欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2017, Vol. 43 ›› Issue (08): 1254-1258.doi: 10.3724/SP.J.1006.2017.01254

• 研究简报 • 上一篇    

豌豆终止子rbc-T在转基因小麦研究中的应用

王小婷1,2,黄锁2,徐兆师2,李连城2,马有志2,陈明2,*,闵东红1,*   

  1. 1西北农林科技大学农学院 / 旱区作物逆境生物学国家重点实验室，陕西杨凌 712100；2中国农业科学院作物科学研究所 / 农作物基因资源与基因改良国家重大科学工程 / 农业部麦类生物学与作物遗传育种重点实验室，北京100081
  • 收稿日期:2017-01-20 修回日期:2017-04-20 出版日期:2017-08-12 网络出版日期:2017-04-27
  • 通讯作者: 闵东红, E-mail: mdh2493@126.com, Tel: 13609123593; 陈明, E-mail: chenming02@caas.cn, Tel: 010-82108750
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2014ZX08002-003B, 2016ZX08002002, 2016ZX08002005)资助。

Application of Pisum sativum Terminator rbc-T in Wheat Transformation

WANG Xiao-Ting1,2,HUANG Suo2,XU Zhao-Shi2,LI Lian-Cheng2,MA You-Zhi2,CHEN-Ming2,*,MIN Dong-Hong1,*   

  1. 1College of Agronomy, Northwest A&F University / State Key Laboratory of Crop Stress Biology for Arid Areas, Yangling 712100, China; 2 National Key Facility for Crop Gene Resource and Genetic Improvement / Key Laboratory of Biology and Genetic Improvement of Triticeae Crops, Ministry of Agriculture / Institute of Crop Science, Chinese Academy of Agricultural Sciences, Beijing 100081, China
  • Received:2017-01-20 Revised:2017-04-20 Published:2017-08-12 Published online:2017-04-27
  • Contact: Min Donghong, E-mail: mdh2493@126.com, Tel: 13609123593; Chen Ming, E-mail: chenming02@caas.cn, Tel: 010-82108750
  • Supported by:

    This study was supported by the National Major Project for Developing New GM Crops (2014ZX08002-003B, 2016ZX08002002, 2016ZX08002005).

RichHTML

PDF

663

被引次数

1

摘要：

目前常用的转基因载体元件大多为真菌或细菌来源，引发对转基因食品安全性的担忧。本研究克隆了豌豆终止子rbc-T，并用其替换农杆菌终止子nos-T构建转化载体，利用基因枪法将携带Ubi启动子、GUS基因和终止子rbc-T组成的最小表达框片段转化普通小麦，同时以带有nos-T终止子的载体为对照，经过PCR检测筛选获得T2代稳定遗传的转基因小麦株系。GUS组织化学染色及酶活定量分析结果表明，带有nos-T及rbc-T的转基因小麦中均能检测到GUS基因不同程度的表达。因此认为，豌豆终止子rbc-T可以代替农杆菌终止子nos-T用于小麦的转基因研究。

关键词: 胭脂碱合酶终止子(nos-T), 豌豆终止子(rbc-T), 转基因小麦, 报告基因

Abstract:

The fungal- or bacterial-origin vector elements are commonly used in transgenic crops, which raise great concerns about the safety of GM foods. In the study, we isolated the pea (Pisum sativum) terminator, rbc-T, and constructed a transformation vector by replacing the Agrobacterium terminator nos-T with rbc-T. The minimum transformation fragment consisting of Ubiquitin promoter, GUS reporter gene and terminator rbc-T was transformed into common wheat by particle bombardment method. Meanwhile a minimum transformation fragment with ?the Agrobacterium terminator nos-T was used as the control. The T2 transgenic lines were regenerated after PCR validation of the transformation. In the transgenic lines carrying both kinds of terminators, GUS gene expression was detected by GUS histochemical staining and enzyme activity analysis, indicating that the pea terminator rbc-T is applicable as an alternative of the Agrobacterium terminator nos-T in transgenic wheat research.

Key words: Nopaline synthase terminator (nos-T), Pea terminator (rbc-T), Transgenic wheat, GUS reporter gene

[1]李军, 瞿绍洪. 用无终止子的RFP报告基因捕获水稻终止子研究. 浙江农业学报, 2012, 24: 758–763 Li J, Qu S H. PolyA trapping by using terminatorless RFP reporter gene in rice. Acta Agric Zhejiangensis, 2012, 24: 758–763 (in Chinese with English abstract) [2]陈贞. 转基因作物的多重PCR检测体系的研究. 暨南大学硕士学位论文, 广东广州, 2011 Chen Z. Multiplex PCR for Detection of Genetically Modified Crops. MS Thesis of Jinan University, Guangzhou, China, 2011 (in Chinese with English abstract) [3]焦改丽, 孟钊红, 聂安全, 南芝润, 张换样, 李俊峰, 王娇娟, 赵俊侠, 李燕娥, 郭三堆. 花椰菜花叶病毒(CaMV)35S启动子在转基因棉花中的表达. 作物学报, 2004, 30: 1135–1139 Jiao G L, Meng Z H, Nie A Q, Nan Z R, Zhang H Y, Li J F, Wang J J, Zhao J X, Li Y E, Guo S D. Expression of CaMV35S promoter in transgenic cotton. Acta Agron Sin, 2004, 30: 1135–1139 (in Chinese with English abstract) [4]牛东东, 郝育杰, 荣瑞娟, 韦汉福, 兰金苹, 史佳楠, 魏健, 李雪姣, 杨烁, 奚文辉, 武鹏程, 刘丽娟, 吴琳, 刘斯奇, 尹长城, 刘国振. 转基因水稻中GUS蛋白质的检测及其表达特征. 中国农业科学, 2014, 47: 2715–2722 Niu D D, Hao Y J, Rong R J, Wei H F, Lan J P, Shi J N, Wei J, Li X J, Yang S, Xi W H, Wu P C, Liu L J, Wu L, Liu S Q, Yin C C, Liu G Z. Detection of GUS protein and its expression pattern in transgenic rice plants. Sci Agric Sin, 2014, 47: 2715–2722 (in Chinese with English abstract) [5]Dipak K S, Nrisingha D, Indu B M. pSiM24 Is a novel versatile gene expression vector for transient assays as well as stable expression of foreign genes in plants. PLoS One, 2014, 9: e98988. [6]徐琼芳, 李连城, 陈孝, 马有志, 叶兴国, 张增艳, 徐惠君, 辛志勇. 基因枪法获得GNA转基因小麦植株的研究. 中国农业科学, 2001, 34: 5–8 Xu Q F, Li L C, Chen X, Ma Y Z, Ye X G, Zhang Z Y, Xu H J, Xin Z Y. Study on the obtaining of transgenic wheats with GNA alien gene by biolistic particle. Sci Agric Sin, 2001, 34: 5–8 (in Chinese with English abstract) [7]Lipp M, Brodmann P, Pietsch K, Pauwels J, Anklam E. IUPAC collaborative trial study of a method to detect genetically modified soybeans and maize in dried powder. JAOAC Int, 1999, 82: 923–928 [8]Jefferson R A, Kavanagh T A, Bevan M W. GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J, 1987, 6: 3901–3907 [9]Kevan M A G, Angela T M, Stanislav V, Karel B, Richard J I, Jill S G. Analysis of genetically modified plant gene expression using GUS fluorimetry. Mol Biotechnol, 2000, 14: 235–239 [10]Chen Y J, Liu P, Alec A K N, Jennifer A N B, Kevin C, Todd P, Christopher A V. Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods, 2013, 10: 659–664 [11]张发云, 尹维波, 胡赞民. 毛白杨叶绿体基因启动子和终止子的克隆及其功能鉴定. 农业生物技术学报, 2007, 15: 439–445 Zhang F Y, Yin W B, Hu Z M. Cloning and function identification of the promoters and terminator of Populus tomentosa chloroplast genes. J Agric Biotechnol, 2007, 15: 439–445 (in Chinese with English abstract) [12]金双侠, 韩杰, 刘小云, 刘冠泽, 王一娴, 唐文鑫, 张献龙. SDS-蛋白酶法分离棉花cpDNA及psbA基因启动子、终止子克隆. 分子植物育种, 2007, 5: 683–689 Jin S X, Han J, Liu X Y, Liu G Z, Wang Y X, Tang W X, Zhang X L. Chroloplast DNA extraction in cotton by SDS-proteinase method and cloning of the promoter and terminator of psbA gene. Mol Plant Breed, 2007, 5: 683–689 (in Chinese with English abstract) [13]钟山, 罗大极, 吴刚, 徐婧, 汪亚平, 朱作言. 两种不同终止子在转基因鲤鱼中的促生长效应. 遗传, 2009, 31: 831–836 Zhong S, Luo D J, Wu G, Xu J, Wang Y P, Zhu Z Y. The growth promoting activity of two different kinds of reconstructed terminators in transgenic common carp. Hereditas (Beijing), 2009, 31: 831–836 (in Chinese with English abstract)
[1] 石磊, 苗利娟, 黄冰艳, 高伟, 张忠信, 齐飞艳, 刘娟, 董文召, 张新友. 花生AhFAD2-1基因启动子及5'-UTR内含子功能验证及其低温胁迫应答[J]. 作物学报, 2021, 47(9): 1703-1711.
[2] 王睿,朱梦琳,高方远,任鄄胜,陆贤军,任光俊,林拥军. 水稻组织特异型人工合成启动子的设计、构建及功能鉴定[J]. 作物学报, 2017, 43(06): 789-794.
[3] 石磊,苗利娟,齐飞艳,张忠信,高伟,孙子淇,黄冰艳,董文召,汤丰收,张新友*. 花生Δ9-硬脂酰-ACP脱氢酶基因启动子的克隆及功能分析[J]. 作物学报, 2016, 42(11): 1629-1637.
[4] 王永霞,杜新华,许为钢,齐学礼,李艳,王会伟,胡琳. 导入外源玉米C4型NADP-ME基因对小麦光合效能的影响[J]. 作物学报, 2016, 42(04): 600-608.
[5] 申芳嫡,洪彦涛,杜丽璞,徐惠君,马翎健,张增艳. 转细胞凋亡抑制基因OpIAP和p35增强小麦对纹枯病的抗性[J]. 作物学报, 2015, 41(10): 1490-1499.
[6] 杨坤,刘欣,杜丽璞,叶兴国,张增艳. 转AcAMP-sn基因抗全蚀病小麦新种质的创制与鉴定[J]. 作物学报, 2014, 40(01): 22-28.
[7] 刘菲,杨丽华,王爱云,马小飞,杜丽璞,刘欣,李盼松,张增艳,马翎健. 转TiERF1-RC7双价基因小麦的鉴定及其全蚀病抗性[J]. 作物学报, 2013, 39(11): 2094-2098.
[8] 杨丽华,王金凤,杜丽璞,徐惠君,魏学宁,李钊,马翎健,张增艳. 抗全蚀病、根腐病的转PgPGIP1基因小麦的获得与鉴定[J]. 作物学报, 2013, 39(09): 1576-1581.
[9] 党良,宿振起,叶兴国,徐惠君,李钊,邵艳军,张增艳. BvGLP1过表达增强了转基因小麦对根腐病的抗性[J]. 作物学报, 2013, 39(02): 368-372.
[10] 祝秀亮，李钊，杜丽璞，徐惠君，杨丽华，庄洪涛，马翎健，张增艳. 兼抗全蚀病和白粉病小麦新种质的创制与鉴定[J]. 作物学报, 2012, 38(12): 2178-2184.
[11] 党良，王爱云，徐惠君，祝秀亮，杜丽璞，邵艳军，张增艳. 抗根腐病的转GmPGIP3基因小麦扬麦18的获得与鉴定[J]. 作物学报, 2012, 38(10): 1833-1838.
[12] 王金凤，杜丽璞，李钊，黄素萍，叶兴国，冯斗，张增艳. 抗纹枯病、根腐病的转SN1基因小麦的获得与鉴定[J]. 作物学报, 2012, 38(05): 773-779.
[13] 吴琼, 许为钢, 李艳, 齐学礼, 胡琳, 张磊, 韩琳琳. 田间条件下转玉米C4型PEPC基因小麦的光合生理特性[J]. 作物学报, 2011, 37(11): 2046-2052.
[14] 孙永伟, 聂丽娜, 马有志, 徐兆师, 夏兰琴. 小麦穗发芽抗性相关Vp1基因启动子的分离及功能验证[J]. 作物学报, 2011, 37(10): 1743-1751.
[15] 李钊, 庄洪涛, 杜丽璞, 周淼平, 蔡士宾, 徐惠君, 李斯深, 张增艳. 组织特异表达启动子RSS1P在转TiERF1基因小麦中的应用[J]. 作物学报, 2011, 37(10): 1897-1903.
Viewed
Full text


Abstract

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