青天葵NfD53基因的克隆及生物信息学分析

Cloning and Bioinformatic Analysis of NfD53 Gene from Nervilia fordii

本研究克隆青天葵NfD53基因并分析其序列特征,可为今后深入研究Nf D53蛋白功能及独脚金内酯(SLs)对植物侧枝生长发育的影响提供参考依据。以濒危药用植物青天葵叶片为实验材料,利用RT-PCR及RACE技术克隆获得2条NfD53基因,分别命名为NfD53-1和NfD53-2,应用生物信息学软件分析其序列并进行功能预测。结果显示:NfD53-1基因全长3523 bp,编码974个氨基酸;NfD53-2全长3916 bp,编码1190个氨基酸。两者等电点分别为:8.34、6.33;亲水性平均数为:-0.250、-0.263;均为不稳定的亲水性蛋白,无信号肽及切割位点,定位于叶绿体。两蛋白都具有ClpA超家族核心序列,而Nf D53-1蛋白还具有ClpC超家族核心序列,并含有3个非特异性位点(ClpA,ClpC和AAA_(2)),Nf D53-2蛋白含有1个ClpA非特异性位点。蛋白的二级结构中均以无规则卷曲占比最高,分别占47.16%和51.21%;其次是α-螺旋,占38.70%和31.69%。系统进化分析表明,Nf D53-1与番茄等聚为一支,但氨基酸相似度较低(20.27%),Nf D53-2与铁皮石斛聚为一支,同源性较高(66.49%)。本研究为后续进一步探讨该基因的功能及SLs在植物生长发育中的作用提供数据参考。

In this study,NfD53 genes were cloned and the characteristics of their sequences were analyzed,which could provide a reference for further study on the function of Nf D53 proteins and the effects of strigolactone on the growth and development of plant lateral branches.Two D53 genes,named NfD53-1 and NfD53-2,were cloned from the leaves of the endangered medicinal plant Nervilia fordii by RT-PCR and RACE techniques.Bioinformatic analysis of the sequences showed that,the full-length of NfD53-1 was 3 523 bp and encoded 974 amino acids;full-length of NfD53-2 was 3 916 bp and encoded 1 190 amino acids.The isoelectric points of two proteins were8.34 and 6.33,and grand averages of hydropathicity were-0.250 and-0.263,respectively,both were unstable hydrophilic proteins with no signal peptide.These proteins were predicted to locate in chloroplasts.Both of them have ClpA superfamily core sequences.Nf D53-1 also has ClpC superfamily core sequence and contains 3 non-specific sites(ClpA,ClpC and AAA_2),while Nf D53-2 only contains one ClpA non-specific site.The secondary structures of two proteins consist the highest proportions of random coils(47.16% and 51.21%),followed by alpha helixes(38.70% and 31.69%).Phylogenetic analysis showed that the Nf D53-1 had the closest relationship with D53 homologous proteins from Solanum lycopersicum(20.27%);while Nf D53-2 was closely related to that of Dendrobium officinale(66.49%).The tertiary structures of these two proteins were also predicted.This study provided reference for further research on the function of Nf D53 s and the role of SLs in growth and development of the endangered plant N.fordii.