Welcome to Acta Agronomica Sinica,

Acta Agron Sin ›› 2010, Vol. 36 ›› Issue (4): 596-601.doi: 10.3724/SP.J.1006.2010.00596


Construction and Transformation of RNAi Vector of MsZFN Gene from Alfalfa (Medicago sativa L.)

QIN Zhi-Hui1,2,CHAO Yue-Hui1,YANG Qing-Chuan1,*,KANG Jun-Mei1,SUN Yan3,WANG Ping-Qing2*,LONG Rui-Cai1   

  1. 1Institute of Animal Sciences,Chinese Academy of Agricultural Sciences,Beijing100193,China;2Bioentineering College of Chongqing University,Chongqing 400030,China;3College of Animal Science and Technology,China Agriculture University,Beijing 100191,China
  • Received:2009-11-10 Revised:2010-01-07 Online:2010-04-12 Published:2010-02-05
  • Contact: YANG Qing-Chuan, E-mail: qcyang66@yahoo.com.cn, WANG Ping-Qing, E-mail: wang_pq@21cn.com


Based on the sequence of Medicago sativa Zinc Finger Protein (MsZFN) gene (GenBank accession No. EU624138), two pairs of specific primers containing different enzyme sites were designed. With the template of full-length cDNA , positive-sense strand and antisense strand were obtained, which were separately inserted into the expression vector pART27. The RNAi vector pART-F-R containing a hairpin structure was constructed. Mediated by Agrobacterium tumefaciens, pART-F-R was transformed into alfalfas. PCR testing showed that three transgenic plants were obtained. Result of RT-PCR showed that transgenic alfalfas had lower expression level of MsZFN gene than wild alfalfas. Those results indicated that the RNAi vector pART-F-R containing a hairpin structure was constructed successfully and highly efficient for the simultaneous silence of MsZFN gene.

Key words:   Alfalfa(Medicago sativa L.), Zinc finger protein, RNAi, Transgene

[1]       Arenz C, Schepers U. RNA interference: from an ancient mechanism to a state of the art therapeutic application? Naturwissenschaften, 2003, 90: 345–359

[2]       Lai W S, Kennington E A, Blackshear P J. Tristetraprolin and its family members can promote the cell-free deadenylation of AU-rich element-containing mRNAs by poly (A) ribonuclease. Mol Cell Biol, 23: 3798–3812

[3]       Li J, Jia D, Chen X. HUA1, a regulator of stamen and carpel identities in Arabidopsis, codes for a nuclear RNA binding protein. Plant Cell, 13: 2269–2281

[4]       Delaney K, Xu R Q, Zhang J X, Li Q Q, Yun K Y, Falcone D L, Hunt A G. Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit. Plant Physiol, 140: 1507–1521

[5]       Kong Z, Li M, Yang W, Xu W, Xue Y. A novel nuclearlocalized CCCH-type zinc finger protein, OsDOS, is involved in delaying leaf senescence in rice. Plant Physiol, 2006, 141: 1376–1388

[6]       Dong H K, Shinjiro Y, Soohwan L, Eunkyoo O, Jeongmu P, Atsushi H, Yuji K, Giltsu C. SOMNUS, a CCCH-Type zinc finger protein in Arabidopsis, negatively regulates light-dependent seed germination downstream of PIL5. Plant Cell, 2008, 20: 1260–1277

[7]       Li Z, Thomas T L. PEI1, an embryo-specific zinc finger protein gene required for heart-stage embryo formation in Arabidopsis. Plant Cell, 1998, 10: 383–398

[8]       Sun J Q, Jiang H L, Xu Y X, Li H M, Wu X Y, Xie Q, Li C. The CCCH-type zinc finger proteins AtSZF1 and AtSZF2 regulate salt stress responses in Arabidopsis. Plant Cell Physiol, 2007, 48: 1148–1158

[9]       Chao Y H, Kang J M, Sun Y, Yang Q C, Wang P Q, Wu M S, Li Y, Long R C, Qin Z H. Molecular cloning and characterization of a novel gene encoding zinc finger protein from Medicago sativa L. Mol Biol Rep, 2009, 36: 2315–2321

[10]    Peng H-B(彭辉兵), Quan Z-H(全智华). RNAi: a powerful tool for gene silencing. Med Recapitulate (医学综述), 2007, 13(3): 177–180 (in Chinese)

[11]    Kamath R S, Fraser A G, Dong Y, Poulin G, Durbin R, Gotta M, Kanapin A, Le Bot N, Moreno S, Sohrmann M, Welchman D P, Zipperlen P, Ahringer J. Systematic functional analysis Caenorhaditis elegans genome using RNAi, Nature, 2003, 421: 231–237

[12]    Ashrafi K, Chang F Y, Watts J L, Fraser A G, Kamath R S, Ahringer J, Ruvkun G. Genome-wide RNAi analysis of Caenorhaditis elegans fat regulatory genes. Nature, 2003, 421: 268–272

[13]    Frankish H. Consortium uses RNAi to uncover genes’ function. Lancet, 2003, 361: 584

[14]    Chuang C F, Meyerowitz E M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA, 2000, 97: 4985–4990
Prasanth S G, Prasanth K V, Stillman B. Orc6 involved in DNA replication, chromosome segregation, and cytokinesis. Science, 2002, 297: 1026–1031
[1] WANG Xia, YIN Xiao-Yu, Yu Xiao-Ming, LIU Xiao-Dan. Effects of drought hardening on contemporary expression of drought stress memory genes and DNA methylation in promoter of B73 inbred progeny [J]. Acta Agronomica Sinica, 2022, 48(5): 1191-1198.
[2] LI Jie-Hua, DUAN Qun, SHI Ming-Tao, WU Lu-Mei, LIU Han, LIN Yong-Jun, WU Gao-Bing, FAN Chu-Chuan, ZHOU Yong-Ming. Development and identification of transgenic rapeseed with a novel gene for glyphosate resistance [J]. Acta Agronomica Sinica, 2021, 47(5): 789-798.
[3] MA Huan-Huan, FANG Qi-Di, DING Yuan-Hao, CHI Hua-Bin, ZHANG Xian-Long, MIN Ling. GhMADS7 positively regulates petal development in cotton [J]. Acta Agronomica Sinica, 2021, 47(5): 814-826.
[4] Xiao-Yan DING,Juan ZHAO,Shan-Shan QIAN,Xing-Ying YAN,Yan PEI. Improving Fiber Yield and Quality in the Short Season Cotton Variety Jinmian 11 by Introducing FBP7::iaaM [J]. Acta Agronomica Sinica, 2018, 44(8): 1152-1158.
[5] Shao-Lan LUO,Xiao-Ping LIAN,Min PU,Xiao-Jing BAI,Yu-Kui WANG,Jing ZENG,Song-Mei SHI,He-Cui ZHANG,Li-Quan ZHU. Molecular Cloning, Location and Expression Analysis of Brasscia oleracea Zinc Finger Protein Transcription Factor BoC2H2 [J]. Acta Agronomica Sinica, 2018, 44(11): 1650-1660.
[6] Yun-Fei LIANG, Lin-Cheng ZHANG, Quan-Ming PU, Zhen-Ze LEI, Song-Mei SHI, Yu-Peng JIANG, Xue-Song REN, Qi-Guo GAO. Cloning of BoLH27 Gene from Cabbage and Phenotype Analysis of Transgenic Cabbage [J]. Acta Agronomica Sinica, 2018, 44(03): 397-404.
[7] WANG Sha,HE Yong,LUO Guang-Yu,YAO Min,ZHANG Xu,CHEN Xin-Bo,ZHOU Xiao-Yun. Influence of OsWR2-RNAi on Rice Cuticle Biosynthesis and Drought Resistance [J]. Acta Agron Sin, 2017, 43(03): 315-323.
[8] YANG Xiang-Dong,NIU Lu,ZHANG Wei,YANG Jing,DU Qian,XING Guo-Jie,GUO Dong-Quan,LI Qi-Yun,DONG Ying-Shan. RNAi-mediated SMV-P3 Silencing Increases Soybean Resistance to Soybean Mosaic Virus [J]. Acta Agron Sin, 2016, 42(11): 1647-1655.
[9] SHAN Tian-Lei,HONG Yan-Tao,DU Li-Pu,XU Hui-Jun,WEI Xue-Ning,ZHANG Zeng-Yan*. Development and Characterization of TaMYB86-Overexpressing Transgenic Wheat Lines with Resistance to Common Root Rot [J]. Acta Agron Sin, 2016, 42(10): 1429-1436.
[10] CHAI Jian-Fang*,WANG Hai-Bo*,MA Xiu-Ying,ZHANG Cui-Mian,DONG Fu-Shuang. Effect of ω-Secalin Gene Silencing on Processing Quality of Wheat 1B/1R Translocation Line [J]. Acta Agron Sin, 2016, 42(05): 627-632.
[11] HU Dan-Dan,ZHANG Fan,HUANG Li-Yu,ZHUO Da-Long,ZHANG Fan,ZHOU Yong-Li,SHI Ying-Yao,LI Zhi-Kang. Stress-activated Protein Kinase OsSAPK2 Involved in Regulating Resistant Response to Xanthomonas oryzae pv. oryzae in Rice [J]. Acta Agron Sin, 2015, 41(08): 1191-1200.
[12] REN Qin,WANG Ya-Jun,GUO Zhi-Hong,LI Ji-Ping,XIE Zhong-Kui,WANG Ruo-Yu,WANG Li,HUI Na-Na. Gene Silence of Phytophthora infestans Induced by Plant-mediated RNA Interference in Potato [J]. Acta Agron Sin, 2015, 41(06): 881-888.
[13] JIA Shuang-Wei,GAO Ying,ZHAO Kai-Jun. Cloning and Characterization of Brassica juncea Zinc Finger Protein Transcription Factor Gene Bj26 [J]. Acta Agron Sin, 2014, 40(07): 1174-1181.
[14] YE Yan-Ying,ZENG Gang,CAO Ming-Qing,MA Rong-Cai,WU Cai-Jun,YAO Lei. HC-Pro Gene Segment Mediated Hyper-Resistance to Turnip mosaic virus [J]. Acta Agron Sin, 2014, 40(03): 550-555.
[15] LIU Jiang,MA Yan-Bin,SUN Quan-Xi,WU Xia,LI Xue-Ying,SUN Mei-Hong,LI Yan-E,LI Xin-Zheng,QI Bao-Xiu. Production of Very Long Chain Polyunsaturated Fatty Acids in Cotton [J]. Acta Agron Sin, 2014, 40(01): 86-92.
Full text



No Suggested Reading articles found!