抗除草剂基因EPSPS在棉花杂种优势中的利用及其快速转育
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摘要
棉花是我国重要的经济作物,棉田杂草危害一直是困扰棉农的问题之一。在棉花已有高产优质等优良性状基础上,通过转基因技术转化抗草甘膦基因,增强作物对草甘膦的抗性,以及将抗草甘膦特性作为指示性状应用于杂种生产,已成为植物转基因领域的重要研究内容。
本研究以农艺和品质性状优良的9个棉花新品系为母本,以具有EPSPS基因抗草甘膦新品系为父本,通过不去雄人工授粉配制杂交组合,确定筛选真杂种的适宜草甘膦浓度和最适喷施时间,对F1的产量及品质性状杂种优势进行分析。同时应用本实验室建立的棉花一年多代快速回交转育技术,将EPSPS基因转育到5个常规亲本中,以期获得稳定的转基因材料。主要结果如下:
1.通过喷施不同浓度的草甘膦,确定了有效鉴别父、母本品种抗与非抗草甘膦的最适浓度为2g/L,鉴别杂交种的最适草甘膦浓度为2.25 g/L,即10%草甘膦水剂稀释45倍,最适宜的喷施时间为棉苗三叶期。
2.在田间对杂种F1进行草甘膦喷施处理并利用PCR检测目的基因,结果显示9个组合的PCR阳性率介于33.6%~62.52%之间,其中有4个组合的阳性率达到50%以上。
3.应用高效液相色谱法测定草甘膦处理3 d后不同组合F1的叶片莽草酸含量,结果显示各组合莽草酸含量的高低与其抗草甘膦强弱表现一致,但不同组合受其遗传背景的影响,其抗性强弱亦稍有差异。
4.转EPSPS基因棉花F1杂种优势分析结果表明,F1皮棉产量为1777.1～2441.3 kg/hm2,产量增加显著。产量的增加主要源于铃数的增加,9个组合平均可增加27%,其中组合1增加量达到49%。断裂比强度的竞争优势可高达15%。
5.以5个品系为轮回亲本,以抗草甘膦品系为非轮回亲本进行杂交、回交,利用幼胚培养、嫁接、生长调控等集成的棉花一年多代回交转育技术,获得5个品系的BC4转基因抗除草剂新材料。
Cotton is an important economic crop. Weeds in cotton field is one of the troubles that farmer faced. Base on the excellent characteristics such as high yield and quality, it has become a hot aspect in transgenic plant breeding research to increase the resistant potential of cotton to glyphosate, as well as to utilize glyphosate resistance as an indicator in the heterosis utilization.
In this study, nine combinations were made between nine lines with good agronomic traits and fiber quality and a glyphosate-resistant new line via no-emasculation-pollination method. The optimum concentration and the spraying time of glyphosate were determined, and heterosis of cotton yield and fiber quality traits was analyzed. Meanwhile, EPSPS gene was transformed to five elite cotton lines by using rapid backcross introgression for generation advancement under local conditions in order to obtain BC4 generation. The results are as follows.
1. The optimum concentration of glyphosate was 2g/L and 2.25 g/L to discriminate resistant glyphosate and non-resistant parental varieties or hybrids, respectively. This concentration can kill non-glyphosate-resistant plants, while preserving the resistant glyphosate plants. And the most appropriate time of spraying was three-leaf stage.
2. F1 generation of nine combinations grown in the field were sprayed with the optimum glyphosate solution and tested by PCR method. The results showed that the positive ratio of hybrid of different combinations ranged from 33.6% to 62.52%. Among them, the positive ratio of four crosses was more than 50%.
3. Shikimic acid contents of F1 generation of different combinations were determined using HPLC method. The results showed that the trend of shikimic acid content of different combinations was in consistent with the trend of glyphosate resistance. To a certain extent, the resistance capacity of different crosses was slightly different due to their different genetic background.
4. The heterosis analysis of F1 of different combinations showed that the range of lint yield is from 1777.1 to 2441.3 kg/hm2, which increased significantly. Glyphosate-resistant cotton yield increases were mainly due to the increase in boll number. The average increase ratio of the 9 combinations was 27%, and the highest combination achieved 49%. The fiber strength of competitive advantage could be as high as 15%.
5. Five elite lines were utilized to cross and backcross with resistant glyphosate line via rapid backcross introgression for generation advancement under local conditions including immature embryo culture, grafting and plant growth regulation. In a year, four backcross generations of five crosses were obtained, which will lay a foundation for further breeding the new resistant glyphosate varieties.
本研究以农艺和品质性状优良的9个棉花新品系为母本,以具有EPSPS基因抗草甘膦新品系为父本,通过不去雄人工授粉配制杂交组合,确定筛选真杂种的适宜草甘膦浓度和最适喷施时间,对F1的产量及品质性状杂种优势进行分析。同时应用本实验室建立的棉花一年多代快速回交转育技术,将EPSPS基因转育到5个常规亲本中,以期获得稳定的转基因材料。主要结果如下:
1.通过喷施不同浓度的草甘膦,确定了有效鉴别父、母本品种抗与非抗草甘膦的最适浓度为2g/L,鉴别杂交种的最适草甘膦浓度为2.25 g/L,即10%草甘膦水剂稀释45倍,最适宜的喷施时间为棉苗三叶期。
2.在田间对杂种F1进行草甘膦喷施处理并利用PCR检测目的基因,结果显示9个组合的PCR阳性率介于33.6%~62.52%之间,其中有4个组合的阳性率达到50%以上。
3.应用高效液相色谱法测定草甘膦处理3 d后不同组合F1的叶片莽草酸含量,结果显示各组合莽草酸含量的高低与其抗草甘膦强弱表现一致,但不同组合受其遗传背景的影响,其抗性强弱亦稍有差异。
4.转EPSPS基因棉花F1杂种优势分析结果表明,F1皮棉产量为1777.1～2441.3 kg/hm2,产量增加显著。产量的增加主要源于铃数的增加,9个组合平均可增加27%,其中组合1增加量达到49%。断裂比强度的竞争优势可高达15%。
5.以5个品系为轮回亲本,以抗草甘膦品系为非轮回亲本进行杂交、回交,利用幼胚培养、嫁接、生长调控等集成的棉花一年多代回交转育技术,获得5个品系的BC4转基因抗除草剂新材料。
Cotton is an important economic crop. Weeds in cotton field is one of the troubles that farmer faced. Base on the excellent characteristics such as high yield and quality, it has become a hot aspect in transgenic plant breeding research to increase the resistant potential of cotton to glyphosate, as well as to utilize glyphosate resistance as an indicator in the heterosis utilization.
In this study, nine combinations were made between nine lines with good agronomic traits and fiber quality and a glyphosate-resistant new line via no-emasculation-pollination method. The optimum concentration and the spraying time of glyphosate were determined, and heterosis of cotton yield and fiber quality traits was analyzed. Meanwhile, EPSPS gene was transformed to five elite cotton lines by using rapid backcross introgression for generation advancement under local conditions in order to obtain BC4 generation. The results are as follows.
1. The optimum concentration of glyphosate was 2g/L and 2.25 g/L to discriminate resistant glyphosate and non-resistant parental varieties or hybrids, respectively. This concentration can kill non-glyphosate-resistant plants, while preserving the resistant glyphosate plants. And the most appropriate time of spraying was three-leaf stage.
2. F1 generation of nine combinations grown in the field were sprayed with the optimum glyphosate solution and tested by PCR method. The results showed that the positive ratio of hybrid of different combinations ranged from 33.6% to 62.52%. Among them, the positive ratio of four crosses was more than 50%.
3. Shikimic acid contents of F1 generation of different combinations were determined using HPLC method. The results showed that the trend of shikimic acid content of different combinations was in consistent with the trend of glyphosate resistance. To a certain extent, the resistance capacity of different crosses was slightly different due to their different genetic background.
4. The heterosis analysis of F1 of different combinations showed that the range of lint yield is from 1777.1 to 2441.3 kg/hm2, which increased significantly. Glyphosate-resistant cotton yield increases were mainly due to the increase in boll number. The average increase ratio of the 9 combinations was 27%, and the highest combination achieved 49%. The fiber strength of competitive advantage could be as high as 15%.
5. Five elite lines were utilized to cross and backcross with resistant glyphosate line via rapid backcross introgression for generation advancement under local conditions including immature embryo culture, grafting and plant growth regulation. In a year, four backcross generations of five crosses were obtained, which will lay a foundation for further breeding the new resistant glyphosate varieties.
引文
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[3] Zhu Y, Yu Z L, Lin M. Bioresistance and biodegradation of glyphosate and construction of transgenic plants[M]. Molecular Plant Breeding, 1(4):435–441.
[4]楚宗艳.棉花抗草甘膦基因的表达及功能鉴定[D].北京:中国农业科学院,博士学位论文, 2008.
[5]苏少泉.抗草甘膦作物的创制与发展[J].世界农业, 2004,3:48–50.
[6] Williams G M, Kroes R, Munro I C. Safety evaluation and risk assessment of the herbicide roundup and its active ingredient, glyphosate, for humans[J]. Toxicology and Pharmacology, 2000,31(2): 117–165.
[7]朱玉,于中连,林敏.草甘膦生物抗性和生物降解及其转基因研究[J].分子植物育种, 2003,4(1):435–441.
[8] Amrhein N, Deus B, Gehrke P, et al. The site of the inhibition of the shikimate pathway by glyphosate[J]. Plant Physiology, 1980,66(5),830–834.
[9] Berlin J, White L. Effects of glyphosate on shikimic acid accumulation in tobacco cell cultures with low and high yields of cinnamoyl putrescines[J]. Zeitschrift fuer Naturforschung, Section c Biosciences, 1981,36(2):210–214.
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