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农杆菌介导的AtMYB118基因转化橡胶树易碎胚性愈伤组织体系优化
作者: 毕政鸿1  哲2 * 王志秋1 戴雪梅2 方家林2 黄华孙2 林位夫2 
LARDET
 Ludovic 3  MONTORO Pascal 3 CARRON Marc-Philippe 3 
LECLERCQ
 Julie 3 左建儒4 牟金叶4  杨晓辉4  响5 
单位: (1海南大学 农学院 海口 570228 2 中国热带农业科学院 橡胶研究所/农业部橡胶树生物学与遗传资源利用重点实验室/国家重要热带作物工程技术研究中心  海南 儋州 571737 3 法国农业研究国际合作中心 生物系统部 法国 蒙彼利埃 34398  
关键词: 橡胶树 农杆菌介导的遗传转化 AtMYB118基因 反转录PCR 易碎胚性愈伤组织 转基因植株 
分类号:S794.1
出版年,卷(期):页码:2014,45(7):1137-1146
摘要:

【目的】采用正交设计L25(56)优化农杆菌介导的AtMYB118基因对橡胶树易碎胚性愈伤组织的遗传转化体系,为橡胶树品种遗传改良提供参考。【方法】以75.0 mg/L卡那霉素筛选经过转化的愈伤组织;采用GUS瞬时表达检测方法评价6个因素即预培养时间、农杆菌菌液浓度(OD600)、乙酰丁香酮(AS)浓度、侵染时间、共培养温度和共培养时间对遗传转化的影响。【结果】6个因素显著影响橡胶树长期培养的易碎胚性愈伤组织的转化频率,易碎胚性愈伤组织遗传转化优化条件为:预培养0 d,菌液OD600为0.7,侵染时间7 min,共培养时间5 d,AS 200 μmol/L,共培养温度25 ℃。在此优化条件下可获得最高转化频率。经过4~6个月的筛选,获得17个GUS染色阳性的易碎愈伤组织系。经PCR和反转录PCR分析转化愈伤组织,进一步证实uidA、nptII、AtMYB118 基因已被整合到愈伤组织基因组中并表达。培养获得1539个胚状体,其中164个为转基因胚状体,转化频率为10.6%;最终获得4株转AtMYB118基因植株。【结论】优化获得可行有效的橡胶树易碎胚性愈伤组织遗传转化体系,可为其品种遗传改良提供技术支持。

【Objective】An orthogonal design L25(56) was used to optimize Agrobacterium-mediated AtMYB118 gene genetic transformation system of friable embryogenic calli in Hevea brasiliensis in order to provide references for genetic improvement of its clones. 【Method】Kanamycin at a concentration of 75.0 mg/L was used to select transformed calli. The effects of six factors viz., preculture time, Agrobacterium growth phase (OD600), acetosyringone (AS) concentration, infection time, co-culture temperature and co-culture time on genetic transformation were evaluated by GUS transient expression detection. 【Result】Six factors were found to significantly affect transformation frequency of long-term cultural friable embryogenic calli. The highest transformation efficiency was achieved by 0-day preculture, long-term cultural friable embryogenic calli as recipients infected by Agrobacterium cultures corresponding to OD600=0.7 for 7 min, followed by co-culture for 5 days in a co-culture medium containing 200 μmol/L AS at 25 ℃. After 4-6 months, 17 GUS positive callus lines were achieved. Polymerase chain reaction (PCR) and reverse transcription-polymerase chain reaction (RT-PCR) analysis results of transgenic tissues further confirmed that uidA, nptII and AtMYB118 genes had been inserted into genome of calli and expressed. At last, 164 transgenic embryoids were obtained from 1539 cultured embryoids. A transformation efficiency of 10.6% was achieved for long-term cultural friable embryogenic calli using this protocol. Four AtMYB118 transgenic plantlets were obtained. 【Conclusion】The optimized genetic transformation system using friable embryogenic calli of rubber tree as recipients was effective and available,which would provide technological supports on genetic improvement of clones.

基金项目:
Chinese State International Scientific and Technological Cooperation Program (2008DFA32020);“948” International Scientific and Technological Cooperation Project financially supported by Chinese Ministry of Agriculture (2010-S7); CATAS Fundamental Research Fund (1630022013029)
作者简介:
* for corresponding author,LI Zhe(1971-), PhD, associate researcher, specializing in plant biotechnology and genetic transformation,E-mail: lizhecnhn@163.com. BI Zheng-hong(1986-), majoring in crop genetics and breeding, and plant biotechnology, E-mail: bizhenghong9999999@163.com
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