灰毡毛忍冬Lm4CL基因克隆及表达分析

Clone and expression analysis of Lm4CL in Lonicera macranthoides Hand-Mazz

【目的】克隆灰毡毛忍冬4CL基因,分析其在不同组织间的表达水平及与绿原酸含量的相关性。【方法】以灰毡毛忍冬转录组4CL候选基因为参考,采用RACE技术克隆4CL基因全长,并对其理化性质、保守结构域等进行生物信息学分析;用荧光定量PCR方法分析其在不同组织、不同发育阶段的表达特性;采用HPLC法测定灰毡毛忍冬叶、茎和花中绿原酸含量;构建表达载体pCold-Lm4CL,转化大肠杆菌BL 21(DE3),IPTG诱导表达后SDS-PAGE检测。【结果】从灰毡毛忍冬中分离出两个4CL基因,分别命名为Lm4CL1和Lm4CL2,分别编码538和560个氨基酸;氨基酸序列分析表明,Lm4CLs包含4-香豆酸:辅酶A连接酶中的Box I和Box II保守结构域及大多数保守的活性位点,而Lm4CL2中的Box I和Box II有4个氨基酸发生改变;Lm4CL1是没有跨膜结构域的不稳定蛋白,Lm4CL2是有两个跨膜结构域的稳定蛋白。系统发育分析表明,这两个同源基因处于不同的类别,Lm4CL1属于Ⅰ类,Lm4CL2属于Ⅱ类。通过建立三维结构模型分析了Lm4CL蛋白的结构特征,为了解Lm4CL的结构特征提供了基础信息。表达模式分析结果表明,Lm4CL1基因在不同组织、不同发育阶段的表达水平与Lm4CL2基因明显不同。基因表达量与绿原酸含量相关性分析显示,Lm4CL1基因的表达量与绿原酸含量呈负相关(r=-0.63),Lm4CL2基因的表达量与绿原酸含量呈正相关(r=0.94)。SDSPAGE结果显示,Lm4CL1蛋白的大小与预测的蛋白相对分子质量基本一致。【结论】推测Lm4CL1基因与灰毡毛忍冬木质素的合成有关,Lm4CL2基因与灰毡毛忍冬的绿原酸合成有关。

【Objective】To clone 4CL genes and analyze its expression level in different tissues of Lonicera macranthoides Hand-Mazz and relationship with the content of chlorogenicacid(CGA).【Method】The 4CL genes cDNA full-length sequence was cloned by RACE technology.Characteristics including the physicochemical properties and conserved domain of the 4CLs protein were determined by a series of bioinformatics tools.The 4CL expression pattern was also analyzed by qRT-PCR method,in the same time,CGA content in different petals were analyzed by HPLC.The recombinant plasmid was transformed into E.coli BL21(DE3)cells.With IPTG induction,SDS-PAGE was used to investigate the situation of expression.【Result】We isolated three 4CL genes from L.macranthoides,named as Lm4CL1,Lm4CL2,and they were predicted to encode two proteins of 538 and 560 amino acids respectively.The amino acid sequence analysis showed that Lm4CLs contained most of the conserved active sites and two conserved motifs that called Box Iand Box II of those kinds of enzymes,while Lm4CL2 has 4 aminoacid were altered.Lm4CL1 belong to the unstable proteins and have no transmembrane region,while Lm4CL2 belong to stable protein and has two transmembrane regions.The phylogenetic analysis suggested that the two homologous genes are in different classes that the Lm4CL1 belonged to class I,and Lm4CL2 belonged to class II.The structural characteristics of Lm4CL proteins were analyzed by constructing the three-dimensional structure model.The results provided basic information to understand the structural characteristic of Lm4CLs.Expression patterns of the two Lm4CL genes in different developmental stages were analyzed.The expression level of Lm4CL1 was significantly different from the Lm4CL2.Correlation analysis between gene expression level and CGA content showed that the expression level of Lm4CL1 was negatively correlated with CGA content(r=-0.63),while the expression level of Lm4CL2 was positively correlated with CGA content(r=0.94).SDS-PAGE results showed that a significant protein band was in consistent with molecular weight of the predicted protein.【Conclusion】In view of the above experimental results,we predicted that Lm4CL1 may be involved in lignin synthesis in L.macranthoides,while Lm4CL2 maymore likely be associated with CGA synthesis.