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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

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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

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