陆地棉转录因子Gh WRKY70的克隆及表达分析
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摘要
【目的】棉花是一种重要的经济作物,雄性不育材料的创造和不育相关基因的挖掘一直是棉花育种工作的热点,Gh WRKY70基因的克隆和表达分析为该基因功能的研究奠定了基础。【方法】根据转录组测序结果,利用Primer 5设计GhWRKY70转录因子全长引物,q-PCR验证Gh WRKY70基因在不育和可育花药中的表达差异。【结果】克隆到Gh WRKY70基因全长为918 bp,定名为Gh WRKY70,Gene Bank号为MF278614。该基因编码的蛋白包含1个典型的WRKY结构域,每个WRKY结构域的C端含有1个C2HC类型的锌指蛋白结构。【结论】同源性分析表明,Gh WRKY70属于III类WRKY蛋白,与亚洲棉和可可的WRKY70的亲缘关系最近。转录因子GhWRKY70在洞A不育花药和可育花药中差异表达,表明该基因可能与雄性不育相关。
【Objective】Cotton is one of the most important economic crops. The creation of male sterile materials and the mining of infertility-related genes has been a hot topic in cotton breeding. The cloning and expressing analysis of Gh WRKY70 gene laid the foundation for the study of the gene function research.【Method】Primer 5 was used in designing primers of Gh WRKY70 gene based on the transcriptome sequencing results,q-PCR verified the difference in expression of Gh WRKY70 gene in infertile and fertile anthers.【Result】The full-legth cDNA was cloned with a length of 918 bp, named Gh WRKY70, and Gene Bank No. is MF278614. The protein encoded by this gene contains a typical WRKY domain, and the C-terminus of each WRKY domain contains a C2HC-type zinc finger protein structure. 【Conclusion】Homology analysis shows that it belongs to the class III WRKY protein, and has the closest genetic relationship to Asian cotton and cocoa WRKY70. The upland cotton transcription factor Gh WRKY70 was differentially expressed in sterile anthers and fertile anthers of Dong A, suggesting that this gene may be associated with male sterility.
【Objective】Cotton is one of the most important economic crops. The creation of male sterile materials and the mining of infertility-related genes has been a hot topic in cotton breeding. The cloning and expressing analysis of Gh WRKY70 gene laid the foundation for the study of the gene function research.【Method】Primer 5 was used in designing primers of Gh WRKY70 gene based on the transcriptome sequencing results,q-PCR verified the difference in expression of Gh WRKY70 gene in infertile and fertile anthers.【Result】The full-legth cDNA was cloned with a length of 918 bp, named Gh WRKY70, and Gene Bank No. is MF278614. The protein encoded by this gene contains a typical WRKY domain, and the C-terminus of each WRKY domain contains a C2HC-type zinc finger protein structure. 【Conclusion】Homology analysis shows that it belongs to the class III WRKY protein, and has the closest genetic relationship to Asian cotton and cocoa WRKY70. The upland cotton transcription factor Gh WRKY70 was differentially expressed in sterile anthers and fertile anthers of Dong A, suggesting that this gene may be associated with male sterility.
引文
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[18]Xiao Luo,Xiaoli Sun,Baohui Liu,et al.Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis[J].PLOS ONE,2013,8(8):1-13.
[19]Hongyan Liu,Mingpu Tan,Haijuan Yu,et al.Comparative transcriptome profiling of the fertile and sterile flower buds of a dominant genic male sterile line in sesame(Sesamum indicum L.)[J].BMC Plant Biology,2016,16:250.
[20]Jihong Hu,Guanglong Chen,Hongyuan Zhang,et al.Comparative transcript profiling of alloplasmic male-sterile lines revealed altered gene expression related to pollen development in rice (Oryza sativa L.)[J].BMC Plant Biology,2016,16:175.
[21]张丽霞,贾海燕.一种简便高效大肠杆菌感受态细胞制备方法[J].江苏农业科学,2013,41(12):41-42.
[22]张婷婷,田云,卢向阳.WRKY转录因子在植物生长发育中的调控作用[J].化学与生物工程,2014,31(8):1-5.
[23]Yiyi Wang,Lin Feng,Yuxin Zhu,et al.Comparative genomic analysis of the WRKY III gene family in populus,grape,arabidopsis and rice[J].Biology Direct,2015(10):48.
[24]冯琳.杨树WRKY54基因的克隆与转化研究[D].合肥:安徽农业大学,2015.
[2]Jian Ling,Weijie Jiang,Ying Zhang,et al.Genome-wide analysis of WRKY gene family in Cucumis sativus[J].BMC Genomics,2011,12:471.
[3]Bernard Kinuthia Karanja,Lianxue Fan,et al.Genome-wide characterization of the WRKY gene family in radish (Raphanus sativus L.) reveals its critical functions under different abiotic stresses[J].Plant Cell Rep,2017,36:1757-1773.
[4]Jing Jin,Jingjing Kong,Jianle Qiu,et al.High level of microsynteny and purifying selection affect the evolution of WRKY family in Gramineae[J].Dev Genes Evol,2016,226:15-25.
[5]Dou L,Zhang X,Pang C,et al.Genome-wide analysis of the WRKY gene family in cotton[J].Molecular Genetics and Genomics,2014,289:1103-1121.
[6]周立.棉花(Gossypium hirsutum)WRKY基因分离与鉴定[D].武汉:华中师范大学,2014.
[7]Kamal Kumar,Vikas Srivastava,Savithri Purayannur,et al.WRKY domain-encoding genes of a crop legume chickpea (Cicer arietinum):comparative analysis with Medicago truncatula WRKY family and characterization of group-III gene(s).DNA Research,2016,23(3):225-239.
[8]Bekir ülker,M.Shahid,Mukhtar·Imre E.Somssich.The WRKY70 transcription factor of Arabidopsis in Xuences both the plant senescence and defense signaling pathways[J].Planta,2007,226:125-137.
[9]Wei Wei,Yuxiu Zhang,Lu Han,et al.A novel WRKY transcriptional factor from Thlaspi caerulescens negatively regulates the osmotic stress tolerance of transgenic tobacco[J].Plant Cell Rep.,2008,27:795-803.
[10]Chen J,Nolan T,Ye H,et al.Arabidopsis WRKY46,WRKY54 and WRKY70 Transcription Factors Are Involved in Brassinosteroid-Regulated Plant Growth and Drought Response[J].Plant Cell,2017,29(6):1425.
[11]Sen S,Chakraborty J,Ghosh P,et al.Chickpea WRKY70 regulates the expression of a Homeodomain-Leucine Zipper (HD-Zip) I transcription factor CaHDZ12,which confers abiotic stress tolerance in transgenic tobacco and chickpea[J].Plant & Cell Physiology,2017.
[12]Jiang C H,Huang Z Y,Xie P,et al.Transcription factors WRKY70 and WRKY11 served as regulators in rhizobacterium Bacillus cereus AR156-induced systemic resistance to Pseudomonas syringae pv.tomato DC3000 in Arabidopsis[J].Proceedings of the Prehistoric Society,2016,41(1):156-253.
[13]燕孟娇,石嵘,郝丽芬,等.油菜黑胫病抗性相关基因BnWRKY70的功能研究[J].中国油料作物学报,2018,40(1):127-133.
[14]石嵘.利用VIGS技术进行油菜抗病相关WRKY70和LRR-RLK基因的功能验证[D].呼和浩特:内蒙古大学,2017.
[15]Ruoyu Guo,Feifei Yu,Zheng Gao,et al.GhWRKY3,a novel cotton (Gossypium hirsutum L.) WRKY gene,is involved in diverse stress responses[J].Mol Biol Rep,2011,38:49-58.
[16]Mingquan Ding,Jiadong Chen,Yurong Jiang,et al.Genome-wide investigation and transcriptome analysis of the WRKY gene family in Gossypium[J].Mol Genet Genomics,2015,290:151-171.
[17]Yujuan Zhang,Jie Chen,Jubo Liu,et al.Transcriptome Analysis of Early Anther Development of Cotton Revealed Male Sterility Genes for Major Metabolic Pathways[J].Plant Growth Regul,2015,34:223-232.
[18]Xiao Luo,Xiaoli Sun,Baohui Liu,et al.Ectopic Expression of a WRKY Homolog from Glycine soja Alters Flowering Time in Arabidopsis[J].PLOS ONE,2013,8(8):1-13.
[19]Hongyan Liu,Mingpu Tan,Haijuan Yu,et al.Comparative transcriptome profiling of the fertile and sterile flower buds of a dominant genic male sterile line in sesame(Sesamum indicum L.)[J].BMC Plant Biology,2016,16:250.
[20]Jihong Hu,Guanglong Chen,Hongyuan Zhang,et al.Comparative transcript profiling of alloplasmic male-sterile lines revealed altered gene expression related to pollen development in rice (Oryza sativa L.)[J].BMC Plant Biology,2016,16:175.
[21]张丽霞,贾海燕.一种简便高效大肠杆菌感受态细胞制备方法[J].江苏农业科学,2013,41(12):41-42.
[22]张婷婷,田云,卢向阳.WRKY转录因子在植物生长发育中的调控作用[J].化学与生物工程,2014,31(8):1-5.
[23]Yiyi Wang,Lin Feng,Yuxin Zhu,et al.Comparative genomic analysis of the WRKY III gene family in populus,grape,arabidopsis and rice[J].Biology Direct,2015(10):48.
[24]冯琳.杨树WRKY54基因的克隆与转化研究[D].合肥:安徽农业大学,2015.
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