【摘要】 【目的】鉴定苹果(Malus domestica Borkh.)基因组上132个WRKY基因,为研究苹果WRKY转录因子在非生物和生物胁迫以及生长和发育过程中的调控作用奠定相关理论基础,也为进一步分析苹果WRKY基因提供信息。【方法】利用HMMER 3.0软件,通过WRKY保守域全蛋白序列PF03106用于鉴定苹果WRKY基因。采用Web Logo3、DNAMAN 5.0、Map Inspect、MEME和MEGA5.1等软件对其蛋白序列进行生物信息学分析。采用RT-PCR技术检测苹果WRKY基因的组织表达情况。【结果】鉴定得到132个苹果WRKY基因。分组鉴定和进化树分析结果显示,苹果WRKY蛋白分为I、II和III类型,I组共有24个成员可进一步分为I-C和I-N亚组,其锌指结构是C2H2类型(CX4CX22-23HXH)。II组含有1个WRKY区域共有79个成员,可进一步分为II-a、II-b、II-c、II-d和II-e亚组,分别有8、12、31、14和14个成员,其锌指结构为C2H2类型(CX4-5CX23HXH)。III组共有29个成员,其锌指结构为C2HC类型(CX7CX23-24HXC);WRKY结构域分析显示,其高度保守,绝大多数都含有WRKYGQK七肽和锌指结构;染色体定位分析显示,苹果WRKY分布于苹果17条染色体中,呈不均匀分布。染色体1和9上分布最多,为13个;其次是染色体12,分布12个;染色体2、5和14分布最少,为4个;基因结构分析表明,Md WRKY基因家族多数由2—5个外显子组成,基因结构进化高度保守;保守元件分析表明,Md WRKY基因家族包含10个保守元件:元件1—6为WRKY盒;元件7—10为未知盒。Md WRKY基因家族都包含有WRKY盒,I组中含有2个WRKY盒,II-a和II-b亚组中含有未知元件8,III组中含有未知元件7和9。半定量结果显示,12个Md WRKY均在根、茎、叶、花和果中表达,且呈现出多种相对表达模式。【结论】苹果WRKY基因家族结构高度保守,可能参与调控苹果生长和发育等过程。更多还原

【Abstract】 【Objective】In this study, 132 putative WRKY proteins in the apple(Malus domestica Borkh.) genome were identified, so as to provide a basis for studying the theoretical roles of WRKY genes in the regulation of plant stress responses, growth and development, and to provide valuable information for WRKY genes cloning in apple.【Method】WRKY DNA-binding domain(PF03106) downloaded from Pfam protein families database was employed to identify WRKY genes from apple genome using HMMER 3.0. The obtained amino acid sequences were analyzed with the bioinformatics softwares, including DNAMAN 5.0, Weblogo 3, MEGA 5.1, Map Inspect and MEME. RT-PCR was used to detect WRKY genes expression in different tissues of apple. 【Result】 Totally 132 apple WRKY genes were found in apple genome. The result of group identification and phylogenetic analysis revealed that apple WRKY genes were classified into Group I, Group II and Group III. Twenty-four Md WRKY proteins with two WRKY domains(group I-N and group I-C) containing CX4CX22-23 HXH zinc-finger motif belonged to Group I. Seventy-nine Group II Md WRKY proteins had a single WRKY domain including CX4–5CX23HXH zinc-finger motif and could be further divided into five subgroups(Group II-a: 8 members, Group II-b: 12 members, Group II-c: 31 members, Group II-d: 14 members, and Group II-e: 14 members, respectively), whereas 29 Group III Md WRKY proteins contained a single WRKY domain with CX7CX23–24HXC zinc-finger motif. The results of domain analysis indicated that the WRKY regions contained a highly conserved heptapeptide stretch WRKYGQK at its N-terminus followed by a zinc-finger motif. Chromosome mapping analysis showed that apple WRKY genes were distributed with different densities on 17 chromosomes. The largest number of apple WRKY genes were found on chromosomes 1 and 9(thirteen genes), followed by chromosome 12(twelve genes). Only 4 genes located on chromosomes 2, 5 and 14. The results of gene structure analysis revealed that most of the WRKY gene contained 2-5 exons and WRKY gene structure were highly conserved in apple. Conserved motif analysis showed that the conserved motifs 1-6, which specify the WRKY domain, were observed in all apple WRKY proteins, motif 8 and motifs 7 and 9 as the unknown domain were observed in Group II-a and II-b and Group III, respectively. Two WRKY domains were assigned to Group I. RT-PCR results indicated that 12 Md WRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels.【Conclusion】These results suggested that Md WRKY gene family was highly and structurally conserved, and may be involved into the regulation of growth and development processes in apple.更多还原