miR156a在火炬松、烟草、拟南芥中的表达与病原菌侵染密切相关。为了研究miR156a在杨树与病原菌互作过程中分子机制，以毛果杨全基因组DNA为材料，预测ptr-MIR156a启动子的大概区域，设计特异性PCR引物，克隆了ptr-MIR156a上游启动子区500、1 000和1 500 bp片段，并进行顺式作用元件分析，然后分别构建了绿色荧光蛋白（GFP）报告基因植物表达载体，最后通过原生质体的瞬时表达体系对其进行了活性检测。结果表明，1 500 bp片段活性最高，1 000 bp片段次之，500 bp片段活性最低。
Abstract
MicroRNAs (miRNAs) are endogenous small RNAs that have large regulatory effects development and stress responses in plants. Recently studies show that the expression of miR156a is intensively related with pathogens infection in Loblolly pine, tobacco, Arabidopsis and maize. To understand the interaction mechanism between miR156a and poplar, we predicted ptr-MIR156a promoter region and amplified three upstreaming fragments of ptr-MIR156a from Populus trichocarpa, 500 bp, 1 000 bp, and 1 500 bp, respectively，analyzed their cisacting elements, then cloned them into plant expression vectors, pMDC83 and transferred into poplar protoplasts. Fluorescence results showed that they drove GFP gene expression at different levels. Based on this result, we could transfer theses fragments into plants and study ptr-miR156a expression patterns and regulation mechanism in the future.
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参考文献
[1]Lee R C, Feinbaum R L, Ambros V. The C.elegans heterochronic gene lin4 encodes small RNAs with antisense complementarity to lin14[J]. Cell, 1993, 75(5):843-854.
[2]Bartel D P. MicroRNAs: target recognition and regulatory functions[J]. Cell, 2009, 136(23): 215-233.
[3]Bracht J, Hunter S, Eachus R, et al. Transsplicing and polyadenylation of let7 microRNA primary transcripts[J]. RNA, 2004, 10（10）: 1586-1594.
[4]Cai X, Hagedorn C H, Cullen B R. Human microRNAs are processed from capped, polyadenylated transcripts that can also function as mRNAs[J]. RNA,2004, 10(12): 1957-1966.
[5]Lee Y, Kim M, Han J, et al. MicroRNA genes are transcribed by RNA polymeraseⅡ[J]. EMBO J, 2004, 23(20): 4051-4060.
[6]Lu S F, Sun Y H, Amerson H, et al. MicroRNAs in loblolly pine (Pinus taeda L.) and their association with fusiform rust gall development[J]. Plant Journal,2007,51(6):1077-1098.
[7]Bazzini A A, Hopp H E, Beachy R N, et al. Infection and coaccumulation of tobacco mosaic virus proteins alter microRNA levels, correlating with symptom and plant development[J]. PNAS, 2007, 104(29):12157-12162.
[8]Tagami Y, Inaba N, Kutsuna N, et al. Specific enrichment of miRNAs in Arabidopsis thaliana infected with Tobacco mosaic virus[J]. DNA Research, 2007, 14(5):227-233.
[9]Chen J, Li W X, Xie D X, et al. Viral virulence protein suppresses RNA silencingmediated defense but upregulates the role of microRNA in host gene expression[J]. Plant Cell, 2004, 16(5):1302-1313.
[10]Yoo S D, Cho Y H, Sheen J. Arabidopsis mesophyll protoplasts: a versatile cell system for transient gene expression analysis[J]. Nature Protocols, 2007, 2(7): 1565-1572.
[11]Zhou X F，Ruan J H，Wang G D, et al. Characterization and identification of microRNA core promoters in four model species[J]. PLoS Computational Biology, 2007, 3(3):412-423.
[12]Jansson S，Douglas C J．Populus: a model system for plant biology[J]．Annu Rev Plant Biol，2007，58: 435-458．
[13]DiazDeLeon F, Klotz L K, Lagrimini L M. Nucleotide sequence of the tobacco (Nicotiana tabacum) anionic peroxidase gene[J]. Plant Physiol,1993, 101(3):1117-1118.
[14]Daraselia N D，Tarchevskaya S，Narita J O. The promoter for tomato 3hydroxy3methylglutaryl coenzyme A reductase gene 2 has unusual regulatory elements that direct highlevel expression[J]. Plant Physiol，1996,112：727-733.
[15]Van der Molen G E, Labavitch J M, Strand L L, et al. Pathogeninduced vascular gels: Ethylene as a host intermediate[J]. Physiol Plant, 1983, 59(4): 573-580.
[16]翁宇,戴小枫. 农作物乙烯合成和信号转导途径及其对抗病反应的调控[J]. 分子植物育种, 2008, 6 (4):739-748.
[17]陈英, 黄敏仁, 诸葛强, 等. 植物抗病信号传导途径及其相互作用[J]. 南京林业大学学报:自然科学版, 2002, 26(3): 85-90.
[18] Hennig J, Dewey R E, Cutt J R, et al. Pathogen,salicylic acid and developmental dependent expression of a β1,3glucanase/GUS gene fusion in transgenic tobacco plants[J]. The Plant Journal, 1993,4(3):481-493.
基金
收稿日期：2011-07-19修回日期：2011-09-19基金项目：国家林业局林业公益性行业科研专项项目（200904048）作者简介：陈英(1973—)，副教授。*黄敏仁（通信作者），教授。Email: njmrhuang@njfu.cn。
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