一种水稻真空渗透转化装置及方法的研究
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摘要
探究适用于水稻的真空渗透转化装置及方法,旨在解决目前水稻转基因效率低、转化体变异多、周期长等问题。以抽穗期的‘云资粳41号’幼穗作为受体,应用自制的水稻真空渗透装置对其短暂抽真空渗透浸染导入外源基因。本试验共计转化水稻30株,收获19860粒T0代种子。研究发现不同浓度除草剂对T0代种子的萌发影响不显著,但对T1代材料的生长发育却具有明显抑制作用,因此选择苗期喷施法进行抗性转化子筛选,最佳筛选浓度为20 mg/L。经除草剂抗性筛选,获得抗性苗135株。经PCR分子检测,最终获得阳性苗114株,表明外源DNA已成功导入到受体水稻基因组中。本研究首次应用真空渗透法快速实现了水稻外源基因的转化,为水稻及其他作物的基因转化提供了一种快速、有效的新装置和新途径。
To solve the problems of low efficiency, high mutation and long period in rice genetic transformation,this paper first reported a piece of new equipment for vacuum infiltration transformation and its use method in rice. Young panicles of‘Yunzijing 41'at heading stage were used as receptor in transformation. The young panicles were treated by short time vacuum infiltration with rice vacuum osmotic device, and then the exogenous genes were introduced into young panicles. In this experiment, a total of 30 rice plants were transformed and 19860 T0 seeds were obtained. The results showed that different concentrations of herbicides had no significant effect on the germination of T0 seeds, but it had significant inhibitory effects on the growth and development of T1 plantlets. Therefore, the resistant transformants were selected by spraying herbicide at seedling stage. The optimal screening concentration was 20 mg/L. 135 resistant rice plants were obtained by herbicide resistance screening. 114 positive rice plants were obtained by PCR confirmation. It indicates that the exogenous DNA has been successfully introduced into the recipient rice genome. This study first uses the vacuum infiltration method to successfully introduce the exogenous genes into rice, which can be a rapid and effective new way as well as a piece of equipment for gene transformation in rice and other cereal crops.
To solve the problems of low efficiency, high mutation and long period in rice genetic transformation,this paper first reported a piece of new equipment for vacuum infiltration transformation and its use method in rice. Young panicles of‘Yunzijing 41'at heading stage were used as receptor in transformation. The young panicles were treated by short time vacuum infiltration with rice vacuum osmotic device, and then the exogenous genes were introduced into young panicles. In this experiment, a total of 30 rice plants were transformed and 19860 T0 seeds were obtained. The results showed that different concentrations of herbicides had no significant effect on the germination of T0 seeds, but it had significant inhibitory effects on the growth and development of T1 plantlets. Therefore, the resistant transformants were selected by spraying herbicide at seedling stage. The optimal screening concentration was 20 mg/L. 135 resistant rice plants were obtained by herbicide resistance screening. 114 positive rice plants were obtained by PCR confirmation. It indicates that the exogenous DNA has been successfully introduced into the recipient rice genome. This study first uses the vacuum infiltration method to successfully introduce the exogenous genes into rice, which can be a rapid and effective new way as well as a piece of equipment for gene transformation in rice and other cereal crops.
引文
[1]朱德峰,程式华,张玉屏,等.全球水稻生产现状与制约因素分析[J].中国农业科学,2010,43(3):474-479.
[2] Toriyama K, Arimoto Y, Uchimiya H, et al. Transgenic rice plants after direct gene transfer into protoplasts[J].Nature Biotechnology, 1988(6):1072-1074.
[3]彭昊,王志兴,窦道龙,等.由根癌农杆菌介导将葡萄糖氧化酶基因转入水稻[J].农业生物技术学报,2003,11(1):16-19.
[4]翟文学,李晓冰,朱立煌,等.由农杆菌介导将白叶枯病抗性基因Xa21转入我国的5个水稻品种[J].中国科学(C辑),2000,30(2):200-206.
[5] Ye R, Huang H, Zhou Y, et al. Development of insect-resistant transgenic rice with cry1C free endosperm[J]. Pest Management Science, 2009, 65(9):1015-1020.
[6]邹智,卢长明.影响农杆菌介导遗传转化的植物因子研究进展[J].生物技术通报,2008,1(192):1-9.
[7]刘凡,王国英,曹鸣庆.农杆菌介导的植物原位转基因方法研究进展[J].分子植物育种,2003,1(1):108-116.
[8]张庆祝,韩天富.植物非组培遗传转化方法研究的进展[J].分子植物育种,2004,2(1):85-91.
[9]邹智,卢长明.整株转化法及其在油菜上的应用与展望[J].植物学报,2009,44(2):236-244.
[10] Bechtold N, Ellis J, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[J]. Comptes Rendus de l'Academie des Sciences, 1993,316:1194-1199.
[11] Tjokrokusumo D, Heinrich T, Wylie S, et al. Vacuum infiltration of Petunia hybrida Pollen with Agrobacterium tumerfaciens to achieve Plant transformation[J]. Plant Cell Reports, 2000, 19(8):792-797.
[12] Mahmoudian M, Yücel M,?ktem H. Transformation of lentil(Lens culinaris M.)cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens[J].Plant Molecular Biology Reporter, 2002,20:251-257.
[13]李晓,王学德,朱玉贤,等.农杆菌真空法转化花粉获得棉花转acsB基因植株[J].作物学报,2002,28(5):709-712.
[14]徐恒戬.利用真空渗入法获得转基因抗虫白菜及其转化机理的研究[D].泰安:山东农业大学,2004:27-36.
[15]金万梅,巩振辉,吕元红.芥菜植株原位真空渗入遗传转化方法的研究[J].华北农学报,2004,19(3):8-12.
[16] Xu H J, Wang X F, Zhao H, et al. An intensive understanding of vacuum infiltration transformation of pakchoi(Brassica rapa ssp. chinensis)[J]. Plant Cell Reports, 2008,27(8):1369-1376.
[17] Park B J, Liu Z C, Kanno A, et al. Transformation of radish(Raphanus sativus L.)via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus[J]. Plant Cell Reports, 2005,24(8):494-500.
[18]刘巧真,王永亮,刘卫群,等.真空渗入浸染转化烟草的初步研究[J].河南农业科学,2006(3):48-50.
[19]王道杰,杨翠玲,陆鸣.真空渗透法转化油菜及转化种子的筛选[J].植物学报,2009,44(2):216-222.
[20]尚霄丽,冯建灿,朱道圩,等.真空辅助农杆菌介导中华猕猴桃高效遗传转化体系的建立[J].中国细胞生物学学报,2010,32(1):126-130.
[2] Toriyama K, Arimoto Y, Uchimiya H, et al. Transgenic rice plants after direct gene transfer into protoplasts[J].Nature Biotechnology, 1988(6):1072-1074.
[3]彭昊,王志兴,窦道龙,等.由根癌农杆菌介导将葡萄糖氧化酶基因转入水稻[J].农业生物技术学报,2003,11(1):16-19.
[4]翟文学,李晓冰,朱立煌,等.由农杆菌介导将白叶枯病抗性基因Xa21转入我国的5个水稻品种[J].中国科学(C辑),2000,30(2):200-206.
[5] Ye R, Huang H, Zhou Y, et al. Development of insect-resistant transgenic rice with cry1C free endosperm[J]. Pest Management Science, 2009, 65(9):1015-1020.
[6]邹智,卢长明.影响农杆菌介导遗传转化的植物因子研究进展[J].生物技术通报,2008,1(192):1-9.
[7]刘凡,王国英,曹鸣庆.农杆菌介导的植物原位转基因方法研究进展[J].分子植物育种,2003,1(1):108-116.
[8]张庆祝,韩天富.植物非组培遗传转化方法研究的进展[J].分子植物育种,2004,2(1):85-91.
[9]邹智,卢长明.整株转化法及其在油菜上的应用与展望[J].植物学报,2009,44(2):236-244.
[10] Bechtold N, Ellis J, Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[J]. Comptes Rendus de l'Academie des Sciences, 1993,316:1194-1199.
[11] Tjokrokusumo D, Heinrich T, Wylie S, et al. Vacuum infiltration of Petunia hybrida Pollen with Agrobacterium tumerfaciens to achieve Plant transformation[J]. Plant Cell Reports, 2000, 19(8):792-797.
[12] Mahmoudian M, Yücel M,?ktem H. Transformation of lentil(Lens culinaris M.)cotyledonary nodes by vacuum infiltration of Agrobacterium tumefaciens[J].Plant Molecular Biology Reporter, 2002,20:251-257.
[13]李晓,王学德,朱玉贤,等.农杆菌真空法转化花粉获得棉花转acsB基因植株[J].作物学报,2002,28(5):709-712.
[14]徐恒戬.利用真空渗入法获得转基因抗虫白菜及其转化机理的研究[D].泰安:山东农业大学,2004:27-36.
[15]金万梅,巩振辉,吕元红.芥菜植株原位真空渗入遗传转化方法的研究[J].华北农学报,2004,19(3):8-12.
[16] Xu H J, Wang X F, Zhao H, et al. An intensive understanding of vacuum infiltration transformation of pakchoi(Brassica rapa ssp. chinensis)[J]. Plant Cell Reports, 2008,27(8):1369-1376.
[17] Park B J, Liu Z C, Kanno A, et al. Transformation of radish(Raphanus sativus L.)via sonication and vacuum infiltration of germinated seeds with Agrobacterium harboring a group 3 LEA gene from B. napus[J]. Plant Cell Reports, 2005,24(8):494-500.
[18]刘巧真,王永亮,刘卫群,等.真空渗入浸染转化烟草的初步研究[J].河南农业科学,2006(3):48-50.
[19]王道杰,杨翠玲,陆鸣.真空渗透法转化油菜及转化种子的筛选[J].植物学报,2009,44(2):216-222.
[20]尚霄丽,冯建灿,朱道圩,等.真空辅助农杆菌介导中华猕猴桃高效遗传转化体系的建立[J].中国细胞生物学学报,2010,32(1):126-130.