毛竹TIP基因成员鉴定及其响应胁迫的表达分析

Identification of TIP Genes and Their Expression Patterns under Stresses in Moso Bamboo(Phyllostachys edulis)

液泡膜内在水通道蛋白(TIPs)在调节植物细胞膨压适应逆境胁迫环境过程中发挥着重要作用。研究毛竹TIP基因成员的分子特征及其在不同逆境胁迫条件下的表达模式,对揭示其在毛竹应答胁迫中的功能具有重要意义。利用生物信息学方法在毛竹基因组中共鉴定19个编码完整TIP蛋白的基因(PeTIP1-1~PeTIP1-3、PeTIP2-1~PeTIP2-4、PeTIP3-1~PeTIP3-2、PeTIP4-1~PeTIP4-7和PeTIP5-1~PeTIP5-3);PeTIPs编码氨基酸的长度为238~434 aa,相对分子量为25.06~44.03 kDa;亚细胞位置预测显示,所有PeTIPs均定位于液泡膜上。PeTIPs包含7个保守基序,其中有4个为共有基序,不同成员的Ar/R选择性过滤器具有一定的差异,而Froger's残基均较为保守。系统进化分析显示,来自毛竹、水稻等6个物种的71条TIP氨基酸序列可分为5个分支,各分支中PeTIPs成员依次为3、4、2、7和3个。共线性分析结果表明,PeTIPs成员间共存在10对片段重复,在12个PeTIPs与水稻8个TIP基因间发现18对片段重复,这些重复基因多半发生在PeTIP4s和PeTIP5s中,且基因对的非同义对同义取代比(Ka/Ks)均小于1.0,表明PeTIPs经复制后的功能差异不大。在PeTIPs启动子区域中,发现多种与胁迫、激素响应相关的调控元件。基于叶片RNA-seq数据分析表明,不同PeTIPs响应低温、干旱和强光胁迫的表达模式均存在一定差异,既有显著性变化的成员(如PeTIP1-1和PeTIP1-2),也有几乎无变化的成员(如PeTIP5的成员)。qPCR结果显示,在不同胁迫条件下毛竹叶片和根中的PeTIPs的表达模式不同,多数呈现不同程度的显著上调,亦有在某一处理下基因表达变化不明显(如强光下叶片中的PeTIP1-1、PeTIP1-3和PeTIP4-2;干旱下根中的PeTIP1-1和PeTIP1-2),个别基因呈现下调(如低温下叶片中的PeTIP1-1)。毛竹叶片和根中PeTIPs的表达变化模式差异,说明它们在响应不同环境胁迫中可能发挥着不同的作用。

Tonoplast intrinsic aquaporin proteins(TIPs)play a significant role in regulating cell turgor pressure to adapt to adversity stresses.Studying the molecular characteristics of TIP gene members of moso bamboo(Phllostachys edulis)and their expression patterns under different stress conditions is of great significance to reveal their functions in response to stresses.Bioinformatics methods were used to systematically identify the gene members of TIP family in moso bamboo genome,and a total of 19 genes encoding complete TIP proteins were obtained(PeTIP1-1~PeTIP1-3,PeTIP2-1~PeTIP2-4,PeTIP3-1~PeTIP3-2,PeTIP4-1~PeTIP4-7 and PeTIP5-1~PeTIP5-3).The length of the amino acids encoded by PeTIPs ranged from 238 to 434 aa,and the relative molecular weights were 25.06~44.03 kDa.Subcellular location prediction showed that all PeTIPs were located on the tonoplast.PeTIPs contained 7 conserved motifs,4 of which were shared motifs.The Ar/R selective filters of different PeTIPs had certain differences,while Froger's residues were relatively conservative.Phylogenetic analysis showed that 71 TIP amino acid sequences from 6 species such as moso bamboo and rice can be divided into 5 branches,and the members of PeTIPs in each branch are 3,4,2,7 and 3 in turn.The results of collinearity analysis showed that there were 10 pairs of fragment duplications among members of PeTIPs,and 18 pairs of fragment duplications were found between 12 PeTIPs and 8 TIP genes in rice.Most of these duplication genes occurred in PeTIP4s and PeTIP5s,and the nonsynonymous and synonymous substitution ratios(Ka/Ks)were all less than 1.0,indicating that PeTIPs had little functional difference after replication.A variety of regulatory elements related to stress and hormone responses were found in the promoters of PeTIPs.Analysis based on leaf RNA-seq data showed that the expression patterns of different PeTIPs in response to low temperature,drought and strong light stress were different.There are not only members with significant changes(such as PeTIP1-1 and PeTIP1-2),but also members with almost no changes(As a member of PeTIP5).The qPCR results showed that the expression patterns of PeTIPs in the leaves and roots of moso bamboo were different under different stress conditions,and most of them showed significant up-regulation to varying degrees.There were also gene expression changes that were not obvious under a certain treatment(such as PeTIP1-1,PeTIP1-3 and PeTIP4-2 in leaves under high light,PeTIP1-1 and PeTIP1-2 in roots under drought),and individual genes were down-regulated(e.g.PeTIP1-1 in leaves under low temperature).The different expression patterns of PeTIPs in leaves and roots indicate that they may play different roles in response to different environmental stresses.