欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2010, Vol. 36 ›› Issue (4): 596-601.doi: 10.3724/SP.J.1006.2010.00596

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

紫花苜蓿锌指蛋白基因RNAi表达载体的构建及在苜蓿的转化

秦智慧1,2，晁跃辉1，杨青川1,*，康俊梅1，孙彦3，王凭青2,*，龙瑞才1   

  1. 1中国农业科学院北京畜牧兽医研究所，北京100193；2重庆大学生物工程学院，重庆400030；3中国农业大学100193
  • 收稿日期:2009-11-10 修回日期:2010-01-07 出版日期:2010-04-12 网络出版日期:2010-02-05
  • 通讯作者: 杨青川, E-mail: qcyang66@yahoo.com.cn, 王凭青, E-mail: wang_pq@21cn.com
  • 基金资助:

    本研究由国际科技支撑计划项目（2008BADB3B05）和现代农业产业技术体系建设专项资金资助。

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 Published:2010-04-12 Published online:2010-02-05
  • Contact: YANG Qing-Chuan, E-mail: qcyang66@yahoo.com.cn, WANG Ping-Qing, E-mail: wang_pq@21cn.com

PDF

1918

被引次数

15

摘要：

根据紫花苜蓿锌指蛋白MsZFN基因(GenBank登录号为EU624138)序列，设计两对含有酶切位点的特异性引物，以紫花苜蓿cDNA为模板，分别合成用于构建干扰载体的正反义片段，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。利用农杆菌介导方法，将pART-F-R转化到紫花苜蓿中，经过PCR检测，获得了3株转基因植株。经过RT-PCR检测，证明转基因植株中MsZFN基因表达量与未转基因的植株相比，明显降低。结果表明，构建成功具有发夹结构的RNAi载体pART-F-R，它可有效的抑制紫花苜蓿MsZFN基因。

关键词: 紫花苜蓿, 锌指蛋白, RNAi, 转基因

Abstract:

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] 单露英, 李俊, 李亮, 张丽, 王颢潜, 高佳琪, 吴刚, 武玉花, 张秀杰. 转基因玉米NK603基体标准物质研制[J]. 作物学报, 2022, 48(5): 1059-1070.
[2] 王渭霞, 赖凤香, 胡海燕, 何佳春, 魏琪, 万品俊, 傅强. 超低温11年保存期对转基因作物基体标准样品核酸检测的影响[J]. 作物学报, 2022, 48(1): 238-248.
[3] 李杰华, 端群, 史明涛, 吴潞梅, 柳寒, 林拥军, 吴高兵, 范楚川, 周永明. 新型抗广谱性除草剂草甘膦转基因油菜的创制及其鉴定[J]. 作物学报, 2021, 47(5): 789-798.
[4] 马欢欢, 方启迪, 丁元昊, 池华斌, 张献龙, 闵玲. 棉花GhMADS7基因正调控棉花花瓣发育[J]. 作物学报, 2021, 47(5): 814-826.
[5] 杨琴莉, 杨多凤, 丁林云, 赵汀, 张军, 梅欢, 黄楚珺, 高阳, 叶莉, 高梦涛, 严孙艺, 张天真, 胡艳. 棉花花器官突变体的鉴定及候选基因的克隆[J]. 作物学报, 2021, 47(10): 1854-1862.
[6] 李晓旭, 王蕊, 张利霞, 宋亚萌, 田晓楠, 葛荣朝. 水稻基因OsATS的克隆及功能鉴定[J]. 作物学报, 2021, 47(10): 2045-2052.
[7] 李俊,李亮,李夏莹,宋贵文,沈平,张丽,翟杉杉,柳方方,吴刚,张秀杰,武玉花. 转基因玉米MIR604基体标准物质研制[J]. 作物学报, 2020, 46(4): 473-483.
[8] 衡友强,游西龙,王艳. 费尔干猪毛菜病程相关蛋白SfPR1a基因的异源表达增强了烟草对干旱、盐及叶斑病的抗性[J]. 作物学报, 2020, 46(4): 503-512.
[9] 陈杉彬, 孙思凡, 聂楠, 杜冰, 何绍贞, 刘庆昌, 翟红. 甘薯IbCAF1基因的克隆及耐盐性、抗旱性鉴定[J]. 作物学报, 2020, 46(12): 1862-1869.
[10] 马硕, 焦悦, 杨江涛, 王旭静, 王志兴. 基因组测序技术解析耐除草剂转基因水稻G2-7的分子特征[J]. 作物学报, 2020, 46(11): 1703-1710.
[11] 张双双,王立伟,姚楠,郭光艳,夏玉凤,秘彩莉. 水稻OsUBA基因的表达及其在促进种子萌发和开花中的功能[J]. 作物学报, 2019, 45(9): 1327-1337.
[12] 周向阳,赵亮,狄佳春,陈旭升. 2个抗虫棉的外源Bt基因分子鉴定及其染色体定位[J]. 作物学报, 2019, 45(9): 1440-1445.
[13] 潘丽娟,陈娜,陈明娜,王通,王冕,陈静,杨珍,万勇善,禹山林,迟晓元,刘风珍. 花生AhPEPC1基因抑制表达的转基因后代转录组分析[J]. 作物学报, 2019, 45(7): 993-1001.
[14] 张小芳,董秋平,乔潇,乔亚科,王冰冰,张锴,李桂兰. 基于Cre/loxP系统的无筛选标记转耐低磷转录因子GmPTF1大豆种质创制与分析[J]. 作物学报, 2019, 45(5): 683-692.
[15] 董玉凤 王旭静 宋亚亚 靳 茜 王志兴. 利用基因拆分技术培育耐草甘膦转基因水稻的研究 [J]. 作物学报, 2019, 45(3): 344-353.
Viewed
Full text


Abstract

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