植物抗病基因sncl在超级杂交稻亲本中的遗传转化研究
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摘要
水稻(Oryza sativa L.)是重要的粮食作物之一,全球约40%人口的主态是水稻传统遗传育种技术已为提高水稻的产量和改善品质做出了重大贡献,但由于生态环境恶化和常规育种方法的限制,水稻的遗传改良特别是水稻抗病虫害性状的遗传改良面临着重大挑战。本研究以光温敏核不育系P88S和超级稻父本0293两个杂交水稻亲本为材料,通过农杆菌介导法将厂谱抗病基因sncl导入这两个品种成熟胚诱导的愈伤组织中,筛选分化后以得到高抗病型的杂交亲本种质。
本试验获得的主要研究结果如下：
1、建立了P88S和0293的愈伤组织诱导体系,确定了NMB诱导培养基：N6大量盐分+MS微量盐分+B5维生素+3.0 mg/L 2-4,D+0.2 mg/L 6-BA+500 mg/L脯氨酸+500 mg/L酸水解酪蛋白+30 g/L蔗糖+7～8 g/L琼脂(pH 5.8)为最适诱导和继代的培养基,愈伤组织诱导率均在92%以上；在NMB培养基上两个品种的继代愈伤组织分别增重0.233 g和0.252 g,增重达185%和220%。
2、通过质粒转化试验,获得了含目的基因sncl的农杆菌EHA105菌株；从农杆菌培养方式、浸染方式、浸染后干燥时间以及共培养时间四个方面优化了农杆菌介导的愈伤组织转化体系：选择LB液体培养基进行农杆菌的培养,以先振荡15 min,然后抽真空15 min,再手动振荡5 min的方式进行农杆菌浸染,浸染后干燥4 h,共培养2 d,抗性愈伤组织产率达到了29.78%。
3、通过抗性愈伤组织的筛选和再生,获得了含有目的基因sncl的P88S和0293转化植株。
Rice (Oryza sativa L.) is one of the most important grain crops in the world and is a kind of staple food for about 40% global population. Traditional genetic breeding techniques have made significant contributions to improving the quality and increasing the yield of rice. However, the genetic improvement of rice, especially the improvement of resistance to insects and diseases, has been severely challenged due to the environmental deterioration and the limitation of conventional breeding method. In this study, two parental lines of super hybrid rice, a light and temperature sensitive genie male sterile line P88S and a paternal line 0293 were used as materials to study the genetic transformation of the broad-spectrum disease-resistance gene sncl into the mature embryo callus of the two varieties by Agrobacterium-mediaXed transformation. After screening and differentiation of the callus, the new germplasms with higher resistance to pathogen are expected to be obtained for rice hybridation.
The main results in the experiments are as the follows:
1. The systems for callus induction of P88S and 0293 were established. The NMB induction medium which including N6 Macronutrient component+MS Micronutrient component+vitamin B5+3.0 mg/L 2-4,D+0.2 mg/L 6-BA+500 mg/L proline+500 mg/LCH+30 g/L sucrose+7-8 g/L agar (pH 5.8) is found to be the optimal medium for callus induction and subculture, the callus induction rates of both varieties are above 92%, and the subcultural calluses of the two varieties on NMB medium increase by 185%(up to 0.233 g) and 220%(up to 0.252 g), respectively.
2. The Agrobacterium of EHA105 with the target gene of sncl was obtained through plasmid transformation. The Agrobacterium-mediated callus transformation system was optimized from four aspects of Agrobacterium cultivation mode, dipping method, drying time after dipping and co-culture time. LB liquid is chose as medium for Agrobacterium cultivation, oscillating for 15 min first, then vacuuming for 15 min, and then manual oscillating for 5 min for dipping of callus. After drying for 4 h and co-cultured for 2 d, the resistant callus obtained rate can reach to 29.78% under these optimum conditions.
3. Through screening and regeneration of the disease-resistance callus, the transformed plants of P88S and 0293 which contained sncl gene is obtained and confirmed by PCR check.
本试验获得的主要研究结果如下：
1、建立了P88S和0293的愈伤组织诱导体系,确定了NMB诱导培养基：N6大量盐分+MS微量盐分+B5维生素+3.0 mg/L 2-4,D+0.2 mg/L 6-BA+500 mg/L脯氨酸+500 mg/L酸水解酪蛋白+30 g/L蔗糖+7～8 g/L琼脂(pH 5.8)为最适诱导和继代的培养基,愈伤组织诱导率均在92%以上；在NMB培养基上两个品种的继代愈伤组织分别增重0.233 g和0.252 g,增重达185%和220%。
2、通过质粒转化试验,获得了含目的基因sncl的农杆菌EHA105菌株；从农杆菌培养方式、浸染方式、浸染后干燥时间以及共培养时间四个方面优化了农杆菌介导的愈伤组织转化体系：选择LB液体培养基进行农杆菌的培养,以先振荡15 min,然后抽真空15 min,再手动振荡5 min的方式进行农杆菌浸染,浸染后干燥4 h,共培养2 d,抗性愈伤组织产率达到了29.78%。
3、通过抗性愈伤组织的筛选和再生,获得了含有目的基因sncl的P88S和0293转化植株。
Rice (Oryza sativa L.) is one of the most important grain crops in the world and is a kind of staple food for about 40% global population. Traditional genetic breeding techniques have made significant contributions to improving the quality and increasing the yield of rice. However, the genetic improvement of rice, especially the improvement of resistance to insects and diseases, has been severely challenged due to the environmental deterioration and the limitation of conventional breeding method. In this study, two parental lines of super hybrid rice, a light and temperature sensitive genie male sterile line P88S and a paternal line 0293 were used as materials to study the genetic transformation of the broad-spectrum disease-resistance gene sncl into the mature embryo callus of the two varieties by Agrobacterium-mediaXed transformation. After screening and differentiation of the callus, the new germplasms with higher resistance to pathogen are expected to be obtained for rice hybridation.
The main results in the experiments are as the follows:
1. The systems for callus induction of P88S and 0293 were established. The NMB induction medium which including N6 Macronutrient component+MS Micronutrient component+vitamin B5+3.0 mg/L 2-4,D+0.2 mg/L 6-BA+500 mg/L proline+500 mg/LCH+30 g/L sucrose+7-8 g/L agar (pH 5.8) is found to be the optimal medium for callus induction and subculture, the callus induction rates of both varieties are above 92%, and the subcultural calluses of the two varieties on NMB medium increase by 185%(up to 0.233 g) and 220%(up to 0.252 g), respectively.
2. The Agrobacterium of EHA105 with the target gene of sncl was obtained through plasmid transformation. The Agrobacterium-mediated callus transformation system was optimized from four aspects of Agrobacterium cultivation mode, dipping method, drying time after dipping and co-culture time. LB liquid is chose as medium for Agrobacterium cultivation, oscillating for 15 min first, then vacuuming for 15 min, and then manual oscillating for 5 min for dipping of callus. After drying for 4 h and co-cultured for 2 d, the resistant callus obtained rate can reach to 29.78% under these optimum conditions.
3. Through screening and regeneration of the disease-resistance callus, the transformed plants of P88S and 0293 which contained sncl gene is obtained and confirmed by PCR check.
引文
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