人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默

doi:10.3724/SP.J.1005.2012.00348

遗传 ›› 2012, Vol. 34 ›› Issue (3): 348-355.doi: 10.3724/SP.J.1005.2012.00348

人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默

李文超, 赵淑清

山西大学生物技术研究所,化学生物学与分子工程教育部重点实验室, 太原 030006

收稿日期:2011-07-03 修回日期:2011-08-03 出版日期:2012-03-20 发布日期:2012-03-25
通讯作者: 赵淑清 E-mail:shuqing@sxu.edu.cn
基金资助:
山西省国际科技合作计划(编号：2009081005), 山西省留学人员科技活动项目择优资助项目, 山西省回国留学人员科研资助项目(编号：201003)和太原市科技明星专项(编号：11014902)资助

Specific gene silencing of At1g13770 and At2g23470 by artificial microRNAs in Arabidopsis

LI Wen-Chao, ZHAO Shu-Qing

Key Laboratory of Chemical Biology and Molecular Engineering of Ministry of Education, Institute of Biotechnology, Shanxi Univer-sity, Taiyuan 030006, China

Received:2011-07-03 Revised:2011-08-03 Online:2012-03-20 Published:2012-03-25

摘要/Abstract

摘要： DUF647 (Domain of unknown function 647) 蛋白家族是在真核生物中广泛存在的、高度保守的蛋白家族。拟南芥中该基因家族共有6个成员, 迄今为止拟南芥DUF647家族中4个成员的功能尚不清楚。文章以拟南芥内源MIR319a前体为骨架, 构建了敲减DUF647家族中2个基因At1g13770和At2g23470表达的人工microRNAs(Artifical microRNAs, amiRNAs)。利用WMD(Web microRNA designer)平台设计分别靶向At1g13770和At2g23470基因的amiRNAs序列, 通过重叠PCR置换拟南芥MIR319a前体序列。构建融合amiRNAs前体的植物表达载体pCHF3-amiRNAs, 在农杆菌介导下转化拟南芥。RT-PCR分析表明, amiRNAs能够显著抑制At1g13770和At2g23470基因的表达, 获得了抑制效果明显的转基因株系。At2g23470-amiRNA转基因植株At2g23470转录水平的下调导致育性严重下降。文章为进一步研究这两个基因的功能奠定了良好的基础。

关键词: 拟南芥, DUF647基因家族, 人工microRNA, 基因沉默

Abstract: DUF647 (domain of unknown function 647) protein family is found in diverse eukaryotic organisms and highly conserved in eukaryotes. It has 6 members in Arabidopsis genome. So far, the function of 4 members of Arabidopsis DUF647 family is unknown. In this report, using an endogenous Arabidopsis MIR319a precursor as the backbone, we constructed two artificial microRNAs (amiRNAs) to knock down the expression of two DUF647 family genes At1g13770 and At2g23470. Using the WMD (Web microRNA Designer) platform, we designed two amiRNAs targeting At1g13770 and At2g23470 genes, respectively. Both amiRNAs sequences were engineered into the MIR319a precursor using overlapping PCR and the amiRNAs backbones were transferred into the binary vector pCHF3. The resulting plasmids that harbor amiRNAs stem loop fragments were transformed into Arabidopsis by Agrobacterium-mediated floral diping. Upon constitutive expression of these two amiRNAs, the target genes were efficiently down-regulated in transgenic line. The decreased level of At2g23470 transcript in At2g23470-amiRNA transgenic plants resulted in severe sterility. This work will facilitate the functional analysis of At1g13770 and At2g23470 genes in Arabidopsis growth and development.

Key words: Arabidopsis, DUF647 gene family, artificial microRNAs, gene silencing

李文超，赵淑清. 人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默[J]. 遗传, 2012, 34(3): 348-355.

LI Wen-Chao, ZHAO Shu-Qing. Specific gene silencing of At1g13770 and At2g23470 by artificial microRNAs in Arabidopsis[J]. HEREDITAS, 2012, 34(3): 348-355.

参考文献

[1] Tong HY, Leasure CD, Hou XW, Yuen GG, Briggs W, He ZH. Role of root UV-B sensing in Arabidopsis early seedling development. Proc Natl Acad Sci USA, 2008, 105(52): 21039-21044.
[2] Leasure CD, Tong HY, Yuen GG, Hou XW, Sun XF, He ZH. ROOT UV-B SENSITIVE2 acts with ROOT UV-B SENSITIVE1 in a root ultraviolet B-sensing pathway. Plant Physiol, 2009, 150(4): 1902-1915.
[3] Ge L, Peer W, Robert S, Swarup R, Ye S, Prigge M, Cohen JD, Friml J, Murphy A, Tang D, Estelle M. Arabidopsis ROOT UVB SENSITIVE2/WEAK AUXIN RESPONSE1 is required for polar auxin transport. Plant Cell, 2010, 22(6): 1749-1761.
[4] Bartel DP. MicroRNAs: genomics, biogenesis, mechanism, and function. Cell, 2004, 116(2): 281-297.
[5] Voinnet O. Origin, biogenesis, and activity of plant microRNAs. Cell, 2009, 136(4): 669-687.
[6] 毛颖波, 薛学义, 陈晓亚. 植物小RNA与RNA干扰: 生物学功能与应用前景. 中国科学(C辑:生命科学), 2009, 39(1): 31-43.
[7] Schwab R, Ossowski S, Riester M, Warthmann N, Weigel D. Highly specific gene silencing by artificial microRNAs in Arabidopsis. Plant Cell, 2006, 18(5): 1121-1133.
[8] Warthmann N, Chen H, Ossowski S, Weigel D, Hervé P. Highly specific gene silencing by artificial miRNAs in rice. PLoS One, 2008, 3(3): e1829.
[9] Khraiwesh B, Ossowski S, Weigel D, Reski R, Frank W. Specific gene silencing by artificial microRNAs in Physcomitrella patens: An alternative to targeted gene knockouts. Plant Physiol, 2008, 148(2): 684-693.
[10] Molnar A, Bassett A, Thuenemann E, Schwach F, Karkare S, Ossowski S, Weigel D, Baulcombe D. Highly specific gene silencing by artificial microRNAs in the unicellular alga Chlamydomonas reinhardtii. Plant J, 2009, 58(1): 165-174.
[11] Zhao T, Wang W, Bai X, Qi YJ. Gene silencing by artificial microRNAs in Chlamydomonas. Plant J, 2009, 58(1): 157-164.
[12] Höfgen R, Willmitzer L. Storage of competent cells for Agrobacterium transformation. Nucl Acids Res, 1988, 16(20): 9877.
[13] Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J, 1998, 16(6): 735-743.
[14] Zhao SQ, Li XF, Guo JB. Expression of the Pathogenesis-related gene promoter-GUS reporter fusion in Arabidopsis. Acta Bot Sin, 2003, 46(8): 982-987.
[15] Wesley SV, Helliwell CA, Smith NA, Wang MB, Rouse DT, Liu Q, Gooding PS, Singh SP, Abbott D, Stoutjesdijk PA, Robinson SP, Gleave AP, Green AG, Waterhouse PM. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J, 2001, 27(6): 581-590.
[16] Lu R, Martin-Hernandez AM, Peart JR, Malcuit I, Baul-combe DC. Virus-induced gene silencing in plants. Methods, 2003, 30(4): 296-303.
[17] Alvarez JP, Pekker I, Goldshmidt A, Blum E, Amsellem Z, Eshed Y. Endogenous and synthetic microRNAs stimulate simultaneous, efficient, and localized regulation of multiple targets in diverse species. Plant Cell, 2006, 18(5): 1134-1151.
[18] Niu QW, Lin SS, Reyes JL, Chen KC, Wu HW, Yeh SD, Chua NH. Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol, 2006, 24(11): 1420-1428.
[19] Qu J, Ye J, Fang RX. Artificial microRNA-mediated virus resistance in plants. J Virol, 2007, 81(12): 6690-6699.
[20] Ossowski S, Schwab R, Weigel D. Gene silencing in plants using artificial microRNAs and other small RNAs. Plant J, 2008, 53(4): 674-690.
[21] Schmid M, Davison TS, Henz SR, Pape UJ, Demar M, Vingron M, Schölkopf B, Weigel D, Lohmann JU. A gene expression map of Arabidopsis thaliana development. Nat Genet, 2005, 37(5): 501-506
[22] Goff SA, Ricke D, Lan TH, Presting G, Wang RL, Dunn M, Glazebrook J, Sessions A, Oeller P, Varma H, Hadley D, Hutchison D, Martin C, Katagiri

编辑推荐

Metrics

www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:

人工microRNAs对拟南芥At1g13770和At2g23470基因的特异沉默

Specific gene silencing of At1g13770 and At2g23470 by artificial microRNAs in Arabidopsis

PDF (PC)

可视化

被引次数

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 15

编辑推荐

Metrics

[1]	陈凯, 王灏, 陈燚婷, 符可, 韩之刚, 李聪, 斯金平, 陈东红. 铁皮石斛WOX家族基因在生长发育中的功能分析[J]. 遗传, 2023, 45(8): 700-714.
[2]	林建辉, 刘自广, 张楠, 严容, 张楠, 何鑫淼, 王文涛, 刘娣, 吴娟. 拟南芥ABI5基因对BR胁迫响应及其对下胚轴生长的调节作用[J]. 遗传, 2021, 43(9): 901-909.
[3]	李晓翠, 康凯程, 黄先忠, 范永斌, 宋苗苗, 黄韵杰, 丁佳佳. 小拟南芥MKK基因家族全基因组鉴定及进化和表达分析[J]. 遗传, 2020, 42(4): 403-421.
[4]	梅志超, 位竹君, 于佳慧, 冀凤丹, 解莉楠. 多组学关联分析揭示表观等位基因在拟南芥环境适应性进化中的作用及机制[J]. 遗传, 2020, 42(3): 321-331.
[5]	李泽琴,李锦涛,邴杰,张根发. 拟南芥APX家族基因在植物生长发育与非生物逆境胁迫响应中的作用分析[J]. 遗传, 2019, 41(6): 534-547.
[6]	刘振宁, 袁黎, VenkatesanSundaresan, 余小林. 拟南芥CKI1基因上游转录调控因子筛选及鉴定[J]. 遗传, 2019, 41(5): 430-438.
[7]	武迪, 黄林周, 高谨, 王永红. 植物重力反应的分子调控机制[J]. 遗传, 2016, 38(7): 589-602.
[8]	陈兆进, 丁传雨, 郑远. 拟南芥QUA1基因在光信号途径中的表达与功能分析[J]. 遗传, 2016, 38(5): 436-443.
[9]	樊锦涛,蒋琛茜,邢继红,董金皋. 拟南芥R2R3-MYB家族第22亚族的结构与功能[J]. 遗传, 2014, 36(10): 985-994.
[10]	李捷陈旭罗莉琼于晶明凤. ANAC092参与调控花药发育的功能初探[J]. 遗传, 2013, 35(7): 913-922.
[11]	郭敏霞傅永福. 拟南芥SUMO底物的研究进展[J]. 遗传, 2013, 35(6): 727-734.
[12]	王增光柴国华王芝瑶唐贤丰孙长江周功克马三梅. 拟南芥AtGA3OX1和AtGA3OX2基因影响茎秆次生细胞壁增厚的分子机理[J]. 遗传, 2013, 35(5): 655-665.
[13]	李延安，祁林林，孙加强，刘宏宇，李传友. 茉莉酸诱导侧根形成缺陷突变体asa1-1抑制子(soa)的鉴定与遗传分析[J]. 遗传, 2011, 33(9): 1003-1010.
[14]	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[J]. 遗传, 2010, 32(8): 848-856.
[15]	张红宇，徐培洲，杨华，吴先军. 拟南芥的印记基因和印记表达调控[J]. 遗传, 2010, 32(7): 670-676.