欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2013, Vol. 39 ›› Issue (09): 1594-1601.doi: 10.3724/SP.J.1006.2013.01594

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

农杆菌介导的RNAi CP基因在大豆中的转化

章洁琼1,李红艳1,胡小南1,单志慧2,*,唐桂香1,*   

  1. 1浙江大学农业与生物技术学院，浙江杭州 310058；2中国农业科学院油料作物研究所，湖北武汉 430062
  • 收稿日期:2012-11-28 修回日期:2013-05-24 出版日期:2013-09-12 网络出版日期:2013-07-09
  • 通讯作者: 唐桂香, E-mail: tanggx@zju.edu.cn; 单志慧, E-mail: zhihuijimi@163.com
  • 基金资助:

    本研究由农业部转基因生物新品种培育重大专项(20112X08004-004-009)，国家自然科学基金项目(31071443)和浙江省科技厅公益技术项目(2012C32001)资助。

Agrobacterium tumefaciens Mediated Transformation of RNAi CP Gene into Soybean (Glycine max L.)

ZHANG Jie-Qiong1,LI Hong-Yan1,HU Xiao-Nan1,SHAN Zhi-Hui2,*,TANG Gui-Xiang1,*   

  1. 1 Institute of Crop Science, College of Agriculture and Biotechnology, Zhejiang University, Hangzhou 310058, China; 2 Oil Crops Research Institute, Chinese Academy of Agriculture Sciences, Wuhan 430062, China
  • Received:2012-11-28 Revised:2013-05-24 Published:2013-09-12 Published online:2013-07-09
  • Contact: 唐桂香, E-mail: tanggx@zju.edu.cn; 单志慧, E-mail: zhihuijimi@163.com

RichHTML

PDF

1226

被引次数

8

摘要：

花叶病毒(soybean mosaic virus, SMV)病是大豆主要病害之一，生产上常采用种植抗性品种方法来防治。本研究以RNA干扰花叶病毒衣壳蛋白(coat protein, CP)基因为表达载体，Bar基因作为筛选标记基因，成熟子叶节为外植体，采用农杆菌介导法获得了22株T0代转基因大豆生根苗，经草丁膦涂抹、Bar试纸条和PCR法鉴定，获得RNAi CP转基因植株18株；对转基因植株T1代的遗传分析表明，外源基因能够稳定遗传到下一代且符合孟德尔遗传规律；T1代Southern杂交表明，导入的干扰片段为单拷贝；花叶病毒摩擦接种表明RNAi CP转基因大豆植株具有抗花叶病毒特性；摩擦接种后3周，DAS-ELISA检测进一步表明，RNAi CP转基因植株花叶病毒检出率仅为7.69%，而非转基因植株为100%。这表明RNAi花叶病毒CP基因可用于抗大豆花叶病毒的研究。

关键词: 大豆, RNAi CP, 遗传转化, 大豆花叶病毒, 病毒鉴定

Abstract:

 Soybean mosaic virus (SMV) causes a severe disease in soybean, which can be efficiently prevented by planting resistant cultivars. In order to improve the SMV resistance of soybean, an agrobacterium-tumefaciens mediated gene transformation was conducted in this study by using RNAi soybean mosaic virus coat protein (CP)gene as the expression vector, bar gene as the selective marker gene, and cotyledonary-node as the explant to get RNAi transformation soybean. As results, 22 putative transgenic soybean plants were obtained and 18 positive transgenic soybean plants were identified by coating with leaves herbicide, using bar protein quick dip stick and PCR analysis. The segregation ratio of T1 transgenic progeny showed that the transformed gene could be inherited according to the Mendel's law. The T1 southern blot analysis showed that the imported interference fragment was one copy. After SMV friction inoculation, the RNAi CP transgenic soybean plants showed good resistance to SMV. DAS-ELISA analysisat three weeks after SMV inoculation revealed that 100% of the non-transgenic lines SMV while only 7.69% of the RNAi plants were infected by SMV. The results demonstrated that the RNAi CP transgenic soybean plants obtained in the study are valuable resources for improving the SMV resistance of soybean.

Key words: Soybean, RNAi CP, Genetic transformation, Soybean mosaic virus, Virus identification

[1]Gardner M W, Kendrick J B. Soybean mosaic virus. Agric Res, 1921, 22: 123–124



[2]Wang L-Z(王连铮), Wang J-L(王金陵). Soybean Genetics and Breeding (大豆遗传育种学). Beijing: Science Press, 1992. pp 208–304 (in Chinese)



[3]Wang D-G(王大刚), Ma Y(马莹), Liu N(刘宁), Zheng J-J(郑桂杰), Yang Z-L(杨中路), Yang Y-Q(杨永庆), Zhi H-J(智海剑). Inheritance of resistances to soybean mosaic virus strains SC4 and SC8 in soybean. Acta Agron Sin (作物学报), 2012, 38(2): 202–209 (in Chinese with English abstract)



[4]Lindbo J A, Dougherty W G. Plant pathology and RNAi: a brief history. Annu Rev Phytopathol, 2005, 43: 191–204



[5]Kamachi S, Mochizuki A, Nishiguchi M, Tabei Y. Transgenic Nicotiana benthamiana plants resistant to cucumber green mottle mosaic virus based on RNA silencing. Plant Cell Rep, 2007, 26: 1283–1288



[6]Bass B L. Double-stranded RNA as a template for gene silencing. Cell, 2000, 101: 235–238



[7]Sharp P A. RNA interference. Genes Dev, 2001, 15: 485–490



[8]Smith N A, Singh S P, Wang M B, Stoutjesdijk P A, Green A G, Waterhouse P M. Total silencing by intron-spliced hairpin RNAs. Nature, 2000, 407: 319–20



[9]Kalantidis K, Psaradakis S, Tabler M, Tsagris M. The occurrence of CMV-specific short RNAs in transgenic tobacco expressing virus-derived double-stranded RNA is indicative of resistance to the virus. Mol Plant Microbe Interact, 2002, 15: 826–33



[10]Missiou A, Kalantidis K, Boutla A, Tzortzakaki S, Tabler M, Tsagris M. Generation of transgenic potato plants highly resistant to potato virus Y (PVY) through RNA silencing. Mol Breed, 2004, 14: 185–197



[11]Xu Z-H(许宗宏), Hao Q-N(郝青南), Chen L-M(陈李淼), Sun D-C(孙佃臣), Tian X-X(田星星), Shan Z-H(单志慧). Plant expression construct based on RNAi for soybean mosaic virus. Acta Agric Boreali-Sin (华北农学报), 2010, 25: 1–4 (in Chinese with English abstract)



[12]Zhang Z Y, Xing A Q, Staswick P. The use of glufosinate as a selective agent in Agrobacterium-mediated transformation of soybean. Plant Cell Tissue Organ Cult, 1999, 56: 37–46



[13]Paz M M, Martinez J C, Kalvig A B, Fonger T M, Wang K. Improved cotyledonary node method using an alternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation. Plant Cell Rep, 2004, 25: 206–213



[14]Edwards K, Johnstone C, Thompson C. A simple and rapid method for the preparation of plant genomic DNA for PCR analysis. Nucl Acids Res, 1991, 19: 1349



[15]Chen K-S(陈昆松), Li F(李方), Xu C-J(徐昌杰), Zhang S-L(张上隆), Fu C-X(傅承新). An efficient Macro-method of genomic DNA isolation from actinidia chinensis leaves. Hereditas (遗传), 2004, 26(4): 529–531 (in Chinese with English abstract)



[16]Liao L, Chen P, Rajcan I, Buss G R, Tolin S A. Genetic analysis of “8101” soybean containing three genes for resistance to soybean mosaic virus. Crop Sci, 2011, 51: 503–511



[17]Pinto Y M, Kok R A, Bandcombe D C. Resistance to rice yellow mottle virus (RYMV) in cultivated African rice varieties containing RYMV transgenes. Nat Biotechnol, 1999, 17: 702–707



[18]Abbott D, Wang M B, Waterhouse P A. A single copy of virusderived transgene encoding hairpin RNA gives immunity to barley yellow dwarf virus. Mol Plant Pathol, 2000, 1: 347–356



[19]Hayakawa T, Zhu Y, Itoh K, Kimura Y, Izawa T, Shimamoto K, Toriyama S. Genetically engineered rice resistant to rice stripe virus, an insect-transmitted virus. Proc Natl Acad Sci USA, 1992, 89: 9865–9869



[20]Yan Y T, Wang J F, Qiu B S, Tian B. Resistance to Rice stripe virus conferred by expression of coat protein in transgenic indica rice plants regenerated from bombarded suspension culture. Virol Sin, 1997, 12: 260–269



[21]Zhu J-H(朱俊华), Zhu X-P(竺晓平), Wen F-J(温孚江), Bai Q-R(白庆荣), Zhu C-X(朱常香), Song Y-Z(宋云枝). The effect of PVY-CP segment length on RNA-mediated virus resistance. Sci China Ser C: Life Sci (中国科学C辑: 生命科学), 2004, 34(1): 23–30 (in Chinese)



[22]van den Boogaart T, Wen F J, Davies J W, Lomonossoff G P. Replicase-derived resistance against pea early browning virus in Nicotiana benthamiana is an unstable resistance based upon posttranscriptional gene silencing. MPMI, 2001, 14: 196–200
[1] 陈玲玲, 李战, 刘亭萱, 谷勇哲, 宋健, 王俊, 邱丽娟. 基于783份大豆种质资源的叶柄夹角全基因组关联分析[J]. 作物学报, 2022, 48(6): 1333-1345.
[2] 杨欢, 周颖, 陈平, 杜青, 郑本川, 蒲甜, 温晶, 杨文钰, 雍太文. 玉米-豆科作物带状间套作对养分吸收利用及产量优势的影响[J]. 作物学报, 2022, 48(6): 1476-1487.
[3] 王炫栋, 杨孙玉悦, 高润杰, 余俊杰, 郑丹沛, 倪峰, 蒋冬花. 拮抗大豆斑疹病菌放线菌菌株的筛选和促生作用及防效研究[J]. 作物学报, 2022, 48(6): 1546-1557.
[4] 于春淼, 张勇, 王好让, 杨兴勇, 董全中, 薛红, 张明明, 李微微, 王磊, 胡凯凤, 谷勇哲, 邱丽娟. 栽培大豆×半野生大豆高密度遗传图谱构建及株高QTL定位[J]. 作物学报, 2022, 48(5): 1091-1102.
[5] 李阿立, 冯雅楠, 李萍, 张东升, 宗毓铮, 林文, 郝兴宇. 大豆叶片响应CO2浓度升高、干旱及其交互作用的转录组分析[J]. 作物学报, 2022, 48(5): 1103-1118.
[6] 彭西红, 陈平, 杜青, 杨雪丽, 任俊波, 郑本川, 罗凯, 谢琛, 雷鹿, 雍太文, 杨文钰. 减量施氮对带状套作大豆土壤通气环境及结瘤固氮的影响[J]. 作物学报, 2022, 48(5): 1199-1209.
[7] 王好让, 张勇, 于春淼, 董全中, 李微微, 胡凯凤, 张明明, 薛红, 杨梦平, 宋继玲, 王磊, 杨兴勇, 邱丽娟. 大豆突变体ygl2黄绿叶基因的精细定位[J]. 作物学报, 2022, 48(4): 791-800.
[8] 冯亚, 朱熙, 罗红玉, 李世贵, 张宁, 司怀军. 马铃薯StMAPK4响应低温胁迫的功能解析[J]. 作物学报, 2022, 48(4): 896-907.
[9] 李瑞东, 尹阳阳, 宋雯雯, 武婷婷, 孙石, 韩天富, 徐彩龙, 吴存祥, 胡水秀. 增密对不同分枝类型大豆品种同化物积累和产量的影响[J]. 作物学报, 2022, 48(4): 942-951.
[10] 杜浩, 程玉汉, 李泰, 侯智红, 黎永力, 南海洋, 董利东, 刘宝辉, 程群. 利用Ln位点进行分子设计提高大豆单荚粒数[J]. 作物学报, 2022, 48(3): 565-571.
[11] 周悦, 赵志华, 张宏宁, 孔佑宾. 大豆紫色酸性磷酸酶基因GmPAP14启动子克隆与功能分析[J]. 作物学报, 2022, 48(3): 590-596.
[12] 王娟, 张彦威, 焦铸锦, 刘盼盼, 常玮. 利用PyBSASeq算法挖掘大豆百粒重相关位点与候选基因[J]. 作物学报, 2022, 48(3): 635-643.
[13] 董衍坤, 黄定全, 高震, 陈栩. 大豆PIN-Like (PILS)基因家族的鉴定、表达分析及在根瘤共生固氮过程中的功能[J]. 作物学报, 2022, 48(2): 353-366.
[14] 张国伟, 李凯, 李思嘉, 王晓婧, 杨长琴, 刘瑞显. 减库对大豆叶片碳代谢的影响[J]. 作物学报, 2022, 48(2): 529-537.
[15] 宋丽君, 聂晓玉, 何磊磊, 蒯婕, 杨华, 郭安国, 黄俊生, 傅廷栋, 汪波, 周广生. 饲用大豆品种耐荫性鉴定指标筛选及综合评价[J]. 作物学报, 2021, 47(9): 1741-1752.
Viewed
Full text


Abstract

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