核桃CM基因家族鉴定及表达分析

Identification and expression analysis of the CM family gene in walnut(Juglans regia L.)

【目的】鉴定分析核桃(Juglans regia L.)分支酸变位酶(chorismate mutase,CM)家族基因,为解析莽草酸途径(shikimic acid pathway)代谢调控的分子机制提供参考。【方法】基于核桃全基因组数据,通过HMM搜索对CM家族成员进行筛选鉴定,并利用实时荧光定量PCR分析其在核桃不同组织中的表达模式。【结果】核桃基因组内共鉴定到7个CM家族基因,分布于6条染色体上,JrCM6与JrCM7位于同一条染色体,其余基因分别位于不同的染色体上。各基因均含有5~6个外显子,结构保守。预测表明,核桃CM均为亲水性蛋白且定位于叶绿体上,其中JrCM5含有信号肽。除JrCM4外,其余成员均无跨膜结构。蛋白质三维结构预测结果显示核桃CM家族蛋白与模板蛋白AtCM1间具有较高的相似性。系统进化分析显示,植物CM蛋白可分为4个大组,核桃CM蛋白聚集在第Ⅱ和Ⅳ分支上。其中JrCM2和JrCM5聚集在第Ⅱ支系,与胡杨CM同源蛋白Potri.T174200的亲缘关系较近;JrCM6、JrCM7、JrCM4、JrCM1及JrCM3聚集在第Ⅳ支系上;JrCM6、JrCM7和JrCM4与葡萄CM同源蛋白GSVIVG01010091001的亲缘关系最近;而JrCM1和JrCM3则与蝴蝶兰CM同源蛋白PEQU04269的亲缘关系最近。对启动子顺势作用原件的预测表明,除CAATbox和TATA-box外,各基因启动子中共发现光响应、激素响应、胁迫响应、转录因子结合位点、生长和发育响应及昼夜节律等7大类调控元件,其中MYB、ERE、MYC、Box 4、G-box、ABRE、ARE、STRE、O2-site、TCA-element的数量最多,这与CM基因主要受到光照、逆境胁迫调控的结论较为一致。qRT-PCR定量检测结果表明,7个基因在核桃叶、茎、果柄和青皮中均有表达,且其表达量与趋势各不相同故具有组织特异性。【结论】在核桃基因组中,共计鉴定得到7个CM家族基因,该家族蛋白具有理化特征、三维结构上的保守性,启动子区域具有响应多种不同信号的顺式作用元件。研究结果为进一步阐明核桃CM基因在莽草酸途径中的生物学功能奠定了基础。

【Objective】It is estimated that about 20%of the carbon fixed by green plants goes through the shikimic acid pathway(SAP),and provides the carbon skeleton for secondary metabolites such as aromatic amino acids,vitamins,lignins,phenols and aromatic substances.Hence,SAP is regarded as the bridge connecting sugar and secondary metabolism in both plants and microorganisms.Chorismate mutase(CM)is a constitutional enzyme of the SAP pathway,catalyzing the conversion of chorismate to prephenate.In the present study,genes of the CM family were first identified from the genomic data of walnut(Juglans regia L.),aiming to uncover their physicochemical properties and genetic features.【Methods】Protein identification was performed by searching the genomic data using the Hidden Markov Model(HMM)model of the CM family protein.The Arabidopsis thaliana AtCM1 protein was also used as the seed to BLAST against the walnut proteins,in order to further confirm the searching result.The walnut genome data was downloaded from the Ensembl Plants database.The non-redundant sequences were re-confirmed by querying the SMART and CDD databases and used as sequences for downstream analyses.The physicochemical parameters,including gene length,molecular weight,isoelectric point,amino acid number,instability coefficient,signal peptide,chromosome location,transmembrane structure,hydrophobicity and subcellular location,were obtained by calculation and prediction using bioinformatic tools.For display of gene structure,structure information was extracted from the sequence annotation file,and displayed by using TBtools.We also predicted the cis-acting elements existing in the promoter regions by querying the PlantCARE database,and the location of the motifs was displayed using GSDS.The phylogenetic analysis of plant CM proteins was performed using the neighbor-joining(NJ)method with 1000 bootstrap replicates(implemented in the MEGA11 software).For quantification of gene expression by qPCR,the primers of CM genes were designed using the Oligo Architect Online tool,and the walnut 18S gene was selected as the internal reference gene.Real time fluorescent quantitative PCR was then conducted to quantify and analyze the gene expression in different tissues(leaf,stem,fruit stalk,green husk).【Results】After HMM and BLAST search,seven CM proteins were identified from the walnut genome.All the CMs contained the CM-2 domain,which was specific to the family.Therefore,these seven genes were identified as members of the CM family.Analyses of physicochemical properties showed that all 7 CMs were hydrophilic proteins except for JrCM5.No signal peptide was detected in walnut CMs,except for JrCM5.Most of the CMs identified in other species had no signal peptide,such as Petunia hybrida PhCM1 and Salvia miltiorrhiza SmCM1.It was speculated that walnut CMs had similar property and function like other homologues.The prediction of protein subcellular localization showed that walnut CMs were all located in the chloroplast,which was consistent with that of PhCM1.Therefore,the functioning place of the CM homologues could probably be in the chloroplast.The CMs might play active roles in plant defense by affecting the synthesis of secondary metabolites,such as salicylic acid and flavonoids.The gene location analysis showed that these 7 CM genes were distributed on 6 chromosomes.Each gene contained 5-6 exons,and their structure was conservative.All the 7 CMs contained no transmembrane structures,except for JrCM4.The results of protein three-dimensional structure prediction showed that there was a high similarity between the members of the walnut CM family proteins and the template AtCM1 protein.A diverse set of cis-acting motifs were detected in the promoter region of CM genes.The CAAT box and TATA box were constantly found in all the promoters.In addition,a total of 296 elements,which could be grouped into seven major categories were detected.These cis-elements could be grouped into 7 categories,including the light response,hormone response,stress response,transcription factor binding site,growth and development response,and circadian rhythm regulation.The phylogenetic analysis showed that the plant CM proteins could be divided into four groups,and the CMs were clustered in branchⅡandⅣ.Wherein,JrCM2 and JrCM5 were closely related to Populus tomentosa CM Potri.T174200 protein;JrCM6,JrCM7 and JrCM3 were closely related to grape CM GSVIVGO1010091001 protein;JrCM1 and JrCM4 had close relationship with Phalaenopsis Aphrodite CM PEQU04269.The results of qRT-PCR analysis showed that 7 genes were expressed in walnut leaves,stems,fruit stalks and green husks,and their expression was tissue specific.【Conclusion】In this study,seven CM family genes were identified from the walnut genome.The proteins of walnut CM family had physicochemical properties and three-dimensional structures similar to those of their homologues.A variety of cis-acting elements were detected in the promoter region of these 7 genes,implying responsiveness to multiple signals.The expression of CM family genes was universally detected in different tissues.The gene expression profiling of CM genes is not only helpful for better understanding of the generation of secondary metabolites(various amino acids,phenols and antioxidant active substances),but also benefitial for the regulation mechanism study of CM proteins.The biosynthetic pathway of plant polyphenols is very complex,involving a wide range of enzymes,and chorismate mutase is only one of them.At present,the functional mechanism of this family is still not fully elucidated,and we will continue to carry out downstream experiments to uncover the molecular mechanism of the shikimic acid pathway regulation.