中国水稻科学

中国水稻科学 ›› 2011, Vol. 25 ›› Issue (6): 580-586.DOI: 10.3969/j.issn.10017216.2011.06.003

• 研究报告 • 上一篇    下一篇

应用Cre/loxP系统在转化细胞水平上高效删除转基因水稻的标记基因

赵艳1,2,* ,张晓丽2 ,郭龙彪1,,钱前1,*   

  1. 1中国水稻研究所 水稻生物学国家重点实验室,  浙江 杭州 310006； 2 浙江工商大学 食品与生物工程学院, 浙江 杭州 310035；
  • 收稿日期:2010-10-23 修回日期:2011-01-05 出版日期:2011-11-10 发布日期:2011-11-10
  • 通讯作者: 赵艳1,2,* 钱前1,*
  • 基金资助:

    国家自然科学基金资助项目（30871511, 30771317）； 农业部转基因生物新品种培育重大专项（2008ZX0801003, 2009ZX08001022B）； 国家863计划资助项目（2006AA10Z1A9）； 中国博士后基金资助项目（20090450477）。

Excision of Marker Genes in Transgenic Rice at Transformed Cell Level using  Cre/loxP System

ZHAO  Yan 1,2,* , ZHANG  Xiaoli 2, GUO  Longbiao 1， QIAN  Qian 1,*   

  1. 1 State Key Laboratory of Rice Biology， China National Rice Research Institute， Hangzhou 310006， China; 2 College of Food Science and Biotechnology, Zhejiang Gongshang University, Hangzhou 310035, China;
  • Received:2010-10-23 Revised:2011-01-05 Online:2011-11-10 Published:2011-11-10
  • Contact: ZHAO Yan1,2,*,QIAN Qian1,*

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被引次数

12

摘要： 研究了Cre/loxP系统在转化细胞水平上删除转基因水稻中抗性标记基因的可行性和效率。采用农杆菌介导法将Cre/loxP标记基因剪切系统载体pNCG导入水稻细胞，用G418筛选法获得水稻抗性愈伤组织后，在组织培养不同阶段的培养基中添加25 μmol/L雌激素进行Cre基因的诱导表达和标记基因的剪切，PCR检测T0植株中标记基因nptⅡ、重组酶基因Cre和目标基因gusA的整合情况。将扩增结果为gusA（+）/nptⅡ（-）/Cre（-）的转基因植株统计为标记基因剪切成功的植株。结果表明，在抗性愈伤组织培养的预分化前、预分化和分化阶段添加雌激素诱导表达重组酶Cre均能成功切除标记基因序列，标记基因剪切成功率为6.82%~46.43%。在预分化前采用液体培养基添加雌激素诱导处理愈伤组织3 d，T0植株中标记基因的剪切效率高达173.33%，主要原因是雌激素处理提高了愈伤组织绿苗分化率。直接在分化培养基中添加雌激素诱导处理,T0植株中标记基因剪切成功率达46.43%，剪切效率（144.44%）也较高。表明雌激素诱导的Cre/loxP系统能在水稻抗性愈伤组织水平上实现对标记基因序列高效快速删除。

关键词: 水稻, 雌激素诱导, Cre/loxP重组酶系统, 转化细胞, 标记基因删除

Abstract: The feasibility and the efficiency of excision of marker genes in transgenic rice were studied  at transformed cell level  by  using the  Cre/loxP system. The vector pNCG including estrogeninduced Cre/loxP marker gene excision system was introduced to rice cell via Agrobacteriummediated transformation. After the resistant calli  were screened out under G418 selection， the Cre gene expression was induced to excise the marker gene by adding 25 μmol/L estrogen to the medium at different stages of tissue culture. The integration of marker gene nptⅡ, recombinase gene Cre and target gene gusA in regenerated transgenic rice plants was analyzed  by PCR. The plantlet  of gusA（+）/nptⅡ（-）/Cre（-） in PCR test were calculated as transgenic plants  with  excision  successful. Results showed that marker gene sequences could be excised successfully by estrogen induction at three tissue culture stages including before predifferentiation, predifferentiation and differentiation. The frequency of marker gene excision ranged from 6.82% to 46.43%. When transformed calli were treated by estrogen in liquid medium for 3 d before predifferentiation，the efficiency of marker gene excision in T0 transgenic rice plants was as high as 173.33%, mainly because the estrogen treatment improved the green plant regeneration frequency of calli. When estrogen was directly added to differentiation medium for induction treatment, the frequency of marker gene excision in T0 transgenic rice plants was 46.43% with a higher efficiency of marker gene excision （144.44%）. The  results confirmed  that marker gene sequences in transgenic rice plants could be excised efficiently and quickly by using  the Cre/loxP system under estrogeninduced treatment   at resistant calli level.

Key words: rice, estrogeninduction, Cre/loxP system, transformed cell, marker gene excision

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