应用真空渗透法将SFL基因转入水稻的研究
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摘要
【目的】真空渗透法无需组培过程,可明显缩短转化周期,提高转化效率。文章建立了水稻的真空渗透转化体系。【方法】应用自主研制的真空渗透转化装置,对开花前的活体水稻进行短暂抽真空条件下的农杆菌侵染,快速将苦参凝集素蛋白基因(SFL)成功转入水稻。【结果】对大量T0代转化种子进行除草剂抗性筛选、氯酚红和PCR检测,获得一批转基因阳性苗,证明外源基因已整合到水稻基因组中;经苗期喷雾接种和田间人工诱发鉴定,与非转基因对照相比,最终筛选出抗性明显增强的转SFL基因水稻材料,证明SFL基因在水稻中得到表达。【结论】本研究为水稻及其它作物的基因转化提供了一条快速、有效的新途径,同时也为SFL基因的功能研究及抗稻瘟病水稻新品种的培育奠定了基础。
【Objective】The Vacuum infiltration transformation method is simple,fast and effective because of non-tissue culture. This paper first worldwide reported a new transformation method in rice-vacuum infiltration transformation method.【Method】Using a set of special equipment designed by ourselves,the most robust plants in vivo before flowering were vacuum infiltrated insuspension,Sophora Flavevescens Lectin( SFL) was successfully introduced into rice.【Result】A large number of T0 transgenic seeds were harvested. A number of transgenic plants were gained by Basta resistance selection,chlorophenol-red( CPR) and PCR determination suggested that the SFL gene had been integrated into the rice genome. The transgenic plants showed high blast resistance by means of the seedling spray inoculation in the field compared to non-transgenic rice. This implied that SFL gene was expressed in transgenic rice plants.【Conclusion】Vacuum infiltration transformation method provided a rapid and effective new approach for genetic transformation in rice and other cereal crops. At the same time,it also laid the foundation for research on the SFL gene function and breeding of high blast resistant rice varieties in the future.
【Objective】The Vacuum infiltration transformation method is simple,fast and effective because of non-tissue culture. This paper first worldwide reported a new transformation method in rice-vacuum infiltration transformation method.【Method】Using a set of special equipment designed by ourselves,the most robust plants in vivo before flowering were vacuum infiltrated insuspension,Sophora Flavevescens Lectin( SFL) was successfully introduced into rice.【Result】A large number of T0 transgenic seeds were harvested. A number of transgenic plants were gained by Basta resistance selection,chlorophenol-red( CPR) and PCR determination suggested that the SFL gene had been integrated into the rice genome. The transgenic plants showed high blast resistance by means of the seedling spray inoculation in the field compared to non-transgenic rice. This implied that SFL gene was expressed in transgenic rice plants.【Conclusion】Vacuum infiltration transformation method provided a rapid and effective new approach for genetic transformation in rice and other cereal crops. At the same time,it also laid the foundation for research on the SFL gene function and breeding of high blast resistant rice varieties in the future.
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]梁斌,肖放华,黄费元,等.云南野生稻对稻瘟病的抗性[J].中国水稻科学,1999,13(31):183-185.
[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]Chen H,Tang W,Xu C G,et al. Transgenic indica rice plants harboring a synthetic cry2A gene of Bacillus thuringiensis exhibiten hanced resistance against lepidopteran rice pests[J]. Theoretical and Applied Genetics,2005,111(7):1330-1337.
[6]Tang W,Chen H,Xu C G,et al. Development of insect-resistant transgenic indica rice with a synthetic cry1C gene[J]. Molecular Breeding,2006,18(1):1-10.
[7]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.
[8]谢戎,刘成元,李永洪,等.转GO基因水稻杂交后代稻瘟病抗谱分析[J].西南农业学报,2015,28(5):1869-1873.
[9]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,2008,192(1):1-9.
[10]Rakoczy-Trojanowska M. Alternative methods of plant transformation-a short review[J]. Cellular Molecular Biology Letters,2002(7):849-858.
[11]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,L(L):L08-L16.
[12]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.
[13]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.
[14]Bechtold N,Ellis J,and Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[A]. Comptes Rendus de l'Academie des Sciences[C]. Paris,Life Sciences,1993,316:1194-1199.
[15]巩振辉,Mi Lner JJ,何玉科.拟南芥基因转移新方法-真空渗入法的研究[J].西北植物学报,1996,16(3):277-283.
[16]Clough S J,Bent A F. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal,1998,16(6):735-743.
[17]Tjokrokusumo D,Heinrich T,Wylie S,et al. Vacuum infiltration of Petunia hybrida Pollen with Agrobacterium tumerfaciens to a chieve Plant transformation[J]. Plant Cell Reports,2000,19(8):792-797.
[18]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.
[19]李晓,王学德,朱玉贤,等.农杆菌真空法转化花粉获得棉花转acs B基因植株[J].作物学报,2002,28(5):709-712.
[20]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].山东农业大学博士学位论文,2004:27-36.
[21]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.
[22]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.
[23]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.
[24]张军杰,刘凡,赵泓,等.蛋白酶抑制剂基因真空渗入法转化不结球白菜获得抗虫性材料[J].植物保护学报,2006,31(1):17-21.
[25]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.
[26]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.
[27]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.
[28]马志刚,鄢波,黄兴奇,等.苦参凝集素蛋白基因的分离克隆[J].植物学报,2001,43(8):821-825.
[2]梁斌,肖放华,黄费元,等.云南野生稻对稻瘟病的抗性[J].中国水稻科学,1999,13(31):183-185.
[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]Chen H,Tang W,Xu C G,et al. Transgenic indica rice plants harboring a synthetic cry2A gene of Bacillus thuringiensis exhibiten hanced resistance against lepidopteran rice pests[J]. Theoretical and Applied Genetics,2005,111(7):1330-1337.
[6]Tang W,Chen H,Xu C G,et al. Development of insect-resistant transgenic indica rice with a synthetic cry1C gene[J]. Molecular Breeding,2006,18(1):1-10.
[7]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.
[8]谢戎,刘成元,李永洪,等.转GO基因水稻杂交后代稻瘟病抗谱分析[J].西南农业学报,2015,28(5):1869-1873.
[9]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,2008,192(1):1-9.
[10]Rakoczy-Trojanowska M. Alternative methods of plant transformation-a short review[J]. Cellular Molecular Biology Letters,2002(7):849-858.
[11]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,L(L):L08-L16.
[12]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.
[13]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.
[14]Bechtold N,Ellis J,and Pelletier G. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants[A]. Comptes Rendus de l'Academie des Sciences[C]. Paris,Life Sciences,1993,316:1194-1199.
[15]巩振辉,Mi Lner JJ,何玉科.拟南芥基因转移新方法-真空渗入法的研究[J].西北植物学报,1996,16(3):277-283.
[16]Clough S J,Bent A F. Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J]. The Plant Journal,1998,16(6):735-743.
[17]Tjokrokusumo D,Heinrich T,Wylie S,et al. Vacuum infiltration of Petunia hybrida Pollen with Agrobacterium tumerfaciens to a chieve Plant transformation[J]. Plant Cell Reports,2000,19(8):792-797.
[18]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.
[19]李晓,王学德,朱玉贤,等.农杆菌真空法转化花粉获得棉花转acs B基因植株[J].作物学报,2002,28(5):709-712.
[20]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].山东农业大学博士学位论文,2004:27-36.
[21]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.
[22]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.
[23]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.
[24]张军杰,刘凡,赵泓,等.蛋白酶抑制剂基因真空渗入法转化不结球白菜获得抗虫性材料[J].植物保护学报,2006,31(1):17-21.
[25]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.
[26]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.
[27]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.
[28]马志刚,鄢波,黄兴奇,等.苦参凝集素蛋白基因的分离克隆[J].植物学报,2001,43(8):821-825.