CsWRKY22启动子的克隆及响应柑橘溃疡病菌侵染的表达特征
|
摘要
由柑橘黄单胞杆菌致病变种(Xanthomonas citri subsp. citri,Xcc)引起的柑橘溃疡病严重危害着柑橘产业的健康可持续发展。前期研究表明,CsWRKY22可能是溃疡病感病基因。从‘晚锦橙’(Citrus sinensis Osbeck)中克隆了CsWRKY22的两个等位启动子pCsWRKY22-1和pCsWRKY22-2,长度分别为1 428和1406bp。序列分析表明两个等位启动子序列一致性为91.76%。两个等位启动子含有多种参与植物防御反应和生长发育的顺式作用元件,相同元件的数量和位置基本一致,不同的是,在TATA-box下游,pCsWRKY22-1含有2个22 bp长的TA-rich转录增强序列,而pCsWRKY22-2只含有1个。构建CsWRKY22启动子控制GUS报告基因的植物表达载体pC sW RKY22::GUS,并用农杆菌介导法转化‘晚锦橙’,经GFP活性检测和PCR鉴定获得pCsWRKY22-1::GUS和pCsWRKY22-2::GUS转基因植株各8株和4株。GUS组织化学染色、GUS酶活性及定量PCR分析表明,CsWRKY22启动子具有较强的机械伤诱导活性和柑橘溃疡病病原菌诱导活性,同时具有一定水平的基础表达特性。pCsWRKY22-1机械伤诱导活性高于pCsWRKY22-2,而pCsWRKY22-2溃疡病菌诱导活性高于pCsWRKY22-1。
Citrus canker disease,induced by Xanthomonas citri subsp. citri(Xcc),is threating citrus industry in the world. Previous work has shown that the CsWRKY22 may be a susceptible gene for citrus canker. Two allelic promoters of the CsWRKY22 gene,named pCsWRKY22-1 and pCsWRKY22-2,were cloned from Wanjincheng orange(Citrus sinensis Osbeck). Sequencing showed that the lengths of pCsWRKY22-1 and pCsWRKY22-2 promoter obtained were 1 428 and 1 406 bp,respectively. Sequence analysis showed that the similarity of these two allelic promoters was 91.76%. PlantCare predication revealed that the two promoters contain multiple cis-acting elements,which were involved in plant defense response and development. Although some single nucleotide polymorphisms(SNPs)and indels(insert and deletion)were detected in their sequences,no obvious difference was found in numbers and locations of the elements between the two promoters. The obvious difference is that the pCsWRKY22-1 has two transcription enhancers"TA-rich"element just downstream of the"TATA-box"core promoter,while the pCsWRKY22-2 has only one. In order to study the function of CsWRKY22 promoters, the pCsWRKY22-1::GUS and pCsWRKY22-2::GUS vectors,in which GUS expression was controlled by pCsWRKY22-1 and pCsWRKY22-2,respectively,were constructed,and introduced into Wanjincheng orange by Agrobacterium-mediated transformation. Eight pC sW RKY22-1::GUS and four pC sW RKY22-2::GUS transgenic lines were obtained,and their transgenic nature was confirmed by GFP fluorescence and PCR analysis. GUS staining and enzyme activity and qPCR analysis showed that the two CsWRKY22 promoters possessed strong wounding-and Xcc-inducible activities. pCsWRKY22-1 displayed higher woundinginducible expression level than pCsWRKY22-2,while pCsWRKY22-2 had higher Xcc-inducible expression level. The promoters also showed some levels of basic expression in leaf and stem tissues in transgenic plants before inoculation.
Citrus canker disease,induced by Xanthomonas citri subsp. citri(Xcc),is threating citrus industry in the world. Previous work has shown that the CsWRKY22 may be a susceptible gene for citrus canker. Two allelic promoters of the CsWRKY22 gene,named pCsWRKY22-1 and pCsWRKY22-2,were cloned from Wanjincheng orange(Citrus sinensis Osbeck). Sequencing showed that the lengths of pCsWRKY22-1 and pCsWRKY22-2 promoter obtained were 1 428 and 1 406 bp,respectively. Sequence analysis showed that the similarity of these two allelic promoters was 91.76%. PlantCare predication revealed that the two promoters contain multiple cis-acting elements,which were involved in plant defense response and development. Although some single nucleotide polymorphisms(SNPs)and indels(insert and deletion)were detected in their sequences,no obvious difference was found in numbers and locations of the elements between the two promoters. The obvious difference is that the pCsWRKY22-1 has two transcription enhancers"TA-rich"element just downstream of the"TATA-box"core promoter,while the pCsWRKY22-2 has only one. In order to study the function of CsWRKY22 promoters, the pCsWRKY22-1::GUS and pCsWRKY22-2::GUS vectors,in which GUS expression was controlled by pCsWRKY22-1 and pCsWRKY22-2,respectively,were constructed,and introduced into Wanjincheng orange by Agrobacterium-mediated transformation. Eight pC sW RKY22-1::GUS and four pC sW RKY22-2::GUS transgenic lines were obtained,and their transgenic nature was confirmed by GFP fluorescence and PCR analysis. GUS staining and enzyme activity and qPCR analysis showed that the two CsWRKY22 promoters possessed strong wounding-and Xcc-inducible activities. pCsWRKY22-1 displayed higher woundinginducible expression level than pCsWRKY22-2,while pCsWRKY22-2 had higher Xcc-inducible expression level. The promoters also showed some levels of basic expression in leaf and stem tissues in transgenic plants before inoculation.
引文
Ayliffe Michael A,Roberts James K,Mitchell Heidi J,Zhang Ren,Lawrence Gregory J,Ellis Jeffrey G,Pryor Tony J.2002.A plant gene up-regulated at rust infection sites.Plant Physiology,129(1):169-180.
Das A K.2003.Citrus canker-a review.Journal of Applied Horticulture,5(1):52-60.
Duan Min-jie.2016.Function research of the susceptibility gene CsLOB1 in citrus canker resistance[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)段敏杰.2016.感病基因CsLOB1在柑橘溃疡病抗性中的功能研究[硕士论文].重庆:西南大学.
Figueroa P,Browse J.2012.The Arabidopsis JAZ2 promoter contains a G-Box and thymidine-rich module that are necessary and sufficient for jasmonate-dependent activation by MYC transcription factors and repression by JAZ proteins.Plant&Cell Physiology,53(53):330-343.
Garretón V,Carpinelli J,Jordana X,Holuigue L.2002.The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species.Plant Physiology,130(3):1516-1526.
Gu R,Liu X F,Zhao W S,Yan S S,Sun L H,Wu B N,Zhang X L.2018.Functional characterization of the promoter and second intron of CUM1during flower development in cucumber(Cucumis sativus L.).Horticultural Plant Journal,4(3):103-110.
Han Shuang,Liu Rui-xia,Zhang Zhao-he,Chen Su-mei,Jiang Jia-fu,Fang Wei-min,Liao Yuan,Chen Fa-di.2013.Cloning of chlorophyll a/b binding protein CmLhcb1 and Promoter from Chrysanthemum morifolium and expression analysis.Acta Horticulturae Sinica,40(6):1119-1128.(in Chinese)韩霜,刘瑞霞,张兆和,陈素梅,蒋甲福,房伟民,廖园,陈发棣.2013.菊花叶绿素a/b结合蛋白基因CmLhcb1及其启动子的克隆和表达分析.园艺学报,40(6):1119-1128.
Huang Wen-ping,Chen Xin-ping,Wu Tie-hua.2009.Occurrence status and control countermeasures of citrus cnker disease in Chaling County.Hunan Agricultural Sciences,(3):62-63.(in Chinese)黄文平,陈新平,吴铁华.2009.茶陵县柑橘溃疡病发生现状及其防控对策.湖南农业科学,(3):62-63.
Kuiper H A,Kleter G A,Noteborn H P,Noteborn J M,Kok E J.2010.Assessment of the food safety issues related to genetically modified foods.Plant Journal,27(6):503-528.
Mengiste T,Chen X,Salmeron J,Dietrich R.2003.The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis.Plant Cell,15(11):2551-2565.
Morris S H,Adley C C.2001.Irish public perceptions and attitudes to modern biotechnology:an overview with a focus on GM foods.Trends in Biotechnology,19(2):43-48.
Murashige T,Skoog F.1962.A revised medium for rapid growth and bio assays with tobacco tissue cultures.Physiol Plant,15:473-497.
Ning Hui-yu,Jiang Min,Zhang Bing-lin,Zou Hua-wen.2018.cloning and functional analysis of maize ZmLTP3 gene promoter.Jiangsu Agricultural Sciences,46(4):43-46.(in Chinese)宁慧宇,江敏,张丙林,邹华文.2018.玉米ZmLTP3基因启动子的克隆及功能分析.江苏农业科学,46(4):43-46.
Park H C,Kim M L,Kang Y H,Jeon J M,Yoo J H,Kim M C,Park C Y,Jeong J C,Moon B C,Lee J H,Yoon H W,Lee S H,Chung WS,Lim C O,Lee S Y,Hong J C,Cho M J.2004.Pathogen-and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1box that interacts with a GT-1-like transcription factor.Plant Physiology,135(4):2150-2161.
Rojas A,Almoguera C,Carranco R,Scharf K D,Jordano J.2002.Selective activation of the developmentally regulated Ha hsp17.6 G1 promoter by heat stress transcription factors.Plant Physiology,129(3):1207-1215.
Rushton P J,Torres J T,Parniske M,Wernert P,Hahbrock K,Somssich I E.1996.Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.EMBO Journal,15(20):5690-5700.
Rushton P J,Reinst?dler A,Lipka V,Lippok B,Somssich I E.2002.Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen-and wound-induced signaling.Plant Cell,14(4):749-762.
Sarah J G,Rushton P J.2005.Engineering plants with increased disease resistance:how are we going to express it?TRENDS in Biotechnology,23(6):283-290.
Yanagisawa S.2002.The Dof family of plant transcription factors.Trends in Plant Science,7(12):555-560.
Yoon I S,Park D H,Mori H,Imaseki H,Kang B G.1999.Characterization of an auxin-inducible 1-aminocyclopropane-1-carboxylate synthase gene,VR-ACS6,of mungbean[Vigna radiata(L.)Wilczek]and hormonal interactions on the promoter activity in transgenic tobacco.Plant&Cell Physiology,40(4):431.
Zhang Shun-cang.2014.Cloning and characterization of relation genes involved in the regulation of phenolic acids biosynthesis in Salvia miltiorrhiza Bunge[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)张顺仓.2014.丹参酚酸类成分生源合成调控相关基因的克隆与功能研究[博士论文].杨凌:西北农林科技大学.
Zhang Y,Fan W,Kinkema M,Li X,Dong X.1999.Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene.Proceedings of the National Academy of Sciences of the United States of America,6(11):6523-6528.
Zhou Peng-fei.2017.Screening and functional analysis of WRKY transcription factor and pathogenesis-related protein genes associated with citrus canker[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)周鹏飞.2017.柑橘溃疡病相关WRKY转录因子和PR基因的筛选与功能分析[硕士论文].重庆:西南大学.
Zhou Peng-fei,Jia Rui-rui,Chen Shan-chun,Xu Lan-zhen,Peng Ai-hong,Lei Tian-gang,Li Qiang,Chen Min,Bai Xiao-jing,Zou Xiu-ping,HE Yong-rui.2017.Cloning and expression analysis of four citrus WRKY genes responding to Xanthomon asaxonopodis pv.citri.Acta Horticulturae Sinica,44(3):452-462.(in Chinese)周鹏飞,贾瑞瑞,陈善春,许兰珍,彭爱红,雷天刚,李强,陈敏,白晓晶,邹修平,何永睿.2017.柑橘4个WRKY转录因子基因的克隆及其响应柑橘溃疡病菌侵染的表达分析.园艺学报,44(3):452-462.
Zhu Q,Doerner P W,Lamb C J.1993.Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco.Plant Journal,3(2):203-212.
Zou X,Li D,Luo X,Pei Y.2008.An improved procedure for Agrobacterium-mediated transformation of trifoliate orange(Poncirus trifoliata L.Raf.)via indirect organogenesis.Vitro Cellular&Developmental Biology Plant,44(3):169-177.
Zou X,Song E,Peng A,He Y,Xu L,Lei T,Yao L,Chen S.2014.Activation of three pathogen-inducible promoters in transgenic citrus(Citrus sinensis Osbeck)after Xanthomonas axonopodis pv.citri infection and wounding.Plant.Cell Tissue&Organ Culture,117(1):85-98.
Das A K.2003.Citrus canker-a review.Journal of Applied Horticulture,5(1):52-60.
Duan Min-jie.2016.Function research of the susceptibility gene CsLOB1 in citrus canker resistance[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)段敏杰.2016.感病基因CsLOB1在柑橘溃疡病抗性中的功能研究[硕士论文].重庆:西南大学.
Figueroa P,Browse J.2012.The Arabidopsis JAZ2 promoter contains a G-Box and thymidine-rich module that are necessary and sufficient for jasmonate-dependent activation by MYC transcription factors and repression by JAZ proteins.Plant&Cell Physiology,53(53):330-343.
Garretón V,Carpinelli J,Jordana X,Holuigue L.2002.The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species.Plant Physiology,130(3):1516-1526.
Gu R,Liu X F,Zhao W S,Yan S S,Sun L H,Wu B N,Zhang X L.2018.Functional characterization of the promoter and second intron of CUM1during flower development in cucumber(Cucumis sativus L.).Horticultural Plant Journal,4(3):103-110.
Han Shuang,Liu Rui-xia,Zhang Zhao-he,Chen Su-mei,Jiang Jia-fu,Fang Wei-min,Liao Yuan,Chen Fa-di.2013.Cloning of chlorophyll a/b binding protein CmLhcb1 and Promoter from Chrysanthemum morifolium and expression analysis.Acta Horticulturae Sinica,40(6):1119-1128.(in Chinese)韩霜,刘瑞霞,张兆和,陈素梅,蒋甲福,房伟民,廖园,陈发棣.2013.菊花叶绿素a/b结合蛋白基因CmLhcb1及其启动子的克隆和表达分析.园艺学报,40(6):1119-1128.
Huang Wen-ping,Chen Xin-ping,Wu Tie-hua.2009.Occurrence status and control countermeasures of citrus cnker disease in Chaling County.Hunan Agricultural Sciences,(3):62-63.(in Chinese)黄文平,陈新平,吴铁华.2009.茶陵县柑橘溃疡病发生现状及其防控对策.湖南农业科学,(3):62-63.
Kuiper H A,Kleter G A,Noteborn H P,Noteborn J M,Kok E J.2010.Assessment of the food safety issues related to genetically modified foods.Plant Journal,27(6):503-528.
Mengiste T,Chen X,Salmeron J,Dietrich R.2003.The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis.Plant Cell,15(11):2551-2565.
Morris S H,Adley C C.2001.Irish public perceptions and attitudes to modern biotechnology:an overview with a focus on GM foods.Trends in Biotechnology,19(2):43-48.
Murashige T,Skoog F.1962.A revised medium for rapid growth and bio assays with tobacco tissue cultures.Physiol Plant,15:473-497.
Ning Hui-yu,Jiang Min,Zhang Bing-lin,Zou Hua-wen.2018.cloning and functional analysis of maize ZmLTP3 gene promoter.Jiangsu Agricultural Sciences,46(4):43-46.(in Chinese)宁慧宇,江敏,张丙林,邹华文.2018.玉米ZmLTP3基因启动子的克隆及功能分析.江苏农业科学,46(4):43-46.
Park H C,Kim M L,Kang Y H,Jeon J M,Yoo J H,Kim M C,Park C Y,Jeong J C,Moon B C,Lee J H,Yoon H W,Lee S H,Chung WS,Lim C O,Lee S Y,Hong J C,Cho M J.2004.Pathogen-and NaCl-induced expression of the SCaM-4 promoter is mediated in part by a GT-1box that interacts with a GT-1-like transcription factor.Plant Physiology,135(4):2150-2161.
Rojas A,Almoguera C,Carranco R,Scharf K D,Jordano J.2002.Selective activation of the developmentally regulated Ha hsp17.6 G1 promoter by heat stress transcription factors.Plant Physiology,129(3):1207-1215.
Rushton P J,Torres J T,Parniske M,Wernert P,Hahbrock K,Somssich I E.1996.Interaction of elicitor-induced DNA-binding proteins with elicitor response elements in the promoters of parsley PR1 genes.EMBO Journal,15(20):5690-5700.
Rushton P J,Reinst?dler A,Lipka V,Lippok B,Somssich I E.2002.Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen-and wound-induced signaling.Plant Cell,14(4):749-762.
Sarah J G,Rushton P J.2005.Engineering plants with increased disease resistance:how are we going to express it?TRENDS in Biotechnology,23(6):283-290.
Yanagisawa S.2002.The Dof family of plant transcription factors.Trends in Plant Science,7(12):555-560.
Yoon I S,Park D H,Mori H,Imaseki H,Kang B G.1999.Characterization of an auxin-inducible 1-aminocyclopropane-1-carboxylate synthase gene,VR-ACS6,of mungbean[Vigna radiata(L.)Wilczek]and hormonal interactions on the promoter activity in transgenic tobacco.Plant&Cell Physiology,40(4):431.
Zhang Shun-cang.2014.Cloning and characterization of relation genes involved in the regulation of phenolic acids biosynthesis in Salvia miltiorrhiza Bunge[Ph.D.Dissertation].Yangling:Northwest A&F University.(in Chinese)张顺仓.2014.丹参酚酸类成分生源合成调控相关基因的克隆与功能研究[博士论文].杨凌:西北农林科技大学.
Zhang Y,Fan W,Kinkema M,Li X,Dong X.1999.Interaction of NPR1 with basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene.Proceedings of the National Academy of Sciences of the United States of America,6(11):6523-6528.
Zhou Peng-fei.2017.Screening and functional analysis of WRKY transcription factor and pathogenesis-related protein genes associated with citrus canker[M.D.Dissertation].Chongqing:Southwest University.(in Chinese)周鹏飞.2017.柑橘溃疡病相关WRKY转录因子和PR基因的筛选与功能分析[硕士论文].重庆:西南大学.
Zhou Peng-fei,Jia Rui-rui,Chen Shan-chun,Xu Lan-zhen,Peng Ai-hong,Lei Tian-gang,Li Qiang,Chen Min,Bai Xiao-jing,Zou Xiu-ping,HE Yong-rui.2017.Cloning and expression analysis of four citrus WRKY genes responding to Xanthomon asaxonopodis pv.citri.Acta Horticulturae Sinica,44(3):452-462.(in Chinese)周鹏飞,贾瑞瑞,陈善春,许兰珍,彭爱红,雷天刚,李强,陈敏,白晓晶,邹修平,何永睿.2017.柑橘4个WRKY转录因子基因的克隆及其响应柑橘溃疡病菌侵染的表达分析.园艺学报,44(3):452-462.
Zhu Q,Doerner P W,Lamb C J.1993.Stress induction and developmental regulation of a rice chitinase promoter in transgenic tobacco.Plant Journal,3(2):203-212.
Zou X,Li D,Luo X,Pei Y.2008.An improved procedure for Agrobacterium-mediated transformation of trifoliate orange(Poncirus trifoliata L.Raf.)via indirect organogenesis.Vitro Cellular&Developmental Biology Plant,44(3):169-177.
Zou X,Song E,Peng A,He Y,Xu L,Lei T,Yao L,Chen S.2014.Activation of three pathogen-inducible promoters in transgenic citrus(Citrus sinensis Osbeck)after Xanthomonas axonopodis pv.citri infection and wounding.Plant.Cell Tissue&Organ Culture,117(1):85-98.