闽楠中WRKY家族成员鉴定及缺磷胁迫下表达分析

Identification of the PbWRKY Gene Family and Its Expression Analysis under Deficiency of Phosphorus in Phoebe bournei

【目的】在闽楠全基因组范围内鉴定WRKY家族成员,分析基因和蛋白序列结构特点及在闽楠幼苗缺磷胁迫下的表达特征,筛选响应缺磷逆境的WRKY基因,为进一步研究它们在楠木磷饥饿响应分子途径中的功能以及耐低磷胁迫分子辅助育种提供参考。【方法】利用WRKY蛋白序列的隐马尔科夫模型(pfam03106),经hmmsearch在闽楠蛋白数据库中检索,筛选WRKY基因。对获得的PbWRKYs利用Protaram、GSDS2.0、MEGA、Batch CD-Search、TBtools和ClustalX等软件进行基因结构、定位分析,蛋白理化性质、序列特征分析以及进化树构建。提取3年生闽楠植株的根、韧皮部和形成层、木质部和叶的RNA,进行转录组测序,分析PbWRKYs在不同组织中的表达特点。对半年生闽楠植株进行缺磷水培处理,60天后,分别取缺磷处理和对照的叶和根进行磷含量测定和转录组测序,分析磷缺乏条件下PbWRKYs表达特征,并对差异表达基因进行qPCR验证。【结果】鉴定到68个WRKY基因,命名为PbWRKY1-68,在各染色体上均有分布,第3号染色体上数量最多,内含子数目在1~28个之间,蛋白分子质量在19.09~115.56 kDa之间。所有PbWRKY均含有WRKY结构域及其特有的锌指结构。根据WRKY结构域数量及其所含锌指结构类型,分为3个亚类:亚类Ⅰ含2个WRKY结构域,锌指结构类型为C2H2型,共14个成员;亚类Ⅱ含1个WRKY结构域和1个C2H2型锌指结构,共47个成员,进化分析表明该亚类又分为5个经典子组Ⅱa、Ⅱb、Ⅱc、Ⅱd、Ⅱe,但其中PbWRKY37与上述子组进化上距离较远,独立成组;亚类Ⅲ含1个WRKY结构域和1个C2HC型锌指结构,共7个成员。PbWRKYs蛋白所含WRKY结构域氨基酸序列特征大部分为“WRKYGQK”型,但PbWRKY62的WRKY结构域变异为“WRKYGKK”。与其他植物相比,PbWRKYs蛋白中WRKY结构域的变异率较低。PbWRKYs的组织特异性表达模式可分为5类:韧皮部和形成层中相对表达量较低;木质部中表达量高而叶片中表达量较低;韧皮部和形成层表达量较高同时叶片中表达量较低;根中表达量相对较高;叶片中表达量高而根中表达量低。闽楠幼苗缺磷处理60天后,叶和根中表达差异达到2倍以上的PbWRKY基因共21个。它们在叶片中被强烈诱导,可能参与低磷条件下叶片中磷元素的运输及再分配过程;其中,PbWRKY52、PbWRKY55、PbWRKY56、PbWRKY65和PbWRKY665个基因的表达还在根中被抑制,表明它们除了参与低磷条件下叶片中磷元素再分配,还可能参与了根部磷的吸收和转运。【结论】闽楠中WRKY基因家族成员共有68个,其蛋白序列中WRKY结构域较保守。缺磷处理60天时,有21个PbWRKY基因参与磷胁迫响应,大部分基因可能主要在叶片中参与低磷条件下磷的运输和分配。PbWRKY52、PbWRKY55、PbWRKY56、PbWRKY65和PbWRKY66除了在叶片中发挥功能外,还可能参与低磷条件下根部对磷的吸收和转运。

【Objective】The WRKY transcription factor gene family members were identified in the whole genome of Phoebe bournei.Gene structure and protein sequences of them were characterized and their expression was analyzed under phosphorus deficiency.Then,PbWRKY members that were involved in phosphorus starvation response were screened.The result will give information to further study their function in the molecular mechanism of phosphorus deficiency stress and provide foundation to molecular assisted breeding for tolerance to low phosphorus in Phoebe.【Method】The hidden Markov model(pfam03106)of WRKY protein sequence were used to search PbWRKY protein sequence from protein database of P.bournei.Then,the chromosome localization,gene structure,protein sequences and phylogenetic tree of PbWRKYs were analyzed by Protaram,GSDS2.0,MEGA,Batch CD-search,TBtools and ClustalX software.Tissue-specific expression was analyzed based on transcriptome sequence data of roots,phloem and cambium,xylem and mature leaves of 3-year-old P.bournei plants.And,six-month-old seedlings were planted in phosphorus deficiency solution.After 60 days,the leaves and roots of phosphorus deficiency treatment and control were harvested for phosphorus content measuring and transcriptome sequencing.The expression of PbWRKY genes under phosphorus deficiency were analyzed,and the PbWRKY genes expressed differentially were verified by qPCR.【Result】68 WRKY genes,named PbWRKY1-68,were identified in P.bournei.They were distributed on all chromosomes of P.bournei and the most were on chromosome 3.All the PbWRKYs had introns,the number of them ranged from 1 to 28.The molecular weight of them from 19.09 to 115.56 kDa.Each PbWRKY had 1 or 2 WRKY domains and a specific zinc finger motif.Based on the number of WRKY domain and the type of zinc finger structure,PbWRKYs can be divided into 3 subclasses.SubclassⅠcontained 2 WRKY domains and 1 C2H2 type of zinc finger,and 14 genes were included in it.There were 47 genes in subclassⅡwhich had 1 WRKY domains and 1 C2H2 type of zinc finger.And phylogenetic analysis showed this subclass can be classified into 5 subgroups:Ⅱa,Ⅱb,Ⅱc,Ⅱd,Ⅱe,but PbWRKY37 didn’t belong to any subgroup above according to the phylogenetic tree although it was a gene of subclassⅡ.SubclassⅢcontained a WRKY domain and a C2HC type of zinc finger,a total of 7 genes.Most of WRKY domain sequence in PbWRKYs were“WRKYGQK”,but it was“WRKYGKK”in PbWRKY62.Tissue-specific expression analysis showed five expression patterns of PbWRKYs were found in different tissues:low expression in phloem and cambium,high expression in xylem but low expression in leaves,high expression in phloem and cambium and low expression in leaves,high expression in roots,and high expression in leaves but low expression in roots.And some of the WRKY genes which closely related had similar tissue-specific expression,suggesting their functions possibly correlated.After 60 days of phosphorus deficiency treatment,21 PbWRKY genes expressed differently more than 2 folds in leaves and roots compared to the control.All the 21 PbWRKY genes were strongly induced in leaves,implying that they possibly participated in the transportation and distribution of phosphorus in leaves under low phosphorus condition.Among them,the expressions of PbWRKY52,PbWRKY55,PbWRKY56,PbWRKY65 and PbWRKY66 were also inhibited in roots,suggesting that they also may be involved in the transportation of phosphorus in roots simultaneously.【Conclusion】There were 68 PbWRKY genes in Phoebe bournei.The WRKY domain sequence of them was very conservative and the variation was very low.21 PbWRKY genes were involved in the response to phosphorus deficiency after 60 days of phosphorus starvation treatment,and most of them were involved in the transportation and distribution of phosphorus in leaves when plants faced phosphorus starvation.However,PbWRKY52,PbWRKY55,PbWRKY56,PbWRKY65 and PbWRKY66 possibly not only played a role in leaves,but also participated in the absorption and transportation of phosphorus in roots under phosphorus deficiency stress.