菊花离体再生及根癌农杆菌介导CBF1基因遗传转化的研究
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摘要
菊花是中国的十大传统名花之一,世界重要盆栽、切花及地被花卉。但低温、干旱条件严重限制了菊花在我国北方城市绿化中大面积应用。植物转录因子不仅在植物的生长发育、形态建成、代谢调控等方面,而且也在抗旱、抗寒、耐盐碱等方面起重要作用。本研究在建立菊花‘小金黄’品种离体再生体系的基础上将拟南芥诱导转录因子CBF1基因转入菊花中,从而为实现菊花品种抗寒、抗旱性状改良奠定基础。
利用器官发生途径以‘小金黄’为试验材料,研究生长调节物质对菊花离体不定芽再生的影响。结果表明:菊花叶片在添加1.0 mg/L6-BA的MS培养基中的再生频率为27.1%,显著高于在MS中添加TDZ或KT;在MS+ 1.0 mg/L 6-BA中添加不同浓度生长素,菊花叶片再生频率存在显著差异,其中以MS+ 1.0 mg/L 6-BA+ 0.6 mg/LNAA的叶片不定芽再生频率最高,即为96.9%,高于2,4-D和IBA。菊花‘小金黄’品种最适宜的生根培养基为MS+ NAA 0.4 mg/L。
利用根癌农杆菌介导法对菊花‘小金黄’品种的叶片进行遗传转化,研究影响菊花转化的若干因素,建立一套高效的遗传转化体系。结果表明:2天的预培养,OD600值约为0.5的农杆菌菌液添加50 mg/L乙酰丁香酮,侵染时间3 min,2天的共培养,从分化培养基获得的再生不定芽在生根培养基MS + 0.4 mg/L NAA+ 10 mg/L Km+ 100 mg/L Cef上进行延迟筛选,有利于获得较高的转化效率,共获得49个Km抗性株系。
通过根癌农杆菌介导的叶盘法将CaMV35S驱动的CBF1基因导入菊花中,对49个Km抗性株系进行PCR检测,有18个株系扩增出的特异条带与阳性质粒扩增出的特异条带一致,阴性对照没有扩增出任何条带,说明CBF1基因已经整合到菊花‘小金黄’品种的基因组中,而抗性株系的PCR阳性率为36.7%。
Dendranthema morifolium is one of the traditional famous flowers in China, an economically important ornamental crop for cut flower, pot plant and ground flower in many parts of the world. Cold and drought stresses play an important role in the application of ornamental plants in northern China. Transcription factors of plant play an important role not only in growth and development but also in cold, salt and drought stresses. Regeneration system of Dendranthema morifolium was obtained and transcription factors CBF1 was transformed for the regulation of cold and drought stresses responsive genes of Denranthema morifolium.
Establishing an efficient regeneration system is a fundamental to the transgenic study. The effects of growth regulators on the generation of adventitious shoots were studied for Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’. The results showed that 1.0 mg/L 6-BA was more favorable to shoot formation than TDZ or KT, and its regeneration rate was 27.1%. Whereas the combination of 6-BA 1.0 mg/L and NAA 0.6 mg/L on regeneration frequency was much more efficient than others with 96.9%. The optimal rooting medium was MS+ NAA 0.4 mg/L.
In order to establish an efficient genetic transformation system with leaves of Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’, several factors affected genetic transformation mediated by Agrobacterium tumefacien was studied. The results showed that the highest transformation was obtained through the following transformation procedure: after preculture for 2 d, the explants were infected 3 minutes with 50 mg/L acetosyringone and OD600=0.5 Agrobacterium tumefacien culture liquid, and then co-cultivated for 2 d. Resistant shoots from leaf discs were screened on MS+ 0.4 mg/L NAA+ 100 mg/L Cef medium supplement with 10 mg/L Km. Finally, there were 49 resistant shoots alive in the rooting medium with 10 mg/L Km.
An inducible transcription factor CBF1 from Arabidopsis thaliana was genetically transformed by using leaf disc method via Agrobacterium tumefacien into the experimental material Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’, trying to strengthen cold and drought resistance of Dendranthema. Results of polymerase chain reaction(PCR) proved that the CBF1 gene was integrated into the genome of transgenic plants, and eighteen transgenic lines were obtained, the transformation rate was 36.7%。
利用器官发生途径以‘小金黄’为试验材料,研究生长调节物质对菊花离体不定芽再生的影响。结果表明:菊花叶片在添加1.0 mg/L6-BA的MS培养基中的再生频率为27.1%,显著高于在MS中添加TDZ或KT;在MS+ 1.0 mg/L 6-BA中添加不同浓度生长素,菊花叶片再生频率存在显著差异,其中以MS+ 1.0 mg/L 6-BA+ 0.6 mg/LNAA的叶片不定芽再生频率最高,即为96.9%,高于2,4-D和IBA。菊花‘小金黄’品种最适宜的生根培养基为MS+ NAA 0.4 mg/L。
利用根癌农杆菌介导法对菊花‘小金黄’品种的叶片进行遗传转化,研究影响菊花转化的若干因素,建立一套高效的遗传转化体系。结果表明:2天的预培养,OD600值约为0.5的农杆菌菌液添加50 mg/L乙酰丁香酮,侵染时间3 min,2天的共培养,从分化培养基获得的再生不定芽在生根培养基MS + 0.4 mg/L NAA+ 10 mg/L Km+ 100 mg/L Cef上进行延迟筛选,有利于获得较高的转化效率,共获得49个Km抗性株系。
通过根癌农杆菌介导的叶盘法将CaMV35S驱动的CBF1基因导入菊花中,对49个Km抗性株系进行PCR检测,有18个株系扩增出的特异条带与阳性质粒扩增出的特异条带一致,阴性对照没有扩增出任何条带,说明CBF1基因已经整合到菊花‘小金黄’品种的基因组中,而抗性株系的PCR阳性率为36.7%。
Dendranthema morifolium is one of the traditional famous flowers in China, an economically important ornamental crop for cut flower, pot plant and ground flower in many parts of the world. Cold and drought stresses play an important role in the application of ornamental plants in northern China. Transcription factors of plant play an important role not only in growth and development but also in cold, salt and drought stresses. Regeneration system of Dendranthema morifolium was obtained and transcription factors CBF1 was transformed for the regulation of cold and drought stresses responsive genes of Denranthema morifolium.
Establishing an efficient regeneration system is a fundamental to the transgenic study. The effects of growth regulators on the generation of adventitious shoots were studied for Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’. The results showed that 1.0 mg/L 6-BA was more favorable to shoot formation than TDZ or KT, and its regeneration rate was 27.1%. Whereas the combination of 6-BA 1.0 mg/L and NAA 0.6 mg/L on regeneration frequency was much more efficient than others with 96.9%. The optimal rooting medium was MS+ NAA 0.4 mg/L.
In order to establish an efficient genetic transformation system with leaves of Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’, several factors affected genetic transformation mediated by Agrobacterium tumefacien was studied. The results showed that the highest transformation was obtained through the following transformation procedure: after preculture for 2 d, the explants were infected 3 minutes with 50 mg/L acetosyringone and OD600=0.5 Agrobacterium tumefacien culture liquid, and then co-cultivated for 2 d. Resistant shoots from leaf discs were screened on MS+ 0.4 mg/L NAA+ 100 mg/L Cef medium supplement with 10 mg/L Km. Finally, there were 49 resistant shoots alive in the rooting medium with 10 mg/L Km.
An inducible transcription factor CBF1 from Arabidopsis thaliana was genetically transformed by using leaf disc method via Agrobacterium tumefacien into the experimental material Dendranthema morifolium Tzvel. cv.‘Xiaojinhuang’, trying to strengthen cold and drought resistance of Dendranthema. Results of polymerase chain reaction(PCR) proved that the CBF1 gene was integrated into the genome of transgenic plants, and eighteen transgenic lines were obtained, the transformation rate was 36.7%。
引文
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