枇杷WRKY转录因子鉴定与表达分析
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摘要
以枇杷(Eriobotrya japonica)转录组数据为材料,利用生物信息学手段对枇杷WRKY转录因子进行鉴定与特征分析。分离鉴定出33条具典型WRKY结构域的序列,CDS长度分布在492～2 040bp,命名为EjWRKY01～EjWRKY33。分组鉴定和进化树分析结果显示,枇杷WRKY转录因子可分为3大类,其中Ⅰ类数量为6个,Ⅱ类和Ⅲ类的数量分别为22和5个,其中Ⅱ类又进一步分为a～e等5个亚类。WRKY结构域分析显示,WRKY结构域高度保守,绝大多数都含有WRKYGQK七肽和锌指结构。枇杷WRKY转录因子基因组DNA全长895～3 873 bp,编码的蛋白在163～679个氨基酸范围内,具有1～5个内含子。WRKY启动子顺式作用元件预测结果表明,大部分WRKY启动子序列具有典型的TATA和CAAT元件。将获得的所有WRKY基因与已报道的拟南芥WRKY进行进化比对分析发现,所有EjWRKY转录因子并没有位于独立的分支上,表明枇杷的基因序列虽与拟南芥物种有差别,但枇杷WRKY蛋白在调控植物生物和非生物逆境反应、信号传导、生长发育等方面的功能相似。此外,荧光定量PCR结果显示,大部分EjWRKY均在枇杷的根、茎、叶、花和果实中表达,但相对表达水平不同,说明WRKY家族基因在枇杷的生长发育中可能具有不同的功能。
In this study,the data of loquat transcriptome were used to identify and characterize loquat WRKY transcription factors. The results show that this study isolated 33 sequences with typical WRKY structure domains,and the CDS length was distributed in 492–2 040 bp,named EjWRKY01–EjWRKY33.The analysis results of group identification and evolutionary tree show that the loquat WRKY TF can be divided into three categories,including classⅠ,Ⅱ and Ⅲ,the number of each class was 6,22 and 5,respectively. Class Ⅱ is further divided into five subgroups(a–e). The genomic DNA sequence of EjWRKY TF is from 895 to 3 873 bp with 1–5 introns,whose encoded protein was in the range of 163–679 amino acids. The prediction results of cis-acting elements of WRKY promoter show that most WRKY promoter sequences have typical TATA and CAAT elements. The phylogenetic analysis of WRKY TF among Arabidopsis and loquat showed that many WRKY proteins clustered together,displaying close phylogenetic relationships,which indicated that although there were differences among the gene sequences of different species,the functional features of the WRKY proteins in regulating biotic and abiotic stress responses,metabolic regulation,signal transduction,growth and development and morphogenesis were similar. Furthermore,the RT-PCR results showed that most of EjWRKY genes were expressed in root,stem,leaf,flower and fruit of loquat,but the relative expression levels were different,which suggested that WRKY family genes may have different functions in the growth and development of loquat.
In this study,the data of loquat transcriptome were used to identify and characterize loquat WRKY transcription factors. The results show that this study isolated 33 sequences with typical WRKY structure domains,and the CDS length was distributed in 492–2 040 bp,named EjWRKY01–EjWRKY33.The analysis results of group identification and evolutionary tree show that the loquat WRKY TF can be divided into three categories,including classⅠ,Ⅱ and Ⅲ,the number of each class was 6,22 and 5,respectively. Class Ⅱ is further divided into five subgroups(a–e). The genomic DNA sequence of EjWRKY TF is from 895 to 3 873 bp with 1–5 introns,whose encoded protein was in the range of 163–679 amino acids. The prediction results of cis-acting elements of WRKY promoter show that most WRKY promoter sequences have typical TATA and CAAT elements. The phylogenetic analysis of WRKY TF among Arabidopsis and loquat showed that many WRKY proteins clustered together,displaying close phylogenetic relationships,which indicated that although there were differences among the gene sequences of different species,the functional features of the WRKY proteins in regulating biotic and abiotic stress responses,metabolic regulation,signal transduction,growth and development and morphogenesis were similar. Furthermore,the RT-PCR results showed that most of EjWRKY genes were expressed in root,stem,leaf,flower and fruit of loquat,but the relative expression levels were different,which suggested that WRKY family genes may have different functions in the growth and development of loquat.
引文
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Bakshi M,Oelmuller R.2014.WRKY transcription factors:jack of many trades in plants.Plant Signal Behav,9(1):e27700.
Chen Y F,Li L Q,Xu Q,Kong Y H,Wang H,Wu W.2009.The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis.Plant Cell,21(11):3554-3566.
Chen J,Nolan T M,Ye H,Zhang M,Tong H,Xin P,Chu J,Chu C,Li Z,Yin Y.2017.Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses.The Plant Cell,29(6):1425-1439.
Dou L,Zhang X,Pang C,Song M,Wei H,Fan S,Yu S.2014.Genome-wide analysis of the WRKY gene family in cotton.Mol Genet Genomics,289:1103-1121.
Duan G F,Li L J,Liu Q L.2014.A WRKY transcription factor from Malus domestica negatively regulates dehydration stress in transgenic Arabidopsis.Acta Physiol Plant,36:541-548.
Eulgem T,Somssich I E.2007.Networks of WRKY transcription factors in defense signaling.Curr Opin Plant Biol,10(4):366-371.
Fan Xiao-jiang,Guo Xiao-hua,Niu Fang-fang,Yang Bo,Jiang Yuan-qing.2018.Exploring the transcriptional activity and interacting proteins of WRKY61 transcription factors in Arabidopsis thaliana.Bot Boreal-Occident Sin,38(1):1-8.(in Chinese)范晓江,郭小华,牛芳芳,杨博,江元清.2018.拟南芥WRKY61转录因子的转录活性与互作蛋白分析.西北植物学报,38(1):1-8.
Gu Yan-bing,Ji Zhi-rui,Chi Fu-mei,Qiao Zhuang,Xu Cheng-nan,Zhang Jun-xiang,Dong Qing-long,Zhou Zong-shan.2015.Bioinformatics and expression analysis of the WRKY gene family in apple.Scientia Agricultura Sinica,48(16):3221-3238.(in Chinese)谷彦冰,冀志蕊,迟福梅,乔壮,徐成楠,张俊祥,董庆龙,周宗山.2015.苹果WRKY基因家族生物信息学及表达分析.中国农业科学,48(16):3221-3238.
Gu Yan-bing,Ji Zhi-rui,Chi Fu-mei,Qiao Zhuang,Xu Cheng-nan,Zhang Jun-xiang,Zhou Zong-shan,Dong Qing-long.2016.Genome-wide identification and expression analysis of the WRKY gene family in peach.Hereditas(Beijing),38(3):254-270.(in Chinese)谷彦冰,冀志蕊,迟福梅,乔壮,徐成楠,张俊祥,周宗山,董庆龙.2016.桃WRKY基因家族全基因组鉴定和表达分析.遗传,38(3):254-270.
He H,Dong Q,Shao Y H,Jiang H Y,Zhu S W,Cheng B J,Xiang Y.2012.Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa.Plant Cell Rep,31(7):1199-1217.
Huang S,Gao Y,Liu J,Peng X,Niu X,Fei Z,Cao S,Liu Y.2012.Genome-wide analysis of WRKY transcription factors in Solanumly copersicum.Mol Genet Genomics,287(6):495-513.
Huang X S,Li K Q,Xu X Y,Yao Z H,Jin C,Zhang S.2015.Genome-wide analysis of WRKY transcription factors in white pear(Pyrus bretschneideri)reveals evolution and patterns under drought stress.BMC Genomics,16:1104.
Jing Z B,Liu Z D.2018.Genome-wide identification of WRKY transcription factors in kiwifruit(Actinidia spp.)and analysis of WRKY expression in responses to biotic and abiotic stresses.Genes&Genomics,40(4):429-446.
Jiang W,Yu D.2009.Arabidopsis WRKY2 transcription factor mediatesseed germination and postgermination arrest of development by abscisic acid.BMC Plant Biology,9:96.
Lai Z,Vinod K M,Zheng Z,Fan B F,Chen Z X.2008.Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens.BMC Plant Biology,8(1):1-13.
Li S,Fu Q,Chen L,Huang W,Yu D.2011.Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance.Planta,233:1237-1252.
Ling J,Jiang W,Zhang Y,Yu H,Mao Z,Gu X,Huang S,Xie B.2011.Genome-wide analysis of WRKY gene family in Cucumis sativus.BMCGenomics,12(1):471.
Miao Li-xiang,Zhang Yu-chao,Yang Xiao-fang,Jiang Gui-hua.2012.Genome-wide identification and analysis of WRKY transcription factors in Fragaria vesca.Journal of Nuclear Agricultural Sciences,26(8):1124-1131.(in Chinese)苗立祥,张豫超,杨肖芳,蒋桂华.2012.森林草莓全基因组WRKY转录因子基因的鉴定与分析.核农学报,26(8):1124-1131.
Okay S,Derelli E,Unver T.2014.Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress.Mol Genet Genomics,289:765-781.
Pandey S P,Somssich I E.2009.The role of WRKY transcription factors in plant immunity.Plant Physiol,150(4):1648-1655.
Qiao Yong-gang,Chen Liang,Cui Fen-fen,Cao Ya-ping,Wang Yong-fei,Song Yun.2018.Mining and Analysis of WRKY Transcription Factors in Transcriptome-based Lonicera japonica Thunb.Molecular Plant Breeding,http://kns.cnki.net/kcms/detail/46.1068.S.20180626.1121.012.html.(in Chinese)乔永刚,陈亮,崔芬芬,曹亚萍,王勇飞,宋芸.2018.基于转录组金银花WRKY转录因子的挖掘与分析.分子植物育种,http://kns.cnki.net/kcms/detail/46.1068.S.20180626.1121.012.html.
Rushton P J,Somssich I E,Ringler P,Shen Q J.2010.WRKY transcription factors.Trends Plant Sci,15(5):247-258.
Schmutz J,Cannon S B,Schlueter J,Ma J,Mitros T,Nelson W,Hyten D L,Song Q,Thelen J J,Cheng J,Xu D,Hellsten U,May G D,Yu Y,Sakurai T,Umezawa T,Bhattacharyya M K,Sandhu D,Valliyodan B,Lindquist E,Peto M,Grant D,Shu S,Goodstein D,Barry K,Futrell-Griggs M,Abernathy B,Du J,Tian Z,Zhu L,Gill N,Joshi T,Libault M,Sethuraman A,Zhang X C,Shinozaki K,Nguyen H T,Wing R A,Cregan P,Specht J,Grimwood J,Rokhsar D,Stacey G,Shoemaker R C,Jackson S A.2010.Genome sequence of the palaeopolyploid soybean.Nature,463(7278):178-183.
Song Hui,Nan Zhi-biao.2014.Genome-wide identification and analysis of WRKY transcription factors in Medicago truncatula.Hereditas(Beijing),36(2):152-168.(in Chinese)宋辉,南志标.2014.蒺藜苜蓿全基因组中WRKY转录因子的鉴定与分析.遗传,36(2):152-168.
Song Y,Ai C R,Jing S J,Yu D Q.2010.Research progress on functional analysis of rice WRKY genes.Rice Sci,17(1):60-72.
Wang L,Zhu W,Fang L,Sun X,Su L,Liang Z,Wang N,Londo JP,Li S,Xin H.2014.Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.BMC Plant Biology,14:103.
Xiao Pei-lian,Feng Rui-jie,Hou Li-xia,LüXiao-tong,Zhu Dan,Liu Xin.2015.Gene cloning and expression analysis of WRKY18 in Vitis vinifera.Plant Physiology Journal Plant Physiology Journal,51(3):391-398.(in Chinese)肖培连,冯睿杰,侯丽霞,吕晓彤,朱丹,刘新.2015.葡萄WRKY18基因的克隆及表达特性分析.植物生理学报,51(3):391-398.
Xu X P,Chen C H,Fan B F,Chen Z X.2006.Physical and functional interactions between pathogen-induced Arabidopsis WRKY18,WRKY40,WRKY60 transcription factors.Plant Cell,18(5):1310-1326.
Yang Zhi-rong,Wang Xing-chun,Xue Jin-ai,Meng Ling-zhi,Li Run-zhi.2013.Identification and expression analysis of WRKY transcription factors in medicinal plant Catharanthus roseus.Chinese Journal of Biotechnology,29(6):785-802.(in Chinese)杨致荣,王兴春,薛金爱,孟令芝,李润植.2013.药用植物长春花WRKY转录因子的鉴定及表达谱分析.生物工程学报,29(6):785-802.
Bakshi M,Oelmuller R.2014.WRKY transcription factors:jack of many trades in plants.Plant Signal Behav,9(1):e27700.
Chen Y F,Li L Q,Xu Q,Kong Y H,Wang H,Wu W.2009.The WRKY6 transcription factor modulates PHOSPHATE1 expression in response to low Pi stress in Arabidopsis.Plant Cell,21(11):3554-3566.
Chen J,Nolan T M,Ye H,Zhang M,Tong H,Xin P,Chu J,Chu C,Li Z,Yin Y.2017.Arabidopsis WRKY46,WRKY54,and WRKY70 transcription factors are involved in brassinosteroid-regulated plant growth and drought responses.The Plant Cell,29(6):1425-1439.
Dou L,Zhang X,Pang C,Song M,Wei H,Fan S,Yu S.2014.Genome-wide analysis of the WRKY gene family in cotton.Mol Genet Genomics,289:1103-1121.
Duan G F,Li L J,Liu Q L.2014.A WRKY transcription factor from Malus domestica negatively regulates dehydration stress in transgenic Arabidopsis.Acta Physiol Plant,36:541-548.
Eulgem T,Somssich I E.2007.Networks of WRKY transcription factors in defense signaling.Curr Opin Plant Biol,10(4):366-371.
Fan Xiao-jiang,Guo Xiao-hua,Niu Fang-fang,Yang Bo,Jiang Yuan-qing.2018.Exploring the transcriptional activity and interacting proteins of WRKY61 transcription factors in Arabidopsis thaliana.Bot Boreal-Occident Sin,38(1):1-8.(in Chinese)范晓江,郭小华,牛芳芳,杨博,江元清.2018.拟南芥WRKY61转录因子的转录活性与互作蛋白分析.西北植物学报,38(1):1-8.
Gu Yan-bing,Ji Zhi-rui,Chi Fu-mei,Qiao Zhuang,Xu Cheng-nan,Zhang Jun-xiang,Dong Qing-long,Zhou Zong-shan.2015.Bioinformatics and expression analysis of the WRKY gene family in apple.Scientia Agricultura Sinica,48(16):3221-3238.(in Chinese)谷彦冰,冀志蕊,迟福梅,乔壮,徐成楠,张俊祥,董庆龙,周宗山.2015.苹果WRKY基因家族生物信息学及表达分析.中国农业科学,48(16):3221-3238.
Gu Yan-bing,Ji Zhi-rui,Chi Fu-mei,Qiao Zhuang,Xu Cheng-nan,Zhang Jun-xiang,Zhou Zong-shan,Dong Qing-long.2016.Genome-wide identification and expression analysis of the WRKY gene family in peach.Hereditas(Beijing),38(3):254-270.(in Chinese)谷彦冰,冀志蕊,迟福梅,乔壮,徐成楠,张俊祥,周宗山,董庆龙.2016.桃WRKY基因家族全基因组鉴定和表达分析.遗传,38(3):254-270.
He H,Dong Q,Shao Y H,Jiang H Y,Zhu S W,Cheng B J,Xiang Y.2012.Genome-wide survey and characterization of the WRKY gene family in Populus trichocarpa.Plant Cell Rep,31(7):1199-1217.
Huang S,Gao Y,Liu J,Peng X,Niu X,Fei Z,Cao S,Liu Y.2012.Genome-wide analysis of WRKY transcription factors in Solanumly copersicum.Mol Genet Genomics,287(6):495-513.
Huang X S,Li K Q,Xu X Y,Yao Z H,Jin C,Zhang S.2015.Genome-wide analysis of WRKY transcription factors in white pear(Pyrus bretschneideri)reveals evolution and patterns under drought stress.BMC Genomics,16:1104.
Jing Z B,Liu Z D.2018.Genome-wide identification of WRKY transcription factors in kiwifruit(Actinidia spp.)and analysis of WRKY expression in responses to biotic and abiotic stresses.Genes&Genomics,40(4):429-446.
Jiang W,Yu D.2009.Arabidopsis WRKY2 transcription factor mediatesseed germination and postgermination arrest of development by abscisic acid.BMC Plant Biology,9:96.
Lai Z,Vinod K M,Zheng Z,Fan B F,Chen Z X.2008.Roles of Arabidopsis WRKY3 and WRKY4 transcription factors in plant responses to pathogens.BMC Plant Biology,8(1):1-13.
Li S,Fu Q,Chen L,Huang W,Yu D.2011.Arabidopsis thaliana WRKY25,WRKY26,and WRKY33 coordinate induction of plant thermotolerance.Planta,233:1237-1252.
Ling J,Jiang W,Zhang Y,Yu H,Mao Z,Gu X,Huang S,Xie B.2011.Genome-wide analysis of WRKY gene family in Cucumis sativus.BMCGenomics,12(1):471.
Miao Li-xiang,Zhang Yu-chao,Yang Xiao-fang,Jiang Gui-hua.2012.Genome-wide identification and analysis of WRKY transcription factors in Fragaria vesca.Journal of Nuclear Agricultural Sciences,26(8):1124-1131.(in Chinese)苗立祥,张豫超,杨肖芳,蒋桂华.2012.森林草莓全基因组WRKY转录因子基因的鉴定与分析.核农学报,26(8):1124-1131.
Okay S,Derelli E,Unver T.2014.Transcriptome-wide identification of bread wheat WRKY transcription factors in response to drought stress.Mol Genet Genomics,289:765-781.
Pandey S P,Somssich I E.2009.The role of WRKY transcription factors in plant immunity.Plant Physiol,150(4):1648-1655.
Qiao Yong-gang,Chen Liang,Cui Fen-fen,Cao Ya-ping,Wang Yong-fei,Song Yun.2018.Mining and Analysis of WRKY Transcription Factors in Transcriptome-based Lonicera japonica Thunb.Molecular Plant Breeding,http://kns.cnki.net/kcms/detail/46.1068.S.20180626.1121.012.html.(in Chinese)乔永刚,陈亮,崔芬芬,曹亚萍,王勇飞,宋芸.2018.基于转录组金银花WRKY转录因子的挖掘与分析.分子植物育种,http://kns.cnki.net/kcms/detail/46.1068.S.20180626.1121.012.html.
Rushton P J,Somssich I E,Ringler P,Shen Q J.2010.WRKY transcription factors.Trends Plant Sci,15(5):247-258.
Schmutz J,Cannon S B,Schlueter J,Ma J,Mitros T,Nelson W,Hyten D L,Song Q,Thelen J J,Cheng J,Xu D,Hellsten U,May G D,Yu Y,Sakurai T,Umezawa T,Bhattacharyya M K,Sandhu D,Valliyodan B,Lindquist E,Peto M,Grant D,Shu S,Goodstein D,Barry K,Futrell-Griggs M,Abernathy B,Du J,Tian Z,Zhu L,Gill N,Joshi T,Libault M,Sethuraman A,Zhang X C,Shinozaki K,Nguyen H T,Wing R A,Cregan P,Specht J,Grimwood J,Rokhsar D,Stacey G,Shoemaker R C,Jackson S A.2010.Genome sequence of the palaeopolyploid soybean.Nature,463(7278):178-183.
Song Hui,Nan Zhi-biao.2014.Genome-wide identification and analysis of WRKY transcription factors in Medicago truncatula.Hereditas(Beijing),36(2):152-168.(in Chinese)宋辉,南志标.2014.蒺藜苜蓿全基因组中WRKY转录因子的鉴定与分析.遗传,36(2):152-168.
Song Y,Ai C R,Jing S J,Yu D Q.2010.Research progress on functional analysis of rice WRKY genes.Rice Sci,17(1):60-72.
Wang L,Zhu W,Fang L,Sun X,Su L,Liang Z,Wang N,Londo JP,Li S,Xin H.2014.Genome-wide identification of WRKY family genes and their response to cold stress in Vitis vinifera.BMC Plant Biology,14:103.
Xiao Pei-lian,Feng Rui-jie,Hou Li-xia,LüXiao-tong,Zhu Dan,Liu Xin.2015.Gene cloning and expression analysis of WRKY18 in Vitis vinifera.Plant Physiology Journal Plant Physiology Journal,51(3):391-398.(in Chinese)肖培连,冯睿杰,侯丽霞,吕晓彤,朱丹,刘新.2015.葡萄WRKY18基因的克隆及表达特性分析.植物生理学报,51(3):391-398.
Xu X P,Chen C H,Fan B F,Chen Z X.2006.Physical and functional interactions between pathogen-induced Arabidopsis WRKY18,WRKY40,WRKY60 transcription factors.Plant Cell,18(5):1310-1326.
Yang Zhi-rong,Wang Xing-chun,Xue Jin-ai,Meng Ling-zhi,Li Run-zhi.2013.Identification and expression analysis of WRKY transcription factors in medicinal plant Catharanthus roseus.Chinese Journal of Biotechnology,29(6):785-802.(in Chinese)杨致荣,王兴春,薛金爱,孟令芝,李润植.2013.药用植物长春花WRKY转录因子的鉴定及表达谱分析.生物工程学报,29(6):785-802.