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 estrogeninduced Cre/loxP marker gene excision system was introduced to rice cell via Agrobacteriummediated 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 predifferentiation, predifferentiation 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 predifferentiation，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 estrogeninduced treatment at resistant calli level.
ZHAO Yan1,2,*, ZHANG Xiaoli2, GUO Longbiao1， QIAN Qian1,*
. Excision of Marker Genes in Transgenic Rice at Transformed Cell Level using Cre/loxP System[J]. Chinese Journal OF Rice Science, 2011
, 25(6)
: 580
-586
.
DOI: 10.3969/j.issn.10017216.2011.06.003
\[1\]Qian Y. Analysis of advantages and disadvantages on transgenic crops.Biotechnol Inform, 1999, 5: 711.
\[2\]Kai G Y, Zhang L, Zhang H Y. Markerfree: A novel tendency of transgenic plants. Acta Bot Sin, 2002, 44(8): 883888.
\[3\]Goldsbrough A P, Lastrella C N, Yoder J I. Transposition mediated repositioning and subsequent elimination of marker genes from transgenic tomato. Nat Biotechnol, 1993, 11: 12861292.
\[4\]Cotsaftis O, Sallaud C, Breitler J C, et al. Transposonmediated generation of TDNA and markerfree rice plants expressing a Bt endotoxin gene. Mol Breeding, 2002, 10: 165180.
\[5\]McKnight T, Lillis M, Simpson R. Segregation of genes transferred to one plant cell from two separate Agrobacterium strains. Plant Mol Biol, 1987, 8: 439445.
\[6\]Komari T, Hiei Y, Saito Y, et al. Vectors carrying two separate TDNAs for cotransformation of higher plants mediated by Agrobacterium tumefaciens and segregation of transformants free from selection markers. Plant J, 1996, 10: 165174.
\[7\]Zuo J, Niu Q W, Chua N H. An estrogen receptorbased transactivator XVE mediates highly inducible gene expression in transgenic plants. Plant J, 2000, 24: 265273.
\[8\]Zhang Y, Liu H, Li B, et al. Generation of selectable markerfree transgenic tomato resistant to drought, cold and oxidative stress using the Cre/loxP DNA excision system. Transgenic Res, 2009, 5(18): 607619.
\[9\]Dale E C, Ow D W. Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA， 1991, 88: 1055810562.
\[10\]Gleave A P, Mitra D S, Mudge S R, et al. Selectable markerfree transgenic plants without sexual crossing: Transient expression of cre recombinase and use of a conditional lethal dominant gene. Plant Mol Biol, 1999, 40: 223235.
\[11\]李霞, 翁海波, 韩绍印, 等. 转基因植物高效删除标记基因的实用型双元转化载体. 生物工程学报, 2006, 22(4): 550554.
\[12\]Sreekala C, Wu L, Gu K. Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep, 2005, 24: 8694.
\[13\]赵艳, 钱前, 王慧中, 等. 基因枪转化的bar基因表达盒在水稻中的重组结构. 中国水稻科学, 2007, 21(5): 469471.
\[14\]曾凡锁， 詹亚光. 转基因植物中外源基因的整合特性及其研究策略. 植物学通报, 2004, 21(5): 565577.
\[15\]陈惠, 赵原， 种康. 一种改进的水稻成熟胚愈伤组织高效基因转化系统. 植物学通报, 2008, 25(3): 322331.
\[16\]Taketo A. DNA transfection of Escherichia coli by electroporation. Biochim Biophys Acta， 1988, 949(3): 318324.
\[17\]Jefferson R A. Assaying chimeric genes in plants:The GUS gene fusion system. Plant Mol Biol Rep, 1987, 5: 387405.
\[18\]Kidwell K K, Osborn T C. Simple plant DNA isolation procedures//Beckman J S, Osborn T C. Plant Genomes: Methods for Genetic and Physical Mapping. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1992: 113.
