欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2010, Vol. 36 ›› Issue (09): 1484-1489.doi: 10.3724/SP.J.1006.2010.01484

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫花苜蓿LEA蛋白基因ihpRNA表达载体构建及烟草转化

白永琴1,康俊梅1,孙彦2,杨青川1,*,李燕1   

  1. 1 中国农业科学院北京畜牧兽医研究所，北京 100193；2 中国农业大学动物科技学院草地研究所，北京 100094
  • 收稿日期:2010-01-08 修回日期:2010-04-20 出版日期:2010-09-12 网络出版日期:2010-05-20
  • 通讯作者: 杨青川, E-mail: qchyang66@yahoo.com.cn
  • 基金资助:
    本研究由国家科技支撑计划(2008BADB3B05)和国家科技支撑计划子课题(2008BADB3B04-1)资助。

Construction of ihpRNA Expression Vectorof MsLEA3-1 Gene from Medicago sativa L. and Genetic Transformation in Tobacco

BAI Yong-Qin1,KANG Jun-Mei1,SUN Yan2,YANG Qing-Chuan1,*,LI Yan1   

  1. 1 Institute of Animal Science, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2 Institute of Grassland Science, China Agricultural University, Beijing 100094, China
  • Received:2010-01-08 Revised:2010-04-20 Published:2010-09-12 Published online:2010-05-20
  • Contact: YANG Qing-Chuan, E-mail: qchyang66@yahoo.com.cn

PDF

1864

被引次数

9

摘要： 根据紫花苜蓿LEA蛋白MsLEA3-1基因(GenBank登录号： EU665182)序列，设计两对含有酶切位点的特异性引物LEAf1/ LEAf2和LEAr1/ LEAr2，以构建好的PMD-LEA质粒为模板，分别合成用于构建干扰载体的正反义片段pMD-F和pMD-R，将正反义片段分别插入表达载体pART27的相应位置，构建成含有发夹结构的RNAi载体pART-F-R，经过Not I酶切鉴定，证明载体构建成功。通过农杆菌介导的方法，以干扰表达载体pART-F-R转化烟草，经过PCR检测，得到16株阳性转基因植株，为MsLEA3-1基因的功能研究奠定基础。

关键词:  紫花苜蓿, MsLEA3-1基因, RNA干扰, ihpRNA表达载体, 烟草转化

Abstract: During long-term evolution, plant has developed various physiological functions and bio-chemical mechanisms to respond the diverse stresses in different environments. Plant cell accumulates a series of proteins to reduce cell dehydration in the period of water shortage, of all proteins, late embriogenesis abundant LEA protein has been paid attention, which is one of the hot topics in plant stress physiology. In the paper, an RNAi expression vector harboring MsLEA3-1 gene fragment from Medicago sativa L. was constructed. On the basis of the sequence of Medicago sativa LEA protein (MsLEA3-1) gene (GenBank accession number: EU665182), two pairs of specific primers containing different enzyme sites were designed. With the template of PMD-LEA plasmid constructed, 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 confirmed by the digestion of restriction enzymes. pART-F-R was transformed into tobacco by Agrobacterium mediated transformation system. PCR testing showed that 16 transgenic plants were obtained.

Key words: Medicago sativa L., MsLEA3-1 gene, RNA interference, ihpRNA expression vector, Tobacco transformation

[1] 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: 6-811 
[3] Li J-R(李加瑞), Zhao W(赵伟), Li Q-Z(李全梓). RNA silencing of waxy gene results in low levels of amylose in the seeds of transgenic wheat. Acta Genet Sin (遗传学报), 2005, 32(8): 846-854 (in Chinese with English abstract) 
[4] Pinto Y M, Kok R A, Bandcombe D C. Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes
[J].Nat Biotechnol.1999, 17:702-707  
[5] Abbott D, Wang M B, Waterhouse P A. A single copy of a virus-derived transgene encoding hairp in RNA gives immunity to barley yellow dwarf virus
[J].Mol Plant Pathol.2000, 1:347-356 
[6] Gal T Z, Glazer I, Koltai H. An LEA group 3 family member is involved in survival of C. elegans during exposure to stress. Fed Eur Biochem Soc, 2004, 577: 21-26  
[7] Dure L, Chlan C. Developmental biochemistry of cottonseed embryogenesis and germination Plant Physiol, 1981, 68: 180-186  
[8] Bai Y-Q(白永琴), Yang Q-C(杨青川). Summary of late embriogenesis abundant protein. Biotechnol Bull (生物技术通报), 2009, (9): 1-7 (in Chinese with English abstract) 
[9] Ann R K, Nikolov Z L, John A H. Production of recombinant proteins in transgenic plants: practical considerations
[J].Bioteclnol Bioeng.1997, 56:473-484 
[10] Fischer R, Emans N. Molecular farming of pharmaceutical proteins
[J].Transgenic Res.2000, 9:279-299 
[11] Gu Q, Han N, Liu J Y, Zhu M Y. Cloning of Helicobacter pylori urease subunit B gene and its expression in tobacco (Nicotiana tabacum L)
[J].Plant Cell Rep.2005, 24:532-539 
[12] Stoutesdijk P A, Hurlestone C, Singh S P, Green A G. High-oleic acid Australia Brassica napus and B. juncea varieties produced by co-suppression of endogenous delta 12-desaturases. Biochem Soc Trans, 2000, 28: 938-940 
[13] Peng H(彭昊), Zhai Y(翟英), Zhang Q(张芊), Zhang Z-G(张志国), Wan S-Y(宛淑艳), Guo Y-P(郭玉朋), Wu J-X(吴金霞), Sun X-H(孙学辉), Sun Y(孙颖), Sun D-Y(孙大业), Lu T-G(路铁钢). Establishment and functional analysis of high efficiency RNA interference system in rice. Sci Agric Sin (中国农业科学), 2006, 39(9): 1729-1735 (in Chinese with English abstract)  
[14] Callis J, Formm M, Walbot V. Introns increases gene expression in cultured maize cells
[J].Genes Dev.1987, 1:1183-2000 
[15] Tanaka A, Mita S, Ohta S, Kyozuka J, Shimamoto K, Nakamura K. Enhancement of foreign gene expression by a dicot intron in rice but not in tobacco is correlated with an increased level of mRNA and an eficient splicing of intron
[J].Nucl Acids Res.1990, 18:6767-6770 
[16] Wesley S V, Helliwell C A, Smith N A, Wang M, 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
[1] 谈欢,刘玉汇,李丽霞,王丽,李元铭,张俊莲. 马铃薯块茎花色素苷合成相关R2R3 MYB蛋白基因的克隆和功能
分析[J]. 作物学报, 2018, 44(7): 1021-1031.
[2] 周香艳, 杨江伟, 唐勋, 文义凯, 张宁, 司怀军. amiRNA技术沉默C-3氧化酶编码基因StCPD对马铃薯抗旱性的影响[J]. 作物学报, 2018, 44(04): 512-521.
[3] 程伟,李和平,何水林,廖玉才. 寄主诱导的基因沉默提高植物真菌病害抗性研究进展[J]. 作物学报, 2017, 43(08): 1115-1121.
[4] 柴建芳*,王海波*,马秀英,张翠绵,董福双. ω-黑麦碱基因沉默对小麦1B/1R易位系加工品质的影响[J]. 作物学报, 2016, 42(05): 627-632.
[5] 周露, 沈贝贝, 白苏阳, 刘喜, 江玲, 翟虎渠, 万建民. 以RNA干扰γ-氨基丁酸转氨酶1基因(OsGABA-T1)表达提高稻米γ-氨基丁酸(GABA)含量[J]. 作物学报, 2015, 41(09): 1305-1312.
[6] 胡丹丹,张帆,黄立钰,卓大龙,张帆,周永力,石英尧,黎志康. 胁迫相关蛋白激酶基因OsSAPK2调控水稻抗白叶枯病反应[J]. 作物学报, 2015, 41(08): 1191-1200.
[7] 尹明智, 官梅, 肖钢, 李栒, 官春云. DOF转录因子AtDof1.7 RNA干扰载体的构建及拟南芥的遗传转化[J]. 作物学报, 2011, 37(07): 1196-1204.
[8] 朱见明, 严学兵, 史莹华, 王成章. 紫花苜蓿光敏色素B基因片段克隆及RNA干扰表达载体的构建[J]. 作物学报, 2011, 37(02): 374-379.
[9] 汪结明，张建，江海洋，朱苏文，范军，程备久. RNA干扰水稻SBE3基因的表达对籽粒淀粉合成及其关键酶活性的影响[J]. 作物学报, 2010, 36(2): 313-320.
[10] 黄丽华，蒋向，李博，李育强，张学文. 薤白EPSP合成酶基因转化烟草提高其草甘膦抗性[J]. 作物学报, 2009, 35(5): 855-860.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备15008789号-2