农杆菌介导籼稻成熟胚高效转化体系的建立
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摘要
水稻（Oryza sativa L.）是重要的粮食作物之一，籼稻和粳稻是水稻的两大亚种，全球80％的稻米生产基于籼稻。当前，利用转基因技术对水稻品种进行全面改良，培育优质、高产、多抗新品种在保障全球粮食安全的同时也是今后水稻育种的发展方向，可见籼稻的遗传转化在品种改良、基因功能检测、创造突变体中发挥着越来越重要的作用，而建立高效、稳定的籼稻遗传转化体系是分子育种和功能基因组学等领域研究的前提。
近年来农杆菌介导法转化水稻技术已日渐成熟，并广泛应用于粳稻转化中。但对籼稻的转化相对困难，主要是其愈伤组织的诱导及分化率都很低。本研究在本实验室已有的粳稻成熟转化技术体系基础上，以籼型品种Q25（白丰B的突变体材料）、IR64、MH86、PA64S、N22和Q4041为材料，开展了影响籼稻遗传转化效率的研究工作，主要结果如下：
1.通过包括基本培养基，脱落酸和生长素类物质三个因素四个水平的正交实验，明确了基因型对培养基的依赖关系，发现MS比较适合Q25、IR64、MH86、PA64S成熟胚愈伤组织的诱导，而N6比较适合N22和Q4041的诱导，通过适当添加外源激素2,4-D和ABA，诱导出了生长状态良好的胚性愈伤组织。
2.通过比较三种常用的选择标记基因和相对应的筛选剂对不同基因型籼稻的抗性愈伤的筛选效果，得出BAR基因在6个籼稻品种的转化中最有应用价值，同时对NPTⅡ抗性基因的筛选剂比较中发现，庆大霉素衍生物（G418）的筛选效果优于硫酸巴龙霉素（P）。
3.在抗性愈伤组织的再生环节上，通过对农杆菌浸泡的同时附加振荡的感染方式以及共培养后25min的干燥处理，提高了籼稻品种N22和Q4041遗传转化的再生频率。
4.通过对籼稻遗传转化各阶段的影响因子的研究，改良了籼稻目标品种N22和Q4041成熟胚愈伤组织为受体材料的遗传转化体系，并将转化率由原来的29%提高到44%。
5.基于本研究的一些结果及结合本实验室多年水稻转化工作的经验积累，对籼稻农杆菌介导的遗传转化中的问题及解决对策进行讨论，并对今后水稻转基因的发展方向提出了初浅的看法。
Rice（Oryza sativa L.）is one of the most important food crops. Increasing of rice yield is anessential factor to secure sufficient food supply for the growing population. Japonica and indica are twosubspecies of rice growing in different regions of the world. About80%of world rice production isbased on indica varieties. It has been suggested that biotechnology, combined with traditional breedingmethods and other technologies, can contribute to the agronomic improvement of indica and make itpossible to achieve the production requirements. The role of genetic transformation of indica playingwill be more and more important in the improvements of exist elite varieties, characterization of genefunction and construction of new rice mutants.
In recent years, Agrobacterium-mediated transformation of rice has been widely used in japonicatransformation. However, the transformation of indica rice is still difficult, mainly because the inductionof embryogenic callus is not routine and the rate of plant regeneration from transformed calli is low,which is determined by the genetic differences between the two subspecies. In this study, a highlyefficient gene transfer method mediated by Agrobacterium tumefaciens was developed for N22andQ4041using mature seeds. Some relevant parameters affecting transformation efficency were alsooptimized in different varieties such as Q25, IR64, MH86and PA64S. The main results are as follows:
1. By analyzing3factors (the basic medium, ABA and hormones) and4levels of them on theinduction effect, we found that MS medium was more suitable for callus induction of Q25, IR64, MH86,PA64S and N6medium was suitable for N22and Q4041. We also found that the exogenous hormones2,4-D and ABA were essential substances for rice embryogenic callus induction.
2. By comparing the selection effects of3common marker genes to resistant calli induction, theresult showed that the BAR gene is the most effective selectable marker in tested indica genotypes. Inaddition, comparison of two agents, G418and Paromomycin, we found that G418was more efficientthan Paromomycin (P).
3. In the resistant calli regeneration, by choosing Agrobacterium-infection methods, soakingthrough Agrobacterium infection, shaking meantime, and adjusting drying time to25min afterco-culture of the calli, we improved the regeneration frequency of the rice varieties N22and Q4041.
4. We optimized Agrobacterium-mediated transformation system of N22and Q4041, andimproved the transformation frequency from29%to44%.
5. Based on the results of this study and well-established japonica rice transformation system inour lab, we propose some strategies to improve efficiency of indica transformation and also put forwardthe future development of transgenic rice.
近年来农杆菌介导法转化水稻技术已日渐成熟，并广泛应用于粳稻转化中。但对籼稻的转化相对困难，主要是其愈伤组织的诱导及分化率都很低。本研究在本实验室已有的粳稻成熟转化技术体系基础上，以籼型品种Q25（白丰B的突变体材料）、IR64、MH86、PA64S、N22和Q4041为材料，开展了影响籼稻遗传转化效率的研究工作，主要结果如下：
1.通过包括基本培养基，脱落酸和生长素类物质三个因素四个水平的正交实验，明确了基因型对培养基的依赖关系，发现MS比较适合Q25、IR64、MH86、PA64S成熟胚愈伤组织的诱导，而N6比较适合N22和Q4041的诱导，通过适当添加外源激素2,4-D和ABA，诱导出了生长状态良好的胚性愈伤组织。
2.通过比较三种常用的选择标记基因和相对应的筛选剂对不同基因型籼稻的抗性愈伤的筛选效果，得出BAR基因在6个籼稻品种的转化中最有应用价值，同时对NPTⅡ抗性基因的筛选剂比较中发现，庆大霉素衍生物（G418）的筛选效果优于硫酸巴龙霉素（P）。
3.在抗性愈伤组织的再生环节上，通过对农杆菌浸泡的同时附加振荡的感染方式以及共培养后25min的干燥处理，提高了籼稻品种N22和Q4041遗传转化的再生频率。
4.通过对籼稻遗传转化各阶段的影响因子的研究，改良了籼稻目标品种N22和Q4041成熟胚愈伤组织为受体材料的遗传转化体系，并将转化率由原来的29%提高到44%。
5.基于本研究的一些结果及结合本实验室多年水稻转化工作的经验积累，对籼稻农杆菌介导的遗传转化中的问题及解决对策进行讨论，并对今后水稻转基因的发展方向提出了初浅的看法。
Rice（Oryza sativa L.）is one of the most important food crops. Increasing of rice yield is anessential factor to secure sufficient food supply for the growing population. Japonica and indica are twosubspecies of rice growing in different regions of the world. About80%of world rice production isbased on indica varieties. It has been suggested that biotechnology, combined with traditional breedingmethods and other technologies, can contribute to the agronomic improvement of indica and make itpossible to achieve the production requirements. The role of genetic transformation of indica playingwill be more and more important in the improvements of exist elite varieties, characterization of genefunction and construction of new rice mutants.
In recent years, Agrobacterium-mediated transformation of rice has been widely used in japonicatransformation. However, the transformation of indica rice is still difficult, mainly because the inductionof embryogenic callus is not routine and the rate of plant regeneration from transformed calli is low,which is determined by the genetic differences between the two subspecies. In this study, a highlyefficient gene transfer method mediated by Agrobacterium tumefaciens was developed for N22andQ4041using mature seeds. Some relevant parameters affecting transformation efficency were alsooptimized in different varieties such as Q25, IR64, MH86and PA64S. The main results are as follows:
1. By analyzing3factors (the basic medium, ABA and hormones) and4levels of them on theinduction effect, we found that MS medium was more suitable for callus induction of Q25, IR64, MH86,PA64S and N6medium was suitable for N22and Q4041. We also found that the exogenous hormones2,4-D and ABA were essential substances for rice embryogenic callus induction.
2. By comparing the selection effects of3common marker genes to resistant calli induction, theresult showed that the BAR gene is the most effective selectable marker in tested indica genotypes. Inaddition, comparison of two agents, G418and Paromomycin, we found that G418was more efficientthan Paromomycin (P).
3. In the resistant calli regeneration, by choosing Agrobacterium-infection methods, soakingthrough Agrobacterium infection, shaking meantime, and adjusting drying time to25min afterco-culture of the calli, we improved the regeneration frequency of the rice varieties N22and Q4041.
4. We optimized Agrobacterium-mediated transformation system of N22and Q4041, andimproved the transformation frequency from29%to44%.
5. Based on the results of this study and well-established japonica rice transformation system inour lab, we propose some strategies to improve efficiency of indica transformation and also put forwardthe future development of transgenic rice.
引文
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