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Acta Agron Sin ›› 2011, Vol. 37 ›› Issue (02): 374-379.doi: 10.3724/SP.J.1006.2011.00374

• RESEARCH NOTES • Previous Articles    

Cloning of Medicago sativa Phychrome B cDNA and Establishment of Its RNA Interference Expression Vector

ZHU Jian-Ming,YAN Xue-Bing,SHI Ying-Hua,WANG Cheng-Zhang*   

  1. Engineering College of Animal Husbandry and Veterinary Sciences, Henan Agricultural University, Zhengzhou 450002, China
  • Received:2010-05-28 Revised:2010-09-22 Online:2011-02-12 Published:2010-12-15
  • Contact: 王成章, E-mail:wangchengzhang@263.net

Abstract: The short day is one of the main factors affecting alfalfa fall dormancy, which is called as the photoperiodic effect. Study on the relationship between the main photoreceptors-PhyB gene and alfalfa fall dormancy may reveal the regulation mechanism of alfalfa fall dormancy radically, and provide a scientific reference for the application of alfalfa varieties differing in fall dormancy in forage production. The objective of this study was to establish an RNAi expression vector harboring PhyB gene of Medicago sativa. Two pairs of specific primers containing different enzyme sites were designed on the basis of PhyB gene sequence of alfalfa variety “Vernal” (GenBank accession number: GQ379903.1). With the template of total RNA, positive sense strand and antisense strand were amplified by RT-PCR and cloned into pGEM-T Easy vector to obtain recombinant vectors pGEMB-1 and pGEMB-2, then based on the intermediate vector pHANNIBAL and the plant binary expression vector pART27, we constructed the RNAi expression vector pART27-RNAi containing a hairpin structure by many times of enzyme digestion and connection. The results provide a foundation for further studying the relationship between alfalfa dormancy and PhyB.

Key words: Medicago sativa, Fall dormancy, Photoreceptor, Phytochrome, RNA interference

[1]Wang C-Z(王成章), Han J-F(韩锦峰), Hu X-F(胡喜峰), Zhang C-M(张春梅), Pan X-J(潘晓建). Effects of photoperiod treatment of different fall-dormant alfalfa on abscisis acid (ABA) levels. Acta Agron Sin (作物学报), 2005, 31(10): 1370–1372 (in Chinese)
[2]Wang C Z, Ma B L, Yan X B, Han J F, Guo Y X, Wang Y H, Li P. Yields of alfalfa varieties with different fall-dormancy levels in a temperate environment. Agron J, 2009, 101: 1146–1152
[3]Li X-L(李向林), Wan L-Q(万里强). Alfalfa fall dormancy and its relationship to winter hardiness and yield. Acta Pratac Sin (草业学报), 2004, 13(3): 1–8 (in Chinese with English abstract)
[4]Shih S C, Jung G A, Shelton D C. Effects of temperature and photoperiod on metabolic changes in alfalfa in relation to cold hardiness. Crop Sci, 1967, 7: 385–389
[5]Wang C-Z(王成章), Han J-F(韩锦峰), Hu X-F(胡喜峰), Zhang C-M(张春梅), He Y(何云), Liu Q-W(刘圈炜). Regulation of PhyB and ABA on fall dormancy of different Medicago sativa varieties in photoperiod treatments. Acta Pratac Sin (草业学报), 2006, 15(6): 56–63 (in Chinese with English abstract)
[6]Fankhauser C, Staiger D. Photoreceptors in Arabidopsis thaliana: Light perception, signal transduction and entrainment of the endogenous clock. Planta, 2002, 216: 1–16
[7]Schafer E, Bowler C. Phytochrome-mediated photoperception and signal transduction in higher plants. EMBO Rep, 2002, 3: 1042–1048
[8]Yu S-W(余叔文), Tang Z-C(汤章诚). Plant Physiology and Molecular Biology (植物生理与分子生物学), 2nd edn. Beijing: Science Press, 1998. pp 633–634 (in Chinese)
[9]Furuya M. Molecular properties and biogenesis of phytochrome I and II. Adv Biophys, 1989, 25: 133–167
[10]Quail P H, Boylan M T, Parks B M, Short T W, Xu Y, Wagner D. Phytochromes: photosensory perception and signal transduction. Science, 1995, 268: 675–680
[11]Wagner D, Tepperman J M, Quail P H. Overexpression of phytochrome B induces a short hypocotyl phenotype in transgenic Arabidopsis. Plant Cell, 1991, 3: 1275–1288
[12]Wang C Z, Ma B L, Han J F, Wang Y H, Gao Y G, Hu X F, Zhang C M. Photoperiod effect on phytochrome and abscistic acid in alfalfa varieties differing in fall dormancy. J Plant Nutr, 2008, 31: 1257–1269
[13]Castonguay Y, Nadeau P, Lechasseur P, Chouinard L. Differential accumulation of carbohydrates in alfalfa cultivars of contrasting winterhardiness. Crop Sci, 1995, 35: 509–516
[14]Cuningham S M, Volenec J J, Teuber L R. Plant survival and root and bud composition of alfalfa populations selected for contrasting fall dormancy. Crop Sci, 1998, 38: 962–969
[15]Castonguay Y, Nadeau P. Enzymatic control of soluble carbohydrate accumulation in cold-acclimated crowns of alfalfa. Crop Sci, 1998, 38: 1183–1189
[16]Brummer E C, Shah M M, Luth D. Reexamining the relationship between fall dormancy and winter hardiness in alfalfa. Crop Sci, 2000, 40: 971–977
[17]Haagenson D M, Cunningham S M, Joern B C, Volenec J J. Autumn defoliation effects on alfalfa winter survival, root physiology, and gene expression. Crop Sci, 2003, 43: 1340–1348
[18]Fire A, Xu S Q, Montgomery M K, Kostas S A, Driver S E, Mello C C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391: 806–811
[19]Chen X(陈新), Liu Q-Z(刘庆忠), Li Y-X(李杨昕), Lü H-Z(吕慧贞), Liang Y-Q(梁雅芹), Zhang S-G(张士刚), Shao J-P(邵建萍), Zhang Y-H(张元湖). RNAi expression vector construction of α-farnesene synthase gene from Yali Pear (Pyrus breschneideri Rehd). Lett Biotechnol (生物技术通讯), 2007, 18(5): 786–788 (in Chinese with English abstract)
[20]Waterhouse P M, Graham M W, Wang M B. Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Nat Acad Sci USA, 1998, 95: 13959–13964
[21]Wesley S V, Helliwell C A, Smith N A, Wang M B, Rouse D T, Liu Q, Gooding P S, Singh S P, Abbott D, Stoutjesdijk P A, Robinson S P, Gleave A P, Green A G, Waterhouse P M. Construct design for efficient, effective and high-throughput gene silencing in plants. Plant J, 200l, 27: 581–590
[22]Gleave A P. A versatile binary vector system with a T-DNA organizational structure conducive to efficient integration of cloned DNA into the plant genome. Plant Mol Biol, 1992, 20: 1203–1207
[23]Huang B-Y(黄冰艳), Ji W-Q(吉万全), Guo A-G(郭蔼光), Sadequr R, Li Z-Y(李忠宜). Post-transcriptional gene silencing (PTGS) and its application to crop genetic improvement. China Biotechnol (中国生物工程杂志), 2005, 25(5): 1–5 (in Chinese with English abstract)
[24]Chuang C F, Meyerowitz E M. Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. Proc Nat Acad Sci USA, 2000, 97: 4985–4990
[25]Gong D M, Zhang C Q, Chen X Y, Gong Z Z, Zhu J K. Constitutive activation and transgenic evaluation of the function of an Arabidopsis PKS protein kinase. J Biol Chem, 2002, 277: 42088–42096
[26]Senthil S, Madge Y G, Oliver Y, Terrence L G. RNA interference of soybean isoflavone synthase genes leads to silencing in tissues distal to the transformation site and enhanced susceptibility to Phytophthora sojae. Plant Physiol, 2005, 137: 1345–1353
[27]Li X-P(李小平), Ma Y-Y(马媛媛), Li P-L(李鹏丽), Zhang L-W(张丽文), Wang Y(王勇), Zhang R(张韧), Wang N-N(王宁宁). Knock-down on the expression of rlpk2 gene by RNAi technique can delay soybean leaf senescence. Chin Sci Bull (科学通报), 2005, 50: 1090–1096 (in Chinese)
[28]Hirotaka K, Eri K, Robert W R, Hiroshi K. RNAi knock-down of ENOD40s leads to significant suppression of nodule formation in Lotus japonicus. Plant Cell Physiol, 2006, 47: 1102–1111
[29]Hammond S M, Bernstein E, Beach D, Hannon G J. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells. Nature, 2000, 404: 293–296
[30]Burch-Smith T M, Miler J L. PTGS approaches to large-scale functional genomics in plants. In: Hannon G ed. RNAi: A Guide to Gene Silencing. New York: Cold Spring Harbor Laboratory Press, 2003. pp 243–263
[31]Fire A. RNA-triggered gene silencing. Trends Genet, l999, 15: 358–363
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