苹果WRKY基因家族生物信息学及表达分析

doi:10.3864/j.issn.0578-1752.2015.16.012

摘要/Abstract

摘要： 【目的】鉴定苹果（Malus domesticaBorkh.）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WebLogo 3、DNAMAN 5.0、MapInspect、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（CX₄CX_22-23HXH）。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（CX_4-5CX₂₃HXH）。III组共有29个成员，其锌指结构为C₂HC类型（CX₇CX_23-24HXC）；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，MdWRKY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，MdWRKY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。MdWRKY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MdWRKY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。

关键词: 苹果, WRKY, 转录因子, 生物信息学, 基因家族

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, MapInspect 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 MdWRKY proteins with two WRKY domains (group I-N and group I-C) containing CX₄CX_22-23HXH zinc-finger motif belonged to Group I. Seventy-nine Group II MdWRKY proteins had a single WRKY domain including CX_4–5CX₂₃HXH 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 MdWRKY proteins contained a single WRKY domain with CX₇CX_23–24HXC 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 MdWRKY genes were expressed in roots, stems, leaves, flowers and fruits at various expression levels.【Conclusion】These results suggested that MdWRKY gene family was highly and structurally conserved, and may be involved into the regulation of growth and development processes in apple.

Key words: apple, WRKY, transcription factor, bioinformatics, gene family

谷彦冰，冀志蕊，迟福梅，乔壮，徐成楠，张俊祥，董庆龙，周宗山. 苹果WRKY基因家族生物信息学及表达分析[J]. 中国农业科学, 2015, 48(16): 3221-3238.

GU Yan-bing, JI Zhi-rui, CHI Fu-mei, QIAO Zhuang, XU Cheng-nan, ZHANG Jun-xiang, DONG Qing-long, ZHOU Zong-shan. Bioinformatics and Expression Analysis of the WRKY Gene Family in Apple[J]. Scientia Agricultura Sinica, 2015, 48(16): 3221-3238.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2015.16.012

https://www.chinaagrisci.com/CN/Y2015/V48/I16/3221

参考文献

[1] Eulgem T, Rushton P J, Robatzek S, Somssich I E. The WRKY superfamily of plant transcription factors. Trends in Plant Science, 2000, 5(5): 199-206.

[2] Rushton P J, Somssich I E, Ringler P, Shen Q J. WRKY transcription factors. Trends in Plant Science, 2010, 15(5): 247-258.

[3] Sun C, Palmqvist S, Olsson H, Boren M, Ahlandsberg S, Jansson C. A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar responsive elements of the iso1 promoter. The Plant Cell, 2003, 15(9): 2076-2092.

[4] Zhang Y, Wang L. The WRKY transcription factor superfamily: its origin in eukaryotes and expansion in plants. BMC Evolution Biology, 2005, 5(1): 1.

[5] Chen L, Song Y, Li S, Zhang L, Zou C, Yu D. The role of WRKY transcription factors in plant abiotic stresses. Biochimica et Biophysica Acta (BBA)-Gene Regulatory Mechanisms, 2012, 1819(2): 120-128.

[6] Ishiguro S, Nakamura K. Characterization of a cDNA encoding a novel DNA-binding protein, SPF1, that recognizes SP8 sequences in the 5' upstream regions of genes coding for sporamin and beta-amylase from sweet potato. Molecular and General Genetics, 1994, 244(6): 563-571.

[7] Rushton PJ, Macdonald H, Huttly A K, Lazarus C M, Hooley R. Members of a new family of DNA-binding proteins bind to a conserved cis-element in the promoters of a-Amy2 genes. Plant Molecular Biology, 1995, 29(4): 691-702.

[8] Rushton P J, Torres J T, Parniske M, Wernert P, Hahlbrock K Somssich I E. Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO Journal, 1996, 15(20): 5690-5700.

[9] de Pater S, Greco V, Pham K, Memelink J, Kijine J. Characterization of a zinc-dependent transcriptional activator from Arabidopsis. Nucleic Acids Research, 1996, 24(23): 4624-4631.

[10] Johnson S C, Kolevski B, Smyth D R. Transparent testa glabra 2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. The Plant Cell, 2002, 14(6): 1359-1375.

[11] Lagace M, Matton D P. Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta, 2004, 219(1): 185-189.

[12] Robatzek S, Somssich I E. Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes & Development, 2002, 16(9): 1139-1149.

[13] Xie Z, Zhang Z L, Zou X, Yang G, Komatsu S, Shen Q J. Interactions of two abscisic-acid induced WRKY genes in repressing gibberellin signaling in aleurone cells. The Plant Journal, 2006, 46(2): 231-242.

[14] Dong J, Chen C, Chen Z. Expression profiles of the Arabidopsis WRKY gene superfamily during plant defense response. Plant Molecular Biology, 2003, 51(1): 21-37.

[15] Ulker B, Somssich I E. WRKY transcription factors: from DNA binding towards biological function. Current Opinion in Plant Biology, 2004, 7(5): 491-498.

[16] Ross C A, Liu Y, Shen Q J. The WRKY gene family in rice (Oryza sativa). Journal of Integrative Plant Biology, 2007, 49(6): 827-842.

[17] Huang S, Gao Y, Liu J, Peng X, Niu X, Fei Z, Cao S, Liu Y. Genome-wide analysis of WRKY transcription factors in Solanum lycopersicum. Molecular Genetics and Genomics, 2012, 287(6): 495-513.

[18] Ling J, Jiang W, Zhang Y, Yu H, Mao Z, Gu X, Huang S, Xie B. Genome-wide analysis of WRKY gene family in Cucumis sativus. BMC Genomics, 2011, 12(1): 471.

[19] He H, Dong Q, Shao Y, Jiang H, Zhu S, Cheng B, Xiang Y. Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa. Plant Cell Reports, 2012, 31(7): 1199-1217.

[20] 许瑞瑞, 张世忠, 曹慧, 束怀瑞. 苹果WRKY转录因子家族基因生物信息学分析. 园艺学报, 2012, 39(10): 2049-2060.

Xu R R, Zhang S Z, Cao H, Shu H R. Bioinformatics analysis of WRKY transcription factor genes family in apple. Acta Horticulturae Sinica, 2012, 39(10): 2049-2060. (in Chinese)

[21] Dou L, Zhang X, Pang C, Song M, Wei H, Fan S, Yu S. Genome-wide analysis of the WRKY gene family in cotton. Molecular Genetics and Genomics, 2014, DOI 10.1007/s00438-01.

[22] Li H, Guo D, Yang Z, Tang X, Peng S. Genome-wide identification andcharacterization of WRKY gene family in Hevea brasiliensis. Genomics, 2014, 104(1): 14-23.

[23] Tian Y, Dong Q, Ji Z, Chi F, Cong P, Zhou Z. Genome-wide identification and analysis of the MADS-box gene family in apple. Gene, 2015, 555(2): 277-290.

[24] 董庆龙, 王海荣, 安淼, 余贤美, 王长君. 苹果NADP依赖的苹果酸酶基因克隆、序列和表达分析. 中国农业科学, 2013, 46(9): 1857-1866.

Dong Q L, Wang H R, An M, Yu X M, Wang C J. Cloning, sequence and expression analysis of NADP-malic enzyme genes in apple. Scientia Agricultura Sinica, 2013, 46(9): 1857-1866. (in Chinese)

[25] Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S. MEGA5: Molecular evolutionary genetics analysis using Maximum Likelihood, Evolutionary Distance, and Maximum Parsimony Methods. Molecular Biology and Evolution, 2011, 28(10): 2731-2739.

[26] 董庆龙, 冀志蕊, 迟福梅, 田义, 安秀红, 徐成楠, 周宗山. 苹果MADS-box转录因子的生物信息学及其在不同组织中的表达. 中国农业科学, 2014, 47(6): 1151-1161.

Dong Q L, Ji Z R, Chi F M, Tian Y, An X H, Xu C N, Zhou Z S. Bioinformatics of the MADS-box transcription factor and their expression in different apple tissues. Scientia Agricultura Sinica, 2014, 47(6): 1151-1161. (in Chinese)

[27] Hu L, Liu S. Genome-wide analysis of the MADS-box gene family in cucumber. Genome, 2012, 55: 245-256.

[28] Velasco R, Zharkikh A, Affourtit J, Dhingra A, Cestaro A, Kalyanaraman A, Fontana P, Bhatnagar S K, Troggio M, Pruss D, Salvi S, Pindo M, Baldi P, Castelletti S, Cavaiuolo M, Coppola G, Costa F, Cova V, Dal Ri A, Goremykin V, Komjanc M, Longhi S, Magnago P, Malacarne G, Malnoy M, Micheletti D, Moretto M, Perazzolli M, Si-Ammour A, Vezzulli S, Zini E, Eldredge G, Fitzgerald L M, Gutin N, Lanchbury J, Macalma T, Mitchell J T, Reid J, Wardell B, Kodira C, Chen Z, Desany B, Niazi F, Palmer M, Koepke T, Jiwan D, Schaeffer S, Krishnan V, Wu C, Chu V T, King S T, Vick J, Tao Q, Mraz A, Stormo A, Stormo K, Bogden R, Ederle D, Stella A, Vecchietti A, Kater M M, Masiero S, Lasserre P, Lespinasse Y, Allan A C, Bus V, Chagne D, Crowhurst R N, Gleave A P, Lavezzo E, Fawcett J A, Proost S, Rouze P, Sterck L, Toppo S, Lazzari B, Hellens R P, Durel C E, Gutin A, Bumgarner R E, Gardiner S E, Skolnick M, Egholm M, Van de Peer Y, Salamini F, Viola R. The genome of the domesticated apple (Malus×domestica Borkh.). Nature Genetics, 2010, 42(10): 833-839.

[29] Duan M R, Nan J, LiangY H, Mao P, Lu L, Li L, Wei C, Lai L, Li Y, Su X D. DNA binding mechanism revealed by high resolution crystal structure of Arabidopsis thaliana WRKY1 protein. Nucleic Acids Research, 2007, 35(4): 1145-1154.

[30] Cannon S B, Mitra A, Baumgarten A, Young N D, May G. The roles of segmental and tandem gene duplication in the evolution of large gene families in Arabidopsis thaliana. BMC Plant Biology, 2004, 4(1): 10.

[31] Zhang S, Xu R, Luo X, Jiang Z, Shu H. Genome-wide identification and expression analysis of MAPK and MAPKK gene family in Malus domestica. Gene, 2013, 531(2): 377-387.

[32] Li X, Guo R, Li J, Singer S D, Zhang Y, Yin X, Wang X. Genome- wide identification and analysis of the aldehyde dehydrogenase (ALDH) gene superfamily in apple (Malus×domestica Borkh.). Plant Physiology and Biochemistry, 2013, 71: 268-282.

[33] Su H, Zhang S, Yuan X, Chen C, Wang X F, Hao Y J. Genome-wide analysis and identification of stress-responsive genes of the NAM-ATAF1, 2-CUC2 transcription factor family in apple. Plant Physiology and Biochemistry, 2013, 71: 11-21.

[34] Wang X, Zhang S, Su L, Liu X, Hao Y. A genome-wide analysis of the LBD (LATERAL ORGAN BOUNDARIES Domain) gene family in Malus domestica with a functional characterization of MdLBD11. Plos One, 2013, 8(2): e57044.

[35] Cao Z H, Zhang S Z, Wang R K, Zhang R F, Hao Y J. Genome wide analysis of the apple MYB transcription factor family allows the identification of MdoMYB121 gene confering abiotic stress tolerance in plants. Plos One, 2013, 8(7): e69955.

[36] Yuan H Z, Zhao K, Lei H J, Shen X J, Liu Y, Liao X, Li T H. Genome-wide analysis of the GH3 family in apple (Malus×domestica). BMC Genomics, 2013, 14(1): 297.