胡萝卜4CL基因家族的鉴定及分析

Genome-wide Identification and Analysis of Dc4CL Gene Family in Carrot(Daucus carota)

旨在全基因组水平上鉴定胡萝卜4CL基因,揭示该基因家族成员的理化性质、系统进化、基因结构、组织表达模式及胁迫响应情况,为胡萝卜4CL的功能研究和利用奠定基础。基于AMP结合结构域(PF00501)的隐马尔可夫模型文件,利用HMMER软件对胡萝卜蛋白质数据库进行检索,获得胡萝卜4CL基因(命名为Dc4CL);利用ProtParam分析Dc4CL的理化性质;利用MEGA6对Dc4CL进行系统进化分析;使用GSDS2.0、MEME分析Dc4CL的基因及蛋白结构;利用荧光定量PCR技术对Dc4CL的组织表达模式及胁迫响应情况进行研究。结果表明,胡萝卜基因组中存在12个4CL基因,分别为Dc4CL1~Dc4CL12。Dc4CL基因开放阅读框(ORF)长度为1350~3363 bp,编码蛋白质长度为449~1120 aa,编码蛋白的分子质量和等电点分别为48.92~123.73 ku和5.34~8.82。系统进化分析表明,Dc4CL与其他来自水稻、拟南芥、二穗短柄草、高粱、大白菜的4CL蛋白可分为两个类群。结构分析表明,Dc4CL基因外显子数目为4~11个,其蛋白序列中均含有BoxⅠ(SSGTTGLPKGV)和BoxⅡ(GEICIRG)两个保守序列。荧光定量PCR结果表明,Dc4CL3、Dc4CL12在胡萝卜叶片及肉质根中均有表达,而Dc4CL8主要在叶片中表达,盐胁迫和低温胁迫均使Dc4CL3、Dc4CL8、Dc4CL12的表达降低。总之,胡萝卜的12个Dc4CL基因在影响4CL蛋白功能的关键位点处具有保守性,而在其他位置上存在一定的差异,且部分成员在逆境胁迫应答过程中可能发挥了一定的作用。

From whole genome level,we identified the 4CL gene family in carrot(here called Dc4CL)and analyzed its physicochemical properties,phylogenetic relationships,gene structures,expression profiles in different tissues and various abiotic stresses,so as to lay a foundation for the further functional study and application of 4CL.Based on the hidden Markov model profile of the AMP-binding domain(PF00501),HMMER software was used to identify Dc4CL gene,ProtParam was used to analyze their physicochemical properties,MEGA6 was used to construct the phylogenetic tree and analyze their phylogenetic relationships,GSDS2.0 and MEME was used to analyze their gene and protein structure,respectively.The expression profiles of Dc4CL in different tissues and under various abiotic stresses were analyzed by using real-time quantitative PCR.The results showed that totally 124CL genes(Dc4CL1-Dc4CL12)were identified in the carrot genome.The ORF length of Dc4CL genes was 1350-3363 bp.The length of protein coded by Dc4CL gene ranged from 449 to 1120 aa,and the molecular weight varied from 48.92 to 123.73 ku with the isoelectric point varying from 5.34 to 8.82.A phylogenetic tree composed of Dc4CL and other 4CL proteins from Oryza sativa,Arabidopsis thaliana,Brachypodium distachyon,Sorghum bicolor and Brassica rapa was constructed and all 4CL proteins were classified into two groups.The structural analysis showed that Dc4CL gene possessed 4-11 exons and all Dc4CL proteins contained the conserved sequences-BoxⅠ(SSGTTGLPKGV)and BoxⅡ(GEICIRG).The expression analysis showed that Dc4CL3 and Dc4CL12 were expressed in carrot leaves and roots,while Dc4CL8 was mainly expressed in leaves,and expression level of the three genes all decreased under salt and low temperature stresses.In conclusion,the 12 Dc4CL genes are conservative at the key sites which affect the function of 4CL protein,but there are some differences in other positions,and some members of this gene family might play roles in the response to abiotic stresses.