农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化

李子东^1,3,赵翠珠¹,周玲君²,刘艳玲¹,向凤宁^1*,夏光敏¹

1. 山东大学生命科学学院, 山东济南 250100; 2. 山东文登农业能源办公室, 山东文登 264400; 3. 清华大学医学院, 北京 100084

收稿日期:1900-01-01 修回日期:1900-01-01 出版日期:2006-10-24 发布日期:2006-10-24
通讯作者: 向凤宁

Agrobactrium tumefaciens mediated DREB1A gene transfer to Lolium multiflorum and optimization of its genetic transformation

LI Zi-dong ^1,3, ZHAO Cui-zhu¹, ZHOU Ling-jun², LIU Yan-ling¹, XIANG Feng-ning ^1*, XIA Guang-min¹

1. School of Life Sciences, Shandong University, Jinan 250100, Shandong, China;2. Agriculture Resource Office of Wendeng Shangdong Province, Wendeng 264400, Shandong, China;3. Medical College, Tsinghua University, Beijing 100084, China

Received:1900-01-01 Revised:1900-01-01 Online:2006-10-24 Published:2006-10-24
Contact: XIANG Feng-ning

摘要/Abstract

摘要： 采用携带卡那霉素抗性基因nptII和GUS基因的ubiquitin启动子驱动的表达载体pBI121／DREB1A的根癌农杆菌AGL1, 对多花黑麦草幼胚来源的胚性愈伤组织进行了遗传转化,并优化了各种影响因素。胚性愈伤组织经根癌农杆菌感染和共培养后,用50mg／L巴龙霉素筛选抗性愈伤组织,待抗性愈伤组织在IB分化培养基上分化成苗后用25mg／L卡那霉素进一步筛选再生植株, 获得了部分抗性植株。抗性植株的总DNA用DREB1A基因的特异引物进行PCR检测,转化频率为2.14％,PCR-Southern blot进一步验证了转化植株基因组中含有该外源基因。各种影响转化效率因素的优化实验表明,当转化时菌液浓度的OD600为2.0、侵染时间为1h、共培养时间为2d、共培养温度为21℃及在共培养期间使用乙酰丁香酮等,均可明显提高转化频率。

关键词: 多花黑麦草, 遗传转化, 根癌农杆菌, DREB1A基因

Abstract: The embryo-derived calli from Lolium multiflorum Lam were transformed with Agrobactrium tumefaciens AGL1 harboring intron-DREB1A expression vector pBI121 containing ubiqutin promoter, nptⅡ marker gene and GUS gene. After infection and co-culture with AGL1, the embyogenic calli were selected with 50mg／L paromomycine and plants regenerated from the resistant calli. All plants were selected further with 25mg／L kanamycin, some of which remained green. The genome DNA of the resistant plants was checked with specific primers and probes from the DREB1A gene. The results of PCR and PCR-Southern blot assay indicated that the DREB1A gene was transferred into Lolium multiflorum Lam with a transformation frequency of 2.14％. It was found that the transformation efficiency can be efficiently improved using the following transformation conditions: Agrobacterium cell density of OD600≈2.0, incubation for 1 hour, coculture at 21℃ for 2 days and application of As in coculture.

Key words: transform, Agrobactrium tumefaciens, DREB1A gene, Lolium multiflorum Lam

中图分类号:

李子东,赵翠珠,周玲君,刘艳玲,向凤宁*,夏光敏 . 农杆菌介导的DREB1A基因转化多花黑麦草及其转化体系的优化[J]. J4, 2008, 43(9): 11-17 .

LI Zi-dong ,ZHAO Cui-zhu,ZHOU Ling-jun,LIU Yan-ling,XIANG Feng-ning *,XIA Guang-min . Agrobactrium tumefaciens mediated DREB1A gene transfer to Lolium multiflorum and optimization of its genetic transformation[J]. J4, 2008, 43(9): 11-17 .

参考文献

多维度评价

Viewed

Full text

Abstract

Cited

Shared

Discussed

[1]	杨俊杰1,于惠敏1,侯丙凯2*,王兴智3. 农杆菌介导的双价抗虫基因对番茄遗传转化的研究[J]. J4, 2013, 48(7): 9-13.
[2]	姬丹丹,王鹏,向凤宁*. 大豆外源基因转化体系建立及条件优化研究[J]. J4, 2011, 46(5): 116-122.
[3]	于惠敏1,石竹1,邵洪伟1,李玮1,侯丙凯2*. 农杆菌介导的抗病基因对番茄的遗传转化[J]. J4, 2011, 46(11): 1-4.
[4]	李煦,王荣春,何影,杨爱芳* . 多基因双T-DNA植物表达载体的构建及拟南芥转化[J]. J4, 2007, 42(9): 1-06 .