欢迎访问作物学报，今天是
作物学报

作物学报 ›› 2011, Vol. 37 ›› Issue (10): 1897-1903.doi: 10.3724/SP.J.1006.2011.01897

• 研究简报 • 上一篇    下一篇

组织特异表达启动子RSS1P在转TiERF1基因小麦中的应用

李钊1,2,庄洪涛1，杜丽璞1，周淼平3，蔡士宾3，徐惠君1，李斯深2,*，张增艳1,*   

  1. LI Zhao1,2,ZHUANG Hong-Tang1,DU Li-Pu1,ZHOU Miao-Ping3,CAI Shi-Bin3,XU Hui-Jun1,LI Si-Shen2,*,ZHANG Zeng-Yan1,*
  • 收稿日期:2010-11-05 修回日期:2011-06-25 出版日期:2011-10-12 网络出版日期:2011-07-28
  • 通讯作者: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781; 李斯深, E-mail: ssli@sdau.edu.cn
  • 基金资助:

    本研究由国家转基因生物新品种培育科技重大专项(2008ZX08002-001, 2009ZX08002-006B)资助。

Utilization of Tissue Specific Expressing Promoter RSS1P in TiERF1 Transgenic Wheat

1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 / 农业部作物遗传育种重点实验室，北京 100081; 2山东农业大学农学院，山东泰安 2701082; 3江苏省农业科学院，江苏南京 210014   

  1. 1 National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Crop Genetic and Breeding, Ministry of Agriculture / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China; 2 College of Agronomy, Shandong Agricultural University, Tai’an 271018, China; 3 Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China
  • Received:2010-11-05 Revised:2011-06-25 Published:2011-10-12 Published online:2011-07-28
  • Contact: 张增艳, E-mail: zhangzy@mail.caas.net.cn, Tel: 010-82108781; 李斯深, E-mail: ssli@sdau.edu.cn

PDF

1092

被引次数

6

摘要： 从水稻叶片中克隆了一个韧皮部组织特异表达的水稻蔗糖合酶启动子(RSS1P)，将RSS1P与中间偃麦草乙烯反应因子基因TiERF1相融合构成组织特异表达的TiERF1基因表达盒，取代pAHC20中Ubi::bar基因表达盒，构建成无选择标记的韧皮部组织特异表达的pA20-RSS1P::TiERF1载体。利用基因枪将pA20-RSS1P::TiERF1与pAHC20载体混合、共轰击小麦品种扬麦12的幼胚愈伤组织，获得转RSS1P::TiERF1基因小麦。对该转基因小麦T0和T1代植株进行PCR、PCR-Southern、半定量RT-PCR和荧光定量PCR分析，证实外源RSS1P::TiERF1基因已转入受体，并且具有可遗传性；转入的RSS1P::TiERF1基因仅在根、茎、叶中表达，以根部表达量最高，在种子内不表达。纹枯病抗性鉴定和主要农艺性状考察结果表明，与受体扬麦12相比，转RSS1P::TiERF1基因小麦对纹枯病的抗性有明显提高，与转Ubi::TiERF1基因小麦的抗病性相当，而且转RSS1P::TiERF1基因小麦的农艺性状没有明显改变，说明可以利用RSS1P启动子创造更实用的转基因小麦新种质。

关键词: 水稻蔗糖合酶启动子, 组织特异型表达, 转基因小麦, 中间偃麦草乙烯反应因子

Abstract: A sucrose synthase-1 promoter (RSS1P) was cloned from rice leaf, and subcloned in-fused with 5′-terminate of the gene of ethylene responsive factor of Thinopyrum intermedium (TiERF1) to form the RSS1P::TiERF1 expressing cassette. The expressing cassette replaced the Ubi::bar gene expression cassette of pAHC20 to produce the phloem-specific expressing vector pA20-RSS1P::TiERF1. The selective mark bar was deleted in the pA20-RSS1P::TiERF1 vector, which is safe for the ecosystem. Using the method of bombardment, the vector DNAs of pA20-RSS1P::TiERF1 andpAHC20 with bar gene were co-transformed to young embryo callus of wheat cultivar Yangmai 12. The RSS1P::TiERF1 transgenic plants in T0 and T1 generations were subjected to PCR, PCR-Southern, RT-PCR, and Q-RT-PCR analyses, and Rhizoctonia cerealis resistance tests. The molecular detection results showed that the transgene RSS1P::TiERF1 was introduced into Yangmai 12 and was inheritable. In the RSS1P::TiERF1 transgenic wheat plants, expression of TiERF1 was detected in root, stem, and leaf except seed. The highest expression level was observed in root. Compared to the untransformated Yangmai 12, resistance to R. cerealis in the RSS1P::TiERF1 transgenic wheat plants was obviously improved, which was similar to that in the Ubi::TiERF1 transgenic wheat plants. The agronomic traits of RSS1P::TiERF1 transgenic plants had no obvious changes compared to untransformated Yangmai 12. These results suggest that RSS1P promoter is feasible to be used for developing new transgenic wheat germplasm.

Key words: Rice sucrose synthase-1 promoter, Phloem-specific expression, Transgenic wheat, Ethylene responsive factor of Thinopyrum intermedium

[1]Chen Y-X(陈延熙), Tang W-H(唐文华), Zhang D-H(张敦华), Jian X-Y(简小鹰). A preliminary study on etiology of sharp eye-spot of wheat in China. Acta Phytophyl Sin (植物保护学报), 1986, 13(1): 39?44 (in Chinese with English abstract)
[2]Shi J-R(史建荣), Wang Y-Z(王裕中), Shen S-W(沈素文), Chen H-G(陈怀谷). Pathogenicity of Rhizoctonia cerealis to wheat in Jiangsu province. Jiangsu J Agric Sci (江苏农业学报), 1997, l3(3): 188?190 (in Chinese with English abstract)
[3]Dong N(董娜), Zhang Z-Y(张增艳), Xin Z-Y(辛志勇). Isolation and expression analysis of a pathogen-induced ERF gene in Triticum aestivum L. Sci Agric Sin (中国农业科学), 2008, 41(4): 946?953 (in Chinese with English abstract)
[4]Shi J-R(史建荣), Wang Y-Z(王裕中), Chen H-G(陈怀谷), Shen S-W(沈素文). Screening techniques and evaluation of wheat resistance to sharp eyespot caused by Rhizoctonia cerealis. Acta Phytophyl Sin (植物保护学报), 2000, 27(2): 107?112 (in Chinese with English abstract)
[5]Yang L-J(杨立军), Yang X-J(杨小军), Yu D-Z(喻大昭), Wang S-N(王绍南). Resistance evaluation of wheat cultivars (lines) to Rhizoctonia cerealis Varder Hoeven and screening of its resistance resources. Plant Prot (植物保护), 2001, 27(2): 4?7 (in Chinese with English abstract)
[6]Wan Y-X(万映秀), Wang W-X(王文相), Zhang P-Z(张平治), Cao W-X(曹文昕), Zhao L(赵莉). Identification techniques and screening of sharp eyespot resistance of wheat. Chin Agric Sci Bull (中国农学通报), 2009, 25(7): 223?226 (in Chinese with English abstract)
[7]Zhang X-C(张小村), Li S-S(李斯深), Zhao X-H(赵新华), Li R-J(李瑞军). Genetic analysis on resistance to sharp eyespot by using fifteen populations of recombinant inbred lines in wheat. J Triticeae Crops (麦类作物学报), 2004, 24(3): 13?16 (in Chinese with English abstract)
[8]Tang T(汤颋), Ren L-J(任丽娟), Cai S-B(蔡士宾), Wu J-Z(吴纪中), Lu W-Z(陆维忠), Chen J-M(陈建民), Ma H-X(马鸿翔). Study on QTL mapping of sharp eyespot resistance (Rhizoctonia cerealis) in wheat ARz. J Triticeae Crops (麦类作物学报), 2004, 24(4): 11?16 (in Chinese with English abstract)
[9]Cai S-B(蔡士宾), Ren L-J(任丽娟), Yan W(颜伟), Wu J-Z(吴纪中), Chen H-G(陈怀谷), Wu X-Y(吴小有), Zhang X-Y(张仙义). Germplasm development and mapping of resistance to sharp eyespot (Rhizoctonia cerealis) in wheat. Sci Agric Sin (中国农业科学), 2006, 39(5): 928?934 (in Chinese with English abstract)
[10]Liang H X, Lu Y, Liu H X, Wang F D, Xin Z Y, Zhang Z Y. A novel activator-type ERF of Thinopyrum intermedium, TiERF1, positively regulates defence responses. J Exp Bot, 2008, 59: 3111?3120
[11]Lu Y(路妍), Zhang Z-Y(张增艳), Ren L-J(任丽娟), Liu B-Y(刘宝业), Liao Y(廖勇), Xu H-J(徐惠君), Du L-P(杜丽璞), Ma H-X(马鸿翔), Ren Z-L(任正隆), Jing J-X(井金学), Xin Z-Y(辛志勇). Molecular analyses on Rs-AFP2 transgenic wheat plants and their resistance to Rhizoctonia cerealis. Acta Agron Sin (作物学报), 2009, 35(4): 640?646 (in Chinese with English abstract)
[12]Chen L, Zhang Z Y, Liang H X, Liu H X, Du L P, Xu H J, Xin Z Y. Overexpression of TiERF1 enhances resistance to sharp eyespot in transgenic wheat. J Exp Bot, 2008, 59: 4195?4204
[13]Oñate-Sánchez L, Anderson J P, Young J, Singh K B. AtERF14, a member of the ERF family of transcription factors, plays a nonredundant role in plant defense. Plant Physiol, 2007, 143: 400?409
[14]Shi Y, Wang M B, Powell K S, Van Damme E, Hilder V A, Gatehouse A M R, Boulter D, Gatehouse J A. Use of the rice sucrose synthase-1 promoter to direct phloem-specific expression of β-glucuronidase and snowdrop lectin genes in transgenic tobacco plants. J Exp Bot, 1994, 45: 623?631
[15]Li Y-C(李永春), Zhang X-Y(张宪银), Xue Q-Z(薛庆中). Cloning and specific expression of rice sucrose synthase gene promoter in transgenic rice plants. Acta Agron Sin (作物学报), 2002, 28(5): 586?590 (in Chinese with English abstract)
[16]Murray M G, Thompson W F. Rapid isolation of high molecular weight plant DNA. Nucl Acids Res, 1980, 8: 4321?4325
[17]Wang M B, Boulter D, Gatehouse J A. A complete sequence of the rice sucrose synthase-1 (RSs1) gene. Plant Mol Biol, 1992, 19: 881?885
[18]Christensen A H, Quail P H. Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res, 1996, 5: 213?218
[19]Xu H-J(徐惠君), Pang J-L(庞俊兰), Ye X-G(叶兴国), Du L-P(杜丽璞), Li L-C(李连城), Xin Z-Y(辛志勇), Ma Y-Z(马有志), Chen J-P(陈剑平), Chen J(陈炯), Cheng S-H(程顺和), Wu Y-H(吴宏亚). Study on the gene transferring of Nib8 into wheat for its resistance to the yellow mosaic virus by bombardment. Acta Agron Sin (作物学报), 2001, 27(6): 684?689 (in Chinese with English abstract)
[20]Sharp P J, Kries M, Shewry P R, Gale M D. Location of β-amylase sequences in wheat and its relatives. Theor Appl Genet, 1988, 75: 286?290
[21]Zhang Z-Y(张增艳), Xu J-S(许景升), Liu Y-G(刘耀光), Wang X-P(王晓萍), Lin Z-S(林志珊), Xin Z-Y(辛志勇). Isolation of resistance gene candidates by a resistance gene analog of Thinopyrum intermedium and pooled-PCR. Acta Agron Sin (作物学报), 2004, 30(3): 189?195 (in Chinese with English abstract)
[22]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2–ΔΔCT method. Methods, 2001, 25: 402?408
[23]Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database. Nucl Acids Res, 1999, 27: 297?300
[24]Prestridge D S. SIGNAL SCAN: a computer program that scans DNA sequences for eukaryotic transcriptional elements. Comput Appl Biosci, 1991, 7: 203?206
[1] 王小婷,黄锁,徐兆师,李连城,马有志,陈明,闵东红. 豌豆终止子rbc-T在转基因小麦研究中的应用[J]. 作物学报, 2017, 43(08): 1254-1258.
[2] 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.
[3] 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.
[4] 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.
[5] 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.
[6] 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.
[7] 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.
[8] 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.
[9] 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.
[10] 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.
[11] 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.
[12] 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.
[13] 路妍，张增艳，任丽娟，刘宝业，廖勇，徐惠君，杜丽璞，马鸿翔，任正隆，井金学，辛志勇. 转Rs-AFP2基因小麦的分子分析及其纹枯病抗性[J]. 作物学报, 2009, 35(4): 640-646.
[14] 王瑞霞;高庆荣;崔德才;刘正斌;乔晓琳;李洪利. 转反义PLDγ基因小麦的分子检测及农艺性状分析[J]. 作物学报, 2005, 31(10): 1354-1358.
[15] 徐琼芳;田芳;陈孝;侯文胜;李连城;马有志;徐惠君;辛志勇. 转基因抗虫小麦中sgna基因的遗传分析及抗虫性鉴定[J]. 作物学报, 2004, 30(05): 475-480.
Viewed
Full text


Abstract

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