柑橘无标记转基因转化体系的建立  

Marker-free transgenic system of Citrus

作　　者：李琳洁[1,2,3] 杨莉[1,2,3] 李芳[1,2,3] 谢玉明[1] 曾柏全[4] 邓子牛[1,2,3] 

机构地区：[1]湖南农业大学园艺园林学院,湖南长沙410128 [2]湖南省作物种质创新与资源利用重点实验室,湖南长沙410128 [3]国家柑橘改良中心长沙分中心,湖南长沙410128 [4]中南林业科技大学生命科学与技术学院,湖南长沙410004

出　　处：《湖南农业大学学报（自然科学版）》2010年第6期649-652,共4页Journal of Hunan Agricultural University(Natural Sciences)

基　　金：国家农业部"948"专项(2010-Z47);国家转基因专项(JY04-B-02);作物种质创新与资源利用重点实验室科学基金开放项目(62162021200014)

摘　　要：采用1个结合化学诱导系统XVE和位点专一性重组系统Cre/LoxP的标记基因剔除载体,对柑橘进行无标记转基因研究.此载体可通过β-雌二醇诱导表达系统控制重组酶系统Cre基因表达来剔除标记基因nptⅡ.将侵染后糖橙节间茎段接种在(MS+3mg/LBA)培养基上,共培养(暗培养)3d后转移到筛选培养基(MS+75mg/LKan+500mg/LCef)中.在筛选抗性再生芽后,切下带1～2mm外植体的抗性芽,转移至含有β-雌二醇的化学诱导培养基(MS+3mg/LBA+500mg/L头孢霉素+2μmol/Lβ-雌二醇)上诱导,15d后用荧光显微镜观察,成功观测到绿色荧光.进行试管嫁接,30d后,提取接穗基因组DNA进行PCR扩增,检测到DNA的切除现象.研究结果表明此化学诱导切除标记基因系统应用在柑橘上是可行的.The marker-free transgenic Citrus was investigated by combining application of a chemical regulated induction system XVE with,Cre/loxP-mediated site-specific DNA recombination in a single transformation to remove carriers.The system can remove marker gene（nptⅡ） by expression of Cre recombinase system,which was tightly controlled by a system upon induction by -estradiol.The infected internodal stem segments of ＇Succari＇ sweet orange as explants were cultured on co-cultivation medium（MS＋ 3 mg/L BA）,After 3 d,they were transformed onto selection medium（MS＋75 mg/L Kan＋500 mg/L Cef）.After selection,regeneration buds grow,with 1~2 mm explants were cut and transferred onto chemical-inducible medium（MS＋ 3 mg/L BA ＋ 500 mg/L Cef＋ 2 μmol/L β-estradiol）.After 15 d of continuous culture,the express of gfp could be observed successfully by fluorescence microscope.Finally,Molecular detection indicated that the DNA recombination and excision in transgenic Citrus had already occurred after 30 d grafted in vitro.The feasibility that this chemical-inducible excising marker gene system could be applied onto Citrus was proved and therefore,an efficient marker-free transgenic system in Citrus could be established.

关 键 词：柑橘 无标记转基因体系 化学诱导 标记基因 Β-雌二醇 

分 类 号：Q943.2[生物学—植物学]