花粉介导法获得耐草甘膦玉米植株及其耐性研究

doi:10.13560/j.cnki.biotech.bull.1985.2016.11.018

摘要/Abstract

摘要： 为获得耐草甘膦转基因玉米植株,进而研究其除草剂耐性及外源基因的遗传特性,以质粒pBI101-aroA-M12为外源基因供体,以玉米自交系昌‘7-2'为受体,采用花粉介导法将外源基因导入玉米自交系中。PCR扩增、Southern杂交分析的结果证明获得了54个转基因植株;对其后代株系的分子检测及田间生物学鉴定,结果证明目的基因可以稳定遗传给后代植株,且赋予和提高了转基因植株的除草剂耐性。目的基因在转化体中的遗传规律研究,结果显示导入的目的基因在F₂受体植株中以3∶1的比例发生遗传分离,符合孟德尔单因子显性遗传分离规律。ELISA分析和蛋白质浓度测定的结果证实目的基因在转化植株中得到表达,表达量介于40.5-112.6 ng/g 叶片鲜重之间,目的基因表达量与该植株的除草剂耐性性状间呈极显著相关,r=0.942 3（P<0.01）。最终研究获得了4个高草甘膦耐性的转基因纯合株系。

关键词: 玉米, aroA-M12基因, 草甘膦耐性, 花粉介导, 遗传分析

Abstract: In order to develop transgenic maize plants tolerant to glyphosate and further study their herbicide tolerance and the genetic characteristics of exogenous gene in transgenic plants,plasmid pBI101-aroA-M12 harboring aroA-M12 gene was transformed into maize inbred Chang ‘7-2' plants by pollen-mediated transformation method. Fifty-four transgenic plants were obtained by PCR detection and Southern blot analysis. The detection results of molecular analysis and bioassay in the field showed that target aroA-M12gene was integrated into the genome of transgenic plants and could stably inherit to next generation. Moreover,the aroA-M12 gene conferred the glyphosate-tolerance to transgenic plants. The result of genetic rule of target gene in transgenic plants suggested that the target gene was segregated according to the ratio of 3∶1 in T₂ generation transgenic plants（lines）and it was consistent with the Mendel law of single-factor heredity. The results of ELISA analysis and protein concentration determination of transgenic plants confirmed that the target gene was expressed in transgenic plants,and the protein amount of gene expression were 40.5-112.6 ng/g leaf fresh weight,there was a significant correlation between protein amount of expression of each transgenic plant and corresponding glyphosate-tolerance of that plant,correlation coefficients r = 0.942 3（P<0.01）. Ultimately,4 pure lines of transgene with high glyphosate-tolerance were obtained from this experiment.

Key words: maize, aroA-M12 gene, glyphosate-tolerance, pollen-mediated transformation, genetic analysis

杨慧珍, 任志强, 肖建红, 卜华虎, 刘惠民. 花粉介导法获得耐草甘膦玉米植株及其耐性研究[J]. 生物技术通报, 2016, 32(11): 152-161.

YANG Hui-zhen, REN Zhi-qiang, XIAO Jian-hong, BU Hua-hu, LIU Hui-min. Development of Transgenic Maize Plants Tolerant to Glyphosate via Pollen-mediated Transformation and Their Glyphosate Tolerance[J]. Biotechnology Bulletin, 2016, 32(11): 152-161.

参考文献

[1] 张林生, 俞嘉宁, 曹让, 等. 转基因植物在农业上的应用[J]. 西北植物学报, 2002, 22（4）：1011-1017.
[2] Gianessi LP. Economic and herbicide use impacts of glyphosate-resistant crops[J]. Pest Manag Sci, 2005, 61（3）：241-245.
[3] Comai L, Sen LC, Stalker DM. An alter aroA gene product confer resistance to the herbicide glyphosate[J]. Science, 1983, 221（4608）：370-371.
[4] Comai L, Facciotti D, Hiatt WR, et al. Expression in plant of a mutant aroA gene from Salmonella typhimurium confer tolerance to glyphosate[J]. Nature, 1985, 317（6039）：741-744.
[5] Duncan K, Lewendon A, Coggins JR. Mutant EPSP synthase genes from tomato, Arabidopsis thaliana, Brassica napus, Glycine max, E. coli K-12 confer tolerance to glyphosate[J]. FEBS Lett, 1984, 170：59-63.
[6] Padgette SR, Kolacz KH, Dekanny X, et al. Development, identification, and characterization of a glyphosate-tolerant soybean line[J]. Crop Sci, 1995, 35（5）：1451-1461.
[7] Nida DL, Kolacz KH, Buehler RE, et al. Glyphosate-tolerant cotton：Gentic characterization and protein expression[J]. J Agr Food Chem, 1996, 44（7）：1960-1966.
[8] 敬凌霞. 抗CP4EPSPS单克隆抗体的制备和生物学特性的研究[D]. 重庆：重庆医科大学, 2007.
[9] 陈海伟, 张鲁华, 陈德富, 等. 除草剂及抗除草剂作物的应用现状与展望[J]. 生物技术通报, 2012（10）：35-40.
[10] Sidhu RS, Hammond BG, Fuchs RL, et al. Glyphosate tolerant corn：the composition and feeding value of grain from glyphosate-tolerant corn is equivalent to that of conventional corn（Zea mays）[J]. J Agric Food Chem, 2000, 48（6）：2305-2312.
[11] Howe AR, Gasser CS, Brown SM, et al. Glyphosate as a selective agent for the production of fertile transgenic maize（Zea mays L. ）plant[J]. Mol Breeding, 2002, 10（3）：153-164.
[12] Bonny S. Genetically modified glyphosate-tolerant soybean in the USA：adoption factors, impacts and prospects. A review[J]. Agronomy for Sustainable Development, 2008, 28（1）：21-32.
[13] Joseph TD, Edward CL, David AM. Effects of landscape composition on spread of an herbicide-resistant weed[J]. Landscape Ecology, 2009, 24（6）：735-747.
[14] Nicola S, Luigi D. Surveying the occurrence of subspontaneous glyphosate-tolerant genetically engineered Brassica napus L. （Brassicaceae）along Swiss railways[J]. Environmental Sciences Europe, 2012, 24：23
[15] Nehl SR, Kraus J, Matzk A, et al. Transgenic varieties：sugarbeet [J]. Sugar Tech, 2010, 12（3-4）：194-200 .
[16] Jones JD, Golds Brough PB, Weller SC. Stability and expression of amplified EPSPS genes in glyphosate resistant tobacco cells and plantlets[J]. Plant Cell Reports, 1996, 15（6）：431-436.
[17] Zhou H, Arroe Smith JW, Fromm ME, et al. Glyphosate -tolerant CP4 and GOX genes as a selectable marker in wheat transformation [J]. Plant Cell Rep, 1995, 15（3-4）：159-163.
[18] Bair DR, Batson W, Watson C, et al. Evaluation of transgenic cotton varieties and a glyphosate application on seedling disease incidence[J]. Mycopathologia, 2004, 158（3）：363-368.
[19] 谢龙旭, 徐培林, 聂燕芳, 等. 抗草甘膦抗虫植物表达载体的构建及其转基因烟草的分析[J]. 生物工程学报, 2003, 19（5）：545-550.
[20] Tong XH, Daud MK, Sun YQ, et al. Physiological and molecular mechanisms of glyphosate tolerance in vitro selected cotton mutant[J]. Pesticide Biochemistry and Physiology, 2009, 94（2 /3）：100-106
[21] 余桂荣, 杜文平, 宋军, 等. 玉米胚性愈伤组织的遗传转化及耐草甘膦植株再生[J]. 中国农业科技导报, 2010, 12（4）：108-113.
[22] 赫福霞, 郎志宏, 陆伟, 等. 以耐草甘膦2mG2-epsps基因为选择标记的玉米转化体系的建立[J]. 生物技术通报, 2008（5）：93-97.
[23] 孙毅, 崔贵梅, 郝曜山, 等. 超声波辅助花粉介导植物转基因方法：中国ZL 201110041484. 0[P]. 2011-2-18.
[24] Sambroo KJ, Fritsch EF, Maniatis T. Molecular Cloning：A Laboratory Manual[M]. 2nd ed. New York：Cold Spring Harbor Laboratory Press, 1989.
[25] 王关林, 方宏筠. 植物基因工程[M]. 第2版. 北京：科学出版社, 2005.
[26] Peng JY, Lister RL, Hodges TK, et al. Inheritance of gusA and neo genes in transgenic rice[J]. Plant Mol Biol, 1995, 27（1）：91-104.
[27] Cheng M, Joyce EF, Pang SZ, et al. Genetic transformation of wheat mediated by Agrobacterium tumefaciens[J]. Plant Physiol, 1997, 115（3）：971-980.
[28] Gao SQ, Xu HJ, Cheng XG, et al. Improvement of wheat drought and salt tolerance by expression of a stress-inducible transcription factor GmDREB of soybean（Glycine max）[J]. Chin Sci Bull, 2005, 50（23）：2714-2723.
[29] Sangtong V, Moran DL, Chik Wamba R, et al. Expression and inheritance of the wheat Glu-1DX5 gene in transgenic maize[J]. Theor Appl Genet, 2002, 105（6/7）：937-945.
[30] Eva S, Sarah W, Duncan K, et al. Molecular characteristics of transgenic wheat and the effect on transgene expression[J]. Transgenic Research, 1998, 7（6）：463-471.
[31] Barro F, Martin A, CabrerAA. Transgene integration and chromosome alterations in two transgenic lines of tritordeum[J]. Chromosome Research, 2003, 11（6）：565-572.
[32] 施利利, 王松文, 张欣, 等. 抗除草剂基因atzA在转基因水稻中的遗传[J]. 南开大学学报：自然科学版, 2008, 41（6）：99-102.
[33] 耿立召, 刘传亮, 李付广. 农杆菌介导法与基因枪轰击法结合在植物遗传转化上的应用[J]. 西北植物学报, 2005, 25（1）：205-210.