作物学报 ›› 2015, Vol. 41 ›› Issue (04): 593-600.
杜皓,丁林云,何曼林,蔡彩平,郭旺珍*
DU Hao,DING Lin-Yun,HE Man-Lin,CAI Cai-Ping,GUO Wang-Zhen*
摘要:
WRKY蛋白属于锌指型转录调控因子,参与植物生长发育及耐逆响应。以陆地棉遗传标准系TM-1为材料,克隆GhWRKY64(KF031101)基因上游1064bp的启动子序列,并对其调控元件及功能进行分析。生物信息学分析表明, 该区域含18个组织器官表达及诱导表达关键元件,分别为6个ROOTMOTIFTAPOX1根特异调控元件,4个CACTFTPPCA1叶肉特异性调控元件,4个OSE2ROOTNODULE病菌诱导元件,2个GTIGMSCAM4盐调控元件和2个W-box胁迫应答响应元件。将该启动子与GUS基因融合,构建pBIW64:GUS植物表达载体,通过农杆菌介导叶盘转化法获得12个转基因烟草株系。选择GUS表达量最高的pBIW64-5进行转基因不同组织器官表达及诱导表达分析。GUS组织化学染色显示,苗期的转基因烟草植株在叶和根部均具有GUS活性,开花期在转基因烟草植株根、叶及叶柄均检测到GUS活性,特别在转基因烟草的根及根尖部分染色更深,在茎和花组织上未检测到GUS活性。对该转基因烟草幼苗进行黄萎病菌诱导处理,与未处理的对照相比,诱导处理后的转基因烟草幼苗其根和叶片GUS染色明显加深。结果表明,GhWRKY64上游1064bp长度的DNA序列含根和叶组织优势表达启动子,且受病原菌诱导上调表达。该启动子可为开展棉花抗黄萎病转基因研究提供调控元件。
[1]Eulgem T, Rushton P, Robatzek S, Somssich IE. The WRKY superfamily of plant transcription factor. Trends Plant Sci, 2000, 5:199–206[2]宋扬, 周军会, 张永强.植物组织特异性启动子研究.生物技术通报, 2007, 6:21–24Song Y, Zhou J H, Zhang Y Q. Research on plant tissue-specific promoter. Biotechnol Bull, 2007, 6:21–24(in Chinese with English abstract)[3]李志邈, 杨悦俭, 杨飞, 叶青静. 番茄根特异表达基因LeGRP2启动子的克隆及其在拟南芥的表达分析. 中国农业科学, 2010,43: 1877–1882Li Z M, Yang Y J, Yang F, Ye Q J. Cloning of LeGRP2 promoter from tomato that shows root-specific expression in Arabidopsis. Sci Agric Sin,2010, 43: 1877–1882 (in Chinese with English abstract)[4]杨学文, 任远, 田曾元, 张生学, 刘允军, 王国英.玉米茎特异启动子克隆及其在转基因烟草中的活性分析. 西北植物学报,2014,34:859–865Yang X W, Ren Y, Tian Z Y, Zhang S X, Liu Y J, Wang G Y. Isolation of a shoot-specific promoter in maize and its activity in transgenic tobacco. Acta BotBor-OccidScin,2014,34:859–865(in Chinese with English abstract)[5]颜彦, 林拥军. 一个水稻鞘叶特异表达启动子的克隆及功能鉴定. 农业生物技术学报, 2013, 21: 757–763Yan Y, Lin Y J. Cloning and functional analysis of a promoter with tissue specific expression in rice (Oryza sativa Lssp. japonica) sheath and lea. JAgricBiotech, 2013, 21: 757–763(in Chinese with English abstract)[6]Ulker B, Somssich I. WRKY transcription factors: from DNA binding towards biological function. CurrOpinPlant Biol, 2004, 7:491–498[7]郝中娜, 王海华, 吴学龙, 郭泽建. 水稻WRKY19基因启动子功能研究初报. 中国农学通报, 2007, 23(6):99–103Hao Z N, Wang H H, Wu X L, Guo Z J. Primary study of function of a rice WRKY19 gene promoter. Chin Agri Sci Bull, 2007, 23 (6):99–103 (in Chinese with English abstract)[8]Paterson A H, Wendel J F, Gundlach H, Guo H, Jenkins J, Jin D,Llewellyn D, Showmaker K C, Shu S, Udall J, Yoo M, Byers R,Chen W, Doron-Faigenboim A, Duke M V, Gong L, Grimwood J,Grover C, Grupp K, Hu G, Lee T, Li J, Lin L, Liu T, Marler B S,Page J T, Roberts A W, Romanel E, Sanders W S, Szadkowski E,Tan X, Tang H, Xu C, Wang J, Wang Z, Zhang D, Zhang L,Ashfari H, Bedon F, Bowers J E, Brubaker C L, Chee P W, Das S,Gingle A R, Haigler C H, Harker D, Hoffmann L V, Hovav R,Jones D C, Lemke C, Mansoor S, Rahman M, Rainville L N,Rambani A, Reddy U K, Rong J, Saranga Y, Scheffler B E,Scheffler J A, Stelly D M, Triplett B A, Deynze A V, Vaslin M FS, Waghmare V N, Walford S, Wright R J, Zaki E A, Zhang T,Dennis E S, Mayer K F X, Peterson D G, Rokhsar D S, Wang X,Schmutz J. Repeated polyploidization of Gossypium genomes and the evolution of spinnable cotton fibres. Nature, 2012, 492:423–427[9]Wang K B, Wang Z W, Li F G, Ye W W, Wang J Y, Song G L, Yue Z, Cong L, Shang H H, Zhu S L, Zou C S, LI Q, Yuan Y L, Lu C R, Wei H L, Gou C Y, Zheng Z Q, Yin Y, Zhang X Y, Liu K, Wang B, Song C, Shi N, Kohel R J, Percy R G, Yu J Z, Zhu Y X, Wang J, Yu S X. The draft genome of a diploid cotton Gossypiumraimondii. Nat Genet, 2012, 44: 1098–1103[10]Cai C P,Niu E L, Du H,Zhao L,Feng Y,Guo W Z. Genome-wide analysis of the WRKY transcriptionfactor gene family in Gossypium raimondii and theexpression of orthologs in cultivated tetraploid cotton.Crop J,2014,2:87–101[11]Paterson A H, Brubaker C, Wendel J F. A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis. Plant Mol Biol Rep, 1993, 11: 122–127[12]马雪梅, 胥晓, 李晓波, 肖向文. 农杆菌介导快速、高效获得转基因烟草纯合株系. 中国烟草学报, 2012,18(4):66–71Ma X M, Xu X, Li X B, Xiao X W. Rapid and efficient production of homozygous transgenic tobacco plants Agrobacterium tumefaciens.Acta TobSci,2012,18(4):66–71 (in Chinese with English abstract)[13]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods, 2001, 25: 402–408[14]Jefferson R A. Assaying chimeric genes in plants: The GUS gene fusion system. Plant MolBiolRep, 1987, 5: 387–405[14]王雪,谭艳平, 周杰, 王春台, 刘学群.一个烟草糖基转移酶启动子在转基因烟草植株中的表达分析.安徽农业科学, 2010, 38:9426–9428Wang X, Tang Y P, Zhou J, Wang C T, Liu X Q.Expression of a tobacco Glycosyl transferasegene’s promoter in transgenic tobacco. JAnhui AgriSci,2010,38:9426–9428(in Chinese with English abstract)[15]付永平, 周海涛, 王丕武.大豆种子特异性启动子的克隆及功能分析.西北农林科技大学学报(自然科学版), 2009, 37(12): 105–111Fu Y P, Zhou H T, Wang P W.Cloning and identification of the seed specific promoter from soybean.JNorthwest SciTech UnivAgricFor(NatSciEdn), 2009, 37(12):105–111 (in Chinese with English abstract)[16]崔同霞, 白江平, 魏桂民, 赵旭, 王蒂, 张金文. 马铃薯SGT3基因表达及其启动子功能分析. 草业学报, 2014, 23(2): 196–206Cui T X, Bai J P, Wei G M, Zhao X, Wang D, Zhang J W.Gene expression and function analysis of SGT3 promoter from Potato. Acta PraSin,2014,23(2): 196–206(in Chinese with English abstract)[17]许兰珍, 彭爱红, 何永睿, 姚利晓, 雷天刚, 刘小丰, 姜国金, 邹修平, 陈善春. 异源韧皮部特异启动子在转基因枳中的表达.园艺学报, 2014, 41: 1–8Xu L Z, Peng A H, He Y R, Yao L X, Lei T G, Liu X F, Jiang G J, Zou X P, Chen S C.Expression analysis of three phloem-specific promoters in transgenic Poncirus trifoliate. Acta HorSin, 2014, 41:1–8(in Chinese with English abstract)[18]房孝良, 刘炜, 安静,王庆国. 水稻种胚特异性启动子OsESP1的克隆及其表达特性. 作物学报, 2011, 37: 1904−1909Fang X L, Liu W, An J, Wang Q G. Isolation and characterization of an embryo-specific promoter OsESP1 from Rice. Acta AgronSin, 2011, 37: 1904−1909(in Chinese with English abstract)[19]Antonio A,Rodrigo R,Luciane M,Naiara P,Osmundo B,Sinara A,SarahM N,Marcio A,João A,Maria C, Maria F. Isolation and functional characterization of a cotton ubiquitination-related promoter and 5?UTR that drives high levels of expression in root and flower tissues.BMC Biotechnol, 2011, 11:115[20]Lightfoot D J, Orford S J, Timmis J N. Identification and characterisation of cotton boll wall-specific gene promoters for future transgenic cotton varieties. Plant MolBiolRep,2013,31: 174–184[21]Zahur M,Maqbool A, Irfan M,Younas M, Barozai K,Qaiser U,Rashid B , Husnain T, Riazuddin S. Functional analysis of cotton small heat shock protein promoter region in response to abiotic stresses in tobacco using Agrobacterium-mediated transient assay. MolBiolRep, 2009, 36:1915–1921[22]Wu A M, Hu J S., Liu J Y. Functional analysis of a cotton cellulose synthase A4 gene promoter in transgenic tobacco plants. Plant Cell Rep, 2009, 28:1539–1548[23]RyuHS,HanM,LeeSK,ChoJI,RyooN,HeuS,LeeYH,BhooSH,WangGL,HahnTR,JeonJS.AcomprehensiveexpressionanalysisoftheWRKYgenesuperfamilyinriceplantsduringdefenseresponse.Plant Cell Rep, 2006, 25:836–847[24]Wu X L, Shiroto Y, Kishitani S, Ito Y, Toriyama K.Enhanced heat and drought tolerance in transgenic rice seedlings over-expressing OsWRKY11 under the control of HSP101 promoter.Plant Cell, 2009, 28:21–30[25]Qiu Y P,Yu D Q.Over-expression of the stress induced OsWRKY45 enhances disease resistance and drought tolerance in Arabidopsis. EnvironExpBot,2009,65:35–47[26]付乾堂, 余迪求. 拟南芥AtWRKY25,AtWRKY26和AtWRKY33在非生物胁迫条件下的表达分析.遗传, 2010, 32:848–856Fu Q T, Yu D Q.Expression profiles of AtWRKY25, AtWRKY26 and AtWRKY33 under abiotic stresses. Hereditas(Beijing),2010, 32:848–856(in Chinese with English abstract)[27]王磊, 高晓清, 朱苓华, 周永力, 黎志康.植物WRKY转录因子家族基因抗病相关功能的研究进展. 植物遗传资源学报, 2011, 12: 80–85Wang L, Gao X Q, Zhu L H, Zhou Y L, Li Z K. Advances in research on function of WRKY transcription factor genes in plant resistance.JPlant GenRes, 2011, 12: 80–85(in Chinese with English abstract)[28]Wu K L, Guo Z J, Wang H H, Li J. The WRKY family of transcription factors in rice and Arabidopsis and their origins. DNA Res, 2005, 12: 9–26[29]范荣伟, 谢华, 姚磊, 马荣才.大白菜BrWRKY33基因上游调控序列的克隆及功能分析.农业生物技术学报, 2010, 18:670–675Fan R W, Xie H, Yao L, Ma R C. Cloning and expression analysis of the up-stream regulatory sequence of transcription factor gene BrWRKY33 from Chinese cabbage. JAgricBiotech,2010, 18:670–675(in Chinese with English abstract)[30]张春晓, 王文棋, 蒋湘宁, 陈雪梅.植物基因启动子研究进展.遗传学报, 2004,31: 1455–1464Zhang C X, Wang W Q, Jiang X N, Chen X M.Review on plant gene promoter. Acta GenetSin, 2004,31:1455–1464(in Chinese with English abstract) |
[1] | 巫燕飞, 胡琴, 周棋, 杜雪竹, 盛锋. 水稻延伸因子复合体家族基因鉴定及非生物胁迫诱导表达模式分析[J]. 作物学报, 2022, 48(3): 644-655. |
[2] | 李文兰, 李文才, 孙琦, 于彦丽, 赵勐, 鲁守平, 李艳娇, 孟昭东. 玉米生长素响应因子家族基因的表达模式分析[J]. 作物学报, 2021, 47(6): 1138-1148. |
[3] | 黄兴, 习金根, 陈涛, 覃旭, 谭施北, 陈河龙, 易克贤. 剑麻苯丙氨酸裂解酶基因的鉴定及表达分析[J]. 作物学报, 2021, 47(6): 1082-1089. |
[4] | 王丹丹, 柳洪鹃, 王红霞, 张鹏, 史春余. 甘薯蔗糖转运蛋白基因IbSUT3的克隆及功能分析[J]. 作物学报, 2020, 46(7): 1120-1127. |
[5] | 衡友强,游西龙,王艳. 费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性[J]. 作物学报, 2020, 46(4): 503-512. |
[6] | 梁思维,姜昊梁,翟立红,万小荣,李小琴,蒋锋,孙伟. 玉米HD-ZIP I亚家族基因鉴定及表达分析[J]. 作物学报, 2020, 46(4): 532-543. |
[7] | 陈杉彬, 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红. 甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定[J]. 作物学报, 2020, 46(12): 1862-1869. |
[8] | 马金姣,兰金苹,张彤,陈悦,郭亚璐,刘玉晴,燕高伟,魏健,窦世娟,杨明,李莉云,刘国振. 过表达OsMPK17激酶蛋白质增强了水稻的耐旱性[J]. 作物学报, 2020, 46(01): 20-30. |
[9] | 王建伟,贺晓岚,李文旭,陈新宏. 小麦近缘属植物1-FFT基因的克隆及功能分析[J]. 作物学报, 2018, 44(6): 814-823. |
[10] | 段方猛, 罗秋兰, 鲁雪莉, 齐娜伟, 刘宪舜, 宋雯雯. 玉米油菜素甾醇生物合成关键酶基因ZmCYP90B1的克隆及其对逆境胁迫的响应[J]. 作物学报, 2018, 44(03): 343-356. |
[11] | 吕有军,杨卫军,赵兰杰,姚金波,陈伟,李燕,张永山. 陆地棉SRO基因家族的鉴定及表达分析[J]. 作物学报, 2017, 43(10): 1468-1479. |
[12] | 王睿,朱梦琳,高方远,任鄄胜,陆贤军,任光俊,林拥军. 水稻组织特异型人工合成启动子的设计、构建及功能鉴定[J]. 作物学报, 2017, 43(06): 789-794. |
[13] | 贺晓岚,王建伟,李文旭,陈真真,赵继新,武军,王中华,陈新宏. 大赖草6-SFT基因的克隆及其转基因烟草抗旱和抗寒性分析[J]. 作物学报, 2016, 42(03): 389-398. |
[14] | 胡利芹,薛飞洋,李微微,王二辉,徐兆师,李连城,周永斌,贾冠清,刁现民,马有志,陈明. 谷子非特异性磷脂酶C基因SiNPC4的克隆及功能分析[J]. 作物学报, 2015, 41(07): 1017-1026. |
[15] | 宋仲戬,张登峰*,李永祥,石云素,宋燕春,王天宇,黎裕. 玉米分子伴侣基因ZmBiP2在逆境下的功能分析[J]. 作物学报, 2015, 41(05): 708-716. |
|