 protein plays an important role in the regulation of plant growth and development. Based on the differential proteome of anther development in the early stage, five genes cDNA, which has full-length of 768 bp, 747 bp, 777 bp, 753 bp and 777 bp respectively, were cloned from the anther of Lycium barbarum L. by RT-PCR technology, and were named Lb14-3-3 a, Lb14-3-3 b, Lb14-3-3 c, Lb14-3-3 d and Lb14-3-3 e. The protein s tructures of five genes encoded by genes of Lb14-3-3 a, Lb14-3-3 b, Lb14-3-3 c,Lb14-3-3 d and Lb14-3-3 e were predicted. These proteins contain amino acids length from 248 to 259 and have a molecular weight from 60.91 to 64.14 kD. The theoretical isoelectric points of proteins are between 4.95 and 4.99,and they are all unstable and hydrophilic proteins. Alpha-helix is the main component of the secondary structure of these five proteins. The 14-3-3 protein tertiary structures are symmetrical structure mainly composed of α-helix,which has a high degree of similarity. The 14-3-3 family protein has neither signal peptide nor obvious transmembrane domain, subcellular localization revealed that Lb14-3-3 a protein localizes on mitochondria, Lb14-3-3 b and Lb14-3-3 e proteins are localized on chloroplasts, but Lb14-3-3 c and Lb14-3-3 d are localized in cytoplasm.Lb14-3-3 a protein is most likely to be phosphorylated, while Lb14-3-3 b is least likely to be phosphorylated. Multi-sequence alignment analysis showed that the similarity among the Lb14-3-3 family proteins was 77.51%, which was highly conserved. Phylogenetic analysis showed that Lb14-3-3 a is far from other Lb14-3-3 family proteins,Lb14-3-3 c and Lb14-3-3 e, Lb14-3-3 d and Lb14-3-3 b are in the same branch, which showed the genetic relationship relatively close. The Lb14-3-3 family protein is closely related to potato, tomato and tobacco. The results laid a molecular basis for elucidating the potential regulatory mechanisms of this protein family during the development of wolfberry anthers.