2个野扁桃自交不亲和SFB基因全长的克隆与序列分析

摘要

In order to lay foundation for genetic improvement of Prunus tenella and further research on selfincompatibility mechanism,two full-length sequences of pollen S-gene SFB were cloned from P tenella anther by RTPCR and RACE techniques.BLAST was used for nucleotide sequence alignment.ORF within SFB was searched by ORF Finder and protein conserved domains were analyzed by CDD.Protein physical and chemical parameters were analyzed by ProtParam.Transmembrane helices in protein were predicted by TMHMM,the presence and location of signal peptide in protein was predicted by SignalP,protein subcellular localization was predicted by SubLoc,and protein secondary structure was analyzed by SOPMA.DANMAN was used for protein multiple sequence alignment,and MEGA6 was used for phylogenetic analysis of SLF/SFB proteins.Protein functions were predicted by ProtFun.The results showed that two cloned SFB genes (PtSFB16 and PtSFB17) belonged to F-box gene family,and the nucleotide sequence identity between the two cloned SFB genes and SFB/SLF genes in many other plant species was 88％;the deduced amino acid sequences of PtSFB16 and PtSFB17 both had typical structure ofF-box protein;length of ORF of PtSFB16 was 1 146 bp,encoding a protein of 381 amino acids;length ofORF of PtSFB17 was 1 131 bp,encoding a protein of 376 amino acids;the two proteins both were slightly-hydrophilic and instable cytoplasmic protein,their secondary structure both mainly presented as α-helix,extended strand and random coil,evolutionary relationships of them were close to some other SFB/SLF proteins in Prunus,homology of cross-species was higher than homology of intraspecies,and their possible functions included biosynthesis of cofactors,energy metabolism and lyase.%为给野扁桃的遗传改良和自交不亲和机制的进一步研究提供理论依据,以新疆野扁桃花药为试材,利用RT-PCR和RACE技术克隆野扁桃自交不亲和性花粉特异性决定因子编码基因,FB全长序列.采用BLAST进行序列比对,ORF Finder寻找开放阅读框,CDD分析蛋白保守结构域,ProtParam分析蛋白理化性质,TMHMM预测蛋白跨膜区,SignalP预测蛋白信号肽,SubLoc预测蛋白亚细胞定位,SOPMA分析蛋白二级结构,DANMAN进行氨基酸多序列比对,MEGA6进行系统进化分析,ProtFun预测蛋白功能.结果表明:克隆到的PtSFB16基因和PtSFB17基因属于F-box基因家族,与其它多种植物的SLF/SFB基因的序列相似度为88％,推导的氨基酸序列均具有F-box蛋白典型结构;PtSFB16基因ORF长1 146 bp,编码381个氨基酸,PtSFB17基因ORF长1 131 bp,编码376个氨基酸;预测2个SFB蛋白均为略显亲水性、不稳定的细胞质蛋白,二级结构均以α-螺旋,延伸链和无规则卷曲为主,SFB/SLF蛋白在蔷薇科李属植物中具有较高的系统进化一致性,种间同源性可能大于种内同源性,SFB蛋白可能的功能主要有辅助因子生物合成、能量代谢和裂合酶.