苹果MdDRB1基因在番茄中异位表达与功能研究Ectopic expression and functional research of Apple MdDRB1 gene in tomato
赵先炎;赵强;刘鑫;郝玉金;由春香;
摘要(Abstract):
【目的】将苹果Md DRB1基因转入番茄中,探究苹果MdDRB1基因在番茄中的异位表达与功能。【方法】采用农杆菌介导法,将苹果基因Md DRB1转入番茄植物体内,用PCR检测到该基因已整合到番茄总DNA中。对获得的5个转基因番茄株系进行半定量RT-PCR分析,挑选MdDRB1基因表达水平由低到高的3个株系,用实时荧光定量PCR检测与生长素有关的mi RNAs及相应靶基因的相对表达量。并对非转基因和转基因番茄进行3μmol·L-1IAA和5μmol·L-1IAA处理,对植株进行向光性检测。【结果】转基因株系中miR164、miR160、mi R393表达量下调,mi R167表达量上调,同时,它们分别对应的番茄中的靶基因SlNAC1、SlARF10和SlARF16、SlTIR1的相对表达量上调,SlARF8相对表达量下调。IAA处理后,转基因株系幼苗的侧根数目比未转基因植株增多,同时,转基因株系幼苗的向光性也增强。【结论】苹果Md DRB1基因在番茄中的异位表达,通过抑制生长素响应相关的mi R164、miR160、miR393和增加miR167的积累水平,部分解除其对下游靶基因SlNAC1、SlARF10和Sl ARF16、SlTIR1以及增加Sl ARF8的转录后基因沉默作用,进而提高了转基因番茄对生长素响应的敏感性。
关键词(KeyWords): 苹果MdDRB1基因;番茄;生长素响应;miRNAs
基金项目(Foundation): 国家自然科学基金(项目号31171946);; 教育部“长江学者和创新团队发展计划”创新团队(IRT1155)
作者(Author): 赵先炎;赵强;刘鑫;郝玉金;由春香;
Email:
DOI: 10.13925/j.cnki.gsxb.20140335
参考文献(References):
- [1]CAO Huiying,ZHANG Lijun,XIA Runxi.Research Progress on micro RNA involved in phytohormone response and biosynthesis[J].Plant Physiology Journal,2013,49(11):1121-1126.曹慧颖,张立军,夏润玺.植物激素相关micro RNA研究进展[J].植物生理学报,2013,49(11):1121-1126.
- [2]WANG Lei,FAN Yunliu.Progress of Micro RNA in plants[J].Journal of Agricultural Science and Technology,2007,9(3):18-23.王磊,范云六.植物微小RNA(micro RNA)研究进展[J].中国农业科技导报,2007,9(3):18-23.
- [3]QIN Cheng,ZHANG Zhiming,LIU Hongjun,GAO Jian,RONG Tingzhao,PAN Guangtang.Advances on micro RNAs regulated flower development of high plant[J].Journal of Agricultural Biotechnology,2011,19(5):938-952.覃成,张志明,刘红军,高健,荣廷昭,潘光堂.调控植物花发育的mi RNAs的研究进展[J].农业生物技术学报,2011,19(5):938-952.
- [4]VAZQUEZ F,GASCIOLLI V,CRéTéP,VAUCHERET H.The nuclear ds RNA binding protein HYL1 is required for micro RNA accumulation and plant development,but not posttranscriptional transgene silencing[J].Current Biology,2004,14(4):346-351.
- [5]ZHANG Shaofeng,LI Xiaorong,SUN Chuanbao,HE Yuke.Epigenetics of plant vernalization regulated by non-coding RNAs[J].Hereditas,2012,34(7):829-834.张绍峰,李晓荣,孙传宝,何玉科.植物非编码RNA调控春化作用的表观遗传[J].遗传,2012,34(7):829-834.
- [6]LU C,FEDOROFF N.A mutation in the Arabidopsis HYL1 gene encoding a ds RNA binding protein affects responses to abscisic acid,auxin,and cytokinin[J].The Plant Cell Online,2000,12(12):2351-2365.
- [7]HIRAGURI A,ITOH R,KONDO N,NOMURA Y,AIZAWA D,MURAI Y,FUKUHARA T.Specific interactions between Dicer-like proteins and HYL1/DRB-family ds RNA-binding proteins in Arabidopsis thaliana[J].Plant Molecular Biology,2005,57(2):173-188.
- [8]KURIHARA Y,TAKASHI Y,WATANABE Y.The interaction between DCL1 and HYL1 is important for efficient and precise processing of pri-mi RNA in plant micro RNA biogenesis[J].RNA,2006,12(2):206-212.
- [9]YANG J H,HAN S J,YOON E K,LEE W S.Evidence of an auxin signal pathway,micro RNA167-ARF8-GH3,and its response to exogenous auxin in cultured rice cells[J].Nucleic Acids Research,2006,34(6):1892-1899.
- [10]SONG L,HAN M H,LESICKA J,FEDOROFF N.Arabidopsis primary micro RNA processing proteins HYL1 and DCL1 define a nuclear body distinct from the Cajal body[J].Proceedings of the National Academy of Sciences,2007,104(13):5437-5442.
- [11]DONG Z,HAN M H,FEDOROFF N.The RNA-binding proteins HYL1 and SE promote accurate in vitro processing of primi RNA by DCL1[J].Proceedings of the National Academy of Sciences,2008,105(29):9970-9975.
- [12]YU Lin,YU Xuhong,SHEN Ruijuan,HE Yuke.HYL1 gene maintains venation and polarity of leaves[J].Planta,2005,221:231-242.
- [13]YU Xuhong,PENG Jiesong,FENG Xianzhong,YANG Suxin,ZHENG Zhiren,TANG Xiang-rong,SHEN Ruijuan,LIU Pinglin,HE Yuke.Cloning and structural and expressional characterization of Bcp LH gene preferentially expressed in folding leaf of Chinese cabbage[J].Science in China Series C:Life Sciences,2000,43(3):321-329.
- [14]YU Lin.Molecular mechanism involved in gene regulation of leaf curvature[D].Beijing:Graduate school of Chinese academy of sciences,Ph.D.Thesis,2005:93-109.余琳.叶卷曲基因调控机制的研究[D].北京:中国科学院研究生院,2005:93-109.
- [15]YOU Chunxiang,ZHAO Qiang,WANG Xiaofei,XIE Xingbin,FENG Xiaoming,ZHAO Lingling,SHU Huairui,HAO Yujin.A ds RNA-binding protein Md DRB1 associated with mi RNA biogenesis modifies adventitious rooting and tree architecture in apple[J].Plant Biotechnology Journal,2014,12(2):183-192.
- [16]MALLORY A C,BARTEL D P,BARTEL B.Micro RNA-directed regulation of Arabidopsis auxin response factor17 is essential for proper development and modulates expression of early auxin response genes[J].The Plant Cell Online,2005,17(5):1360-1375.
- [17]BONNET E,WUYTS J,ROUZéP,VAN DE PEER Y.Detection of 91 potential conserved plant micro RNAs in Arabidopsis thaliana and oryza sativa identifies important target genes[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(31):11511-11516.
- [18]NAVARRO L,DUNOYER P,JAY F,ARNOLD B,DHARMA-SIRI N,ESTELLE M,JONES J D.A plant mi RNA contributes to antibacterial resistance by repressing auxin signaling[J].Science,2006,312(5772):436-439.
- [19]MILLAR A A,GUBLER F.The Arabidopsis GAMYB-like genes,MYB33 and MYB65,are micro RNA-regulated genes that redundantly facilitate anther development[J].The Plant Cell Online,2005,17(3):705-721..
- [20]WANG Jiawei,WANG Lingjian,MAO Yingbo,CAI Wenjuan,XUE Hongwei,CHEN Xiaoya.Control of root cap formation by micro RNA-targeted auxin response factors in Arabidopsis[J].The Plant Cell Online,2005,17(8):2204-2216.
- [21]GUO Huishan,XIE Qi,FEI Jifeng,CHUA Namhai.Micro RNA directs m RNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development[J].The Plant Cell Online,2005,17(5):1376-1386.
- [22]FENG Xiaoming,QIAO Yu,WANG Shuangshuang,HAO Yujin.Overexpression ofArabidopsis Atmi R393agene alters the sensitivity to auxin in tomato[J].Scientia Horticulturae,2010,124(2):165-169.
- [23]CHEN C,RIDZON D A,BROOMER A J,ZHOU Z,LEE D H,NGUYEN J T,GUEGLER K J.Real-time quantification of micro RNAs by stem–loop RT–PCR[J].Nucleic Acids Research,2005,33(20):e179-e179.
- [24]LIN Dongbo,YANG Yingwu,KHALIL R,XIAN Zhiqiang,HU Guojian,LI Zhengguo.Slmi R393 controls the auxin receptor homologous genes expression,and regulates sensitivity to auxin in tomato root growth[J].Scientia Horticulturae,2013,162:90-99.
- [25]HENDELMAN A,STAV R,ZEMACH H,ARAZI T.The tomato NAC transcription factor Sl NAM2 is involved in flower-boundary morphogenesis[J].Journal of Experimental Botany,2013,64(18):5497-5507.
- [26]SHAN Lanlan.Funetional analysis of cdnas associated with flesh lignification in loquat fruit and small RNAs associated with auxin response faetors in tomato[D].Hangzhou:Zhejiang University,2009.单兰兰.枇杷果肉木质化相关c DNA和番茄ARF相关小RNA的功能分析[D].杭州:浙江大学,2009.
- [27]MIAO Qing,LI Zhengguo,YANG Yingwu,LI Ji,HU Guojian.Isolation and function analysis of Sly-mi R167a promotor in tomato[J].Chinese Journal of Tropical Crops,2011,32(4):653-657.苗青,李正国,杨迎伍,李季,胡国建.番茄Sly-mi R167a启动子的分离及功能分析[J].热带作物学报,2011,32(4):653-657.
- [28]BARTEL D P.Micro RNAs:genomics,biogenesis,mechanism,and function[J].Cell,2004,116(2):281-297.
- [29]HAN M H,GOUD S,SONG L,FEDOROFF,N.The Arabidopsis double-stranded RNA-binding protein HYL1 plays a role in micro RNA-mediated gene regulation[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(4):1093-1098.
- [30]INUKAI Y,SAKAMOTO T,UEGUCHI-TANAKA M,SHIBATA Y,GOMI K,UMEMURA I,MATSUOKA M.Crown rootless1,which is essential for crown root formation in rice,is a target of an auxin response factor in auxin signaling[J].The Plant Cell Online,2005,17(5):1387-1396.
- [31]DHARMASIRI N,DHARMASIRI S,ESTELLE M.The F-box protein TIR1 is an auxin receptor[J].Nature,2005,435(7041):441-445.
- [32]KEPINSKI S,LEYSER O.The Arabidopsis F-box protein TIR1is an auxin receptor[J].Nature,2005,435(7041):446-451.
- [33]WOODWARD A W,BARTEL B.A receptor for auxin[J].The Plant Cell Online,2005,17(9):2425-2429.
- [34]XIE Qi,FRUGIS G,COLGAN D,CHUA Namhai.Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development[J].Genes&Development,2000,14(23):3024-3036.
- [35]MüLLER B,SHEEN J.Cytokinin and auxin interaction in root stem-cell specification during early embryogenesis[J].Nature,2008,453(7198):1094-1097.
- [36]YANG Cunyi,SHEN Hong,LIU Ling,YAN Xiaolong.Roles of mi RNA in plant development and adaptabilities[J].Acta Botanica Boreale-Occidentalia Sinica,2005,25(12):2553-2561.杨存义,沈宏,刘灵,严小龙.mi RNA在植物发育与环境适应性中的作用[J].西北植物学报,2005,25(12):2553-2561.