WRKY基因家族编码产物是一类含有WRKY保守结构域的转录因子(Transcription factors, TFs),在植物的生长发育、胁迫应答等过程中起着重要的作用。目前,已在多种植物中鉴定了WRKY基因家族,但在普通烟草(Nicotiana tabacum L.)中WRKY家族的系统鉴定与分析还未见报道。本研究利用WRKY保守域全蛋白序列PF03106检索普通烟草蛋白序列,获得WRKY家族候选序列共164个。利用多种生物信息学软件对该家族成员进行了系统进化树、保守结构域、亚细胞定位预测、染色体定位和组织表达等分析。分组鉴定和进化树分析结果显示,可将164个候选NtWRKY 蛋白分为Ⅰ、Ⅱ和Ⅲ 3个亚家族。其中,Ⅱ亚家族又可细分为Ⅱ-a、Ⅱ-b、Ⅱ-c、Ⅱ-d和Ⅱ-e共5个亚组。WRKY结构域分析结果显示,多数基因由2~5个外显子组成,各成员间的核心结构域高度保守,含有WRKY盒和锌指结构。亚细胞定位预测结果显示,大部分NtWRKY成员都定位在细胞核中,参与核基因的转录调控;而第Ⅲ亚家族的大部分成员(74%)定位于细胞质基质中,可能参与细胞质基因的转录调控。染色体定位结果显示,共有154个NtWRKY基因能够定位于24条染色体中,且呈不均匀分布,6号染色体上分布最多,含16个候选基因,10号染色体上分布最少,只有1个候选基因。组织表达分析结果显示,大部分NtWRKY基因在烟草根、茎、叶中都有表达,但不同基因间的表达模式存在差异,这暗示了烟草NtWRKY家族的不同成员在功能上可能具有多样性。研究还发现基因NtWRKY26、NtWRKY30、NtWRKY32的表达模式受黑胫病原菌(Phytophthora parasitica var. nicotianae)诱导,可能与烟草-黑胫病菌互作机制相关。本研究不仅为研究普通烟草WRKY 转录因子在调控生长发育过程中的作用奠定了相关理论基础,也为进一步分析普通烟草WRKY基因提供了有价值的信息。
The coding products of WRKY gene family plays important roles in plant growth and development as well as in various stress responses. They have been identified in various plants, but only few in common tobacco (Nicotiana tabacum L.). In this study, 164 putative WRKY proteins in the common tobacco genome were identified by using the conserved WRKY sequence (PF03106) from the Pfam database. Phylogenetic trees, functional domain analysis, chromosomal localization, subcellular localization and tissue expression patterns were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, MG2C and MEME. First of all, phylogenetic trees divided all the candidate genes into three subfamilies: Ⅰ, Ⅱ and Ⅲ, respectively, and subfamily Ⅱ could be further divided into five subgroups: group Ⅱ-a, -b, -c, -d and -e. Secondly, the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK followed by a zinc-finger motif. Most of the NtWRKY genes contained 2-5 exons and a highly conserved gene structure. Thirdly, 154 out of 164 NtWRKY genes were distributed with different densities on 24 chromosomes, and each subfamily with different patterns and frequency. The largest number of NtWRKY genes was found on chromosome VI, and only one on chromosome X. Fourthly, the majority of NtWRKY members located in the nucleus, with 74 percent of subfamily Ⅲ in the extracellular matrix. Lastly, the members in the same subfamily had different spatial and temporal expression profiles, with 11 NtWRKY genes in roots, stems and leaves expressed at various levels. The expression of genes NtWRKY26, NtWRKY30 and NtWRKY32 can be induced by Phytophthora nicotianae. Our research thus provides valuable information for NtWRKY gene cloning and functional characterization in common tobacco.
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