Abstract:Rice (Oryza sativa) is one of the most important food crops in the world. Among the factors harmful to rice production, weeds results in the most severe yield loss. To control weeds by herbicide is convenience, fast and low cost, that becomes the first choice in weeding. To cultivate transgenic non-selective herbicide resistant crops can decrease the kind, dosage and residual of herbicide, and prompt the productive efficiency of land, that plays an important role in commercial crop production. The Mat (methionine sulfone N-acetyltransferase) gene from bacterium Nocardia sp. AB2253 is a novel glufosinate resistant gene reported in 2009, which may be useful to develop herbicide resistant crops. However, the previous studies showed that the original Mat gene was not powerful enough in transgenic rice to provide high level of glufosinate resistance. In this study, the optimized Mat# gene according to codon bias in rice was transformed into an indica rice cultivar 9K (O. sativa ssp. indica) by use of Agrobacterium-mediated method. PCR analysis and Southern blot analysis of transgenic plants showed that the target gene Mat# had been integrated into rice genome with two copies. Glufosinate spraying on T2 generation plants at seedling stage showed that the T2 generation plants were resistant to glufosinate with a tolerance concentration of 1 000 mg/L at least. Progeny segregation statistics showed that the ratio of plant resistant and sensitive to glufosinate in T2 generation was 3.56∶1, which was conform to Mendelian inheritance by Chi-square test. Germination of T3 generation seeds under glufosinate stress showed that the T3 generation plants were resistant to glufosinate with a tolerance concentration of 600 mg/L at least, which indicated the resistance level of transgenic plants with Mat# gene was not lower than that of 9KA2 with Bar gene transformed. The investigation of agronomic traits showed that there was no significant differences in main agronomic traits between the T2 generation plants and non-transgenic controls, which indicated the introduction of alien gene did not have an obvious impact on main agronomic traits. Enzyme activity assay showed that the average activity of N-acetyltransferase in leaf was about 6.6 times of that in non-transgenic control. These results suggestted that the optimized Mat# gene provided higher glufosinate resistance than the original version, which can be used as a selection marker and new herbicide resistant gene resource in transgenic crop breeding.
[1]James C.年全球生物技术转基因作物商业化发展态势[J].中国生物工程杂志, 2014, 34(1):1-10[2]陈芬, 杜洪伟, 陈浩东, 等.干旱响应转录因子基因导入水稻光温敏核不育系的研究[J].生命科学研究, 2008, 12(1):24-28[3]樊庆鲁, 郭加沅, 肖国樱, 等.水稻籼爪重组自交系群体芽期耐旱性鉴定广西植物[J].广西植物, 2009, 29(1):74-77[4]高俊凤.植物生理学实验指导[J]., 2006, :214-215[5]罗伯祥, 肖自友, 肖国樱, 等.转和基因水稻及相关特性分析[J].农业生物技术学报, 2012, 20(1):30-37[6]肖国樱, 陈芬, 孟秋成, 等.我国转基因抗除草剂水稻的生态风险与控制[J].农业生物技术学报, 2015, 23(1):1-11[7]肖国樱, 唐俐, 袁定阳, 等.转基因抗除草剂两系杂交早稻恢复系-的培育研究[J].杂交水稻, 2007, 22(6):57-61[8]肖国樱, 袁隆平.爪哇稻及其亚种间杂种优势的研究[J]., 2009, :94-95[9]杨益军.全球草铵膦市场现状及前景预测分析[J].农药市场信息, 2013, 2013(19):18-19[10]曾崇华, 陈芬, 孟秋成, 等.水稻后代中高组织培养力家系的筛选[J].杂交水稻, 2016, 31(1):51-56[11]张凤娟, 张满良, 朱水芳.一种改进的水稻总的快速提取方法[J].植物检疫, 2004, 18(6):330-332[12]张宏军, 刘学, 张佳, 等.草铵膦的作用机理及其应用[J].农药科学与管理, 2004, 25(4):23-27[13]周浩, 杨益善, 唐俐, 等.草铵膦在转基因抗除草剂杂交稻直播栽培中的应用效果[J].作物研究, 2013, 27(5):427-430[14]Bedford D J, Lewis C G, Buttner M J.Characterization of a gene conferring bialaphos resistance in Streptomyces coelicolor A3(2)[J].Gene, 1991, 104(1):39-45[15]Brookes G, Barfoot P.Environmental impacts of genetically modified (GM) crop use 1996-2013: impacts on pesticide use and carbon emissions[J].GM Crops and Food, 2015, 6(2):103-133[16]Cui Y, Liu Z, Li Y, et al.Application of a novel phosphinothricin N-acetyltransferase (RePAT) gene in developing glufosinate-resistant rice[J].Scientific Reports, 2016, 6:21259-[17]D'Halluin K, Block M D, Denecke J, et al.The Bar gene as selectable and screenable marker in plant engineering[J].Methods in Enzymology, 1992, 216(1):415-426[18]Deng L H, Weng L S, Xiao G Y.Optimization of Epsps gene and development of double herbicide tolerant transgenic PGMS rice[J].Journal of Agricultural Science and Technology, 2014, 16(1):217-228[19]Deng L X, Chen F, Jiang L P, et al.Ectopic expression of GmPAP3 enhances salt tolerance in rice by alleviating oxidative damage[J].Plant Breeding, 2014, 133(3):348-355[20]Ellman G L.Tissue sulfhydryl groups[J].Archives of Biochemistry and Biophysics, 1959, 82(1):70-77[21]Fischhoff D A, Bowdish K S, Perlak F J, et al.Insect tolerant transgenic tomato plants[J].Nature Biotechnology, 1987, 5(8):807-813[22]Kita Y, Hanafy M S, Deguchi M, et al.Generation and characterization of herbicide-resistant soybean plants expressing novel phosphinothricin N-acetyltransferase genes[J].Breeding Science, 2009, 59(3):245-251[23]Oerke E-C.Crop losses to pests[J].The Journal of Agricultural Science, 2006, 144(1):31-43[24]Peng R H, Yao Q H, Xiong A S, et al.Codon-modifications and an endoplasmic reticulum-targeting sequence additively enhance expression of an Aspergillus phytase gene in transgenic canola[J].Plant Cell Reports, 2006, 25(2):124-132[25]Thompson C J, Movva N R, Tizard R, et al.Characterization of the herbicide-resistance gene Bar from Streptomyces hygroscopicus[J].The EMBO Journal, 1987, 6(9):2519-2523[26]Toki S, Hara N, Ono K, et al.Early infection of scutellum tissue with Agrobacterium allows high-speed transformation of rice[J].The Plant Journal, 2006, 47(6):969-976[27]Vaeck M, Reynaerts A, Hfte H, et al.Transgenic plants protected from insect attack[J].Nature, 1987, 328(2):33-37[28]Weng L S, Deng L H, Lai F X, et al.Optimization of the Cry2Aa gene and development of insect-resistant and herbicide-tolerant photoperiod-sensitive genic male sterile rice[J].Czech Journal of Genetics and Plant Breeding, 2014, 50(1):19-25[29]Wohlleben W, Arnold W, Broer I, et al.Nucleotide sequence of the phosphinothricin N-acetyltransferase gene from Streptomyces viridochromogenes Tü494 and its expression in Nicotiana tabacum[J].Gene, 1988, 70(1):25-37[30]Wu G, Yuan M, Wei L, et al.Characterization of a novel cold-adapted phosphinothricin N-acetyltransferase from the marine bacterium Rhodococcus spstrain YM12[J].Journal of Molecular Catalysis B: Enzymatic, 2014, 104:23-28[31]Yun C S, Hasegawa H, Nanamiya H, et al.Novel bacterial N-acetyltransferase gene for herbicide detoxification in land plants and selection maker in plant transformation ,[J].Bioscience, Biotechnology and Biochemistry, 2009, 73(5):1000-1006