中国春小麦肌醇磷脂依赖的磷脂酶C基因的全基因组鉴定及表达分析

Genomic Profiling and Expression Analysis of Phosphatidylinositol-specific PLC Gene Families Among Chinese Spring Wheat

【目的】探知小麦基因组序列中肌醇磷脂依赖的磷脂酶C(PLC)的编码基因,解析小麦中PLC基因的结构与进化特征,揭示Ta PLC基因在小麦各组织中的表达模式及其响应盐胁迫和干旱胁迫过程中的表达规律,以便深入分析小麦Ta PLC基因调节小麦应答盐或干旱胁迫中的生理作用。【方法】基于Ensembl Plants全基因组数据库,以水稻和拟南芥PLC基因为参考序列,检索小麦Ta PLC基因家族(http://plants.ensembl.org/index.html),并利用在线蛋白结构分析工具(Pfam、CDD和SMART)对TaPLC基因进行结构分析和鉴定;运用ExPASy(http://cn.expasy.org/tools)分析Ta PLC基因的分子和生化特征;使用Wo LF PSORT(https://www.genscript.com/wolf-psort.html)预测Ta PLC基因的亚细胞定位;运用MEGA 7.0软件进行系统进化树分析;并利用实时荧光定量PCR分析Ta PLC基因在不同组织以及幼苗期在盐或干旱胁迫下的表达特征。【结果】在小麦基因组中鉴定了11个Ta PLC基因序列,其编码蛋白与其他植物PLC结构相似,均具有X和Y保守催化结构域以及C2结构域;系统进化分析表明11个Ta PLC基因可分为4组,即Ta PLC1A/Ta PLC1D、Ta PLC2A/Ta PLC2B/Ta PLC2D、Ta PLC3A/Ta PLC3B/Ta PLC3D、Ta PLC4A/Ta PLC4B/Ta PLC4D,均为植物PLC基因家族的直系同源基因,在进化方面具有保守性,但也存在一定的分歧;通过与小麦祖先种比对分析,发现B亚基因组中的Ta PLC1在异源六倍体形成前就已经缺失,进一步表明小麦亚基因组间进化的不对称性;亚细胞定位预测Ta PLC基因定位于叶绿体、线粒体或细胞质中;Ta PLC基因家族各成员的启动子普遍存在植物激素响应和应激响应相关的顺式调控元件;实时定量结果表明,不同的Ta PLC基因在不同的组织中有不同的表达水平,其主要在根、叶、穗和籽粒中表达;且多数基因参与小麦对盐和干旱胁迫的响应,无论是在耐旱和耐盐品种中,还是在对照品种中,Ta PLC基因均受到胁迫的诱导表达,并在12 h内迅速达到峰值。【结论】在小麦基因组中共鉴定11个Ta PLC基因,编码典型的植物PLC蛋白,在进化上具有保守性,但亚基因组间也存在进化的不对称性。不同的Ta PLC基因有各自的组织和细胞表达特征,在小麦生长发育过程中起调节作用;Ta PLC基因在应答盐或干旱胁迫中起重要作用。

【Objective】To characterize the gene family encoding phosphatidylinositol-specific phospholipase-C(PI-PLC),the gene number,structure,phylogenetic relationship and expression pattern of TaPLC genes were analyzed.In addition,we explored their relative transcription levels in various tissue and the expression pattern under drought and salt stress to further analysis the physiological role of TaPLC genes in response to abiotic stress.【Method】Based on the genome database(Ensembl Plants,http://plants.ensembl.org/index.html),TaPLC genes were identified from Triticum aestivum,and the gene structures were analyzed by some bioinformatics tools,such as Pfam,CDD and SMART.The protein sequence characteristics of the members in TaPLC family were analyzed with the online server ExPASy(http://cn.expasy.org/tools).The subcellular localization of TaPLC genes were predicted using the WoLF PSORT(https://www.genscript.com/wolf-psort.html).Phylogenic tree analysis was performed by software MEGA 7.0.The real-time PCR was used to detect the gene expression levels in different tissues under abiotic stress.【Result】A total of 11 TaPLC genes members within the heterologous hexaploid wheat genome were identified and analyzed.All TaPLC genes have conserved X-Y catalytic domains and C2 domains,just like other plant PLC genes.Subcellular localization showed that these proteins were located in the chloroplast or mitochondria.Phylogenetic analysis revealed that all of the TaPLC genes were unevenly classified into four numbered,TaPLC1 A/TaPLC1 D,TaPLC2 A/TaPLC2 B/TaPLC2 D,TaPLC3 A/TaPLC3 B/TaPLC3 D,and TaPLC4 A/TaPLC4 B/TaPLC4 D,which were all orthologous genes of plant PLC genes in plants,and highly conserved during evolution.However,the deletion of TaPLC1 in B subgenome indicates asymmetry evolution in different subgenomes.Further analysis of cis-regulatory elements elucidated that these members of TaPLC family shared similar elements,such as the phytohormone and the stress response cis-elements.Each PLC gene has a unique tissue expression pattern,and they are expressed mainly in roots,leaves,spikes,and gains.In addition,PLC genes mediate the response of wheat to salt and drought stresses,which are induced rapidly by salt or drought stress stimulation.【Conclusion】The TaPLC family in Triticum aestivum contains 11 members which have conserved X-Y catalytic domains and C2 domains.Although the TaPLC genes were highly conserved,they were asymmetrically evolved in different subgenomes.They had specific expression pattern in different tissues,and were induced by salt or drought stress,which suggested TaPLC genes play a role in response to salt or drought in wheat.