农杆菌活体转化技术的优化与高抗草甘膦棉种质的创制

发布时间：2018-01-19 01:13

本文关键词： 棉花草甘膦 EPSPS-G6 农杆菌活体转化方法草甘膦抗性产量纤维品质　出处：《浙江大学》2016年硕士论文　论文类型：学位论文

【摘要】：棉花是唯一可由种子生产纤维的农作物,棉花生产需要投入较多劳动力。由于劳动力的转移,我国的人工价格不断上涨,植棉的比较效益和经济效益下降,导致近年我国棉花生产的连年滑坡。我国的棉花生产仍以人工作业为主,其中棉田除草是植棉用工的主要内容之一,占植棉用工的1/3以上。草甘膦(Glyphosate)是一种典型的广谱性、内吸传导型灭生性除草剂,是杂草控制的首选除草剂。本研究将具有自主知识产权的抗草甘膦除草剂基因(EPSPS-G6)构建进植物表达载体,用农杆菌活体转化方法将目的基因转入陆地棉推广品种中棉所49,创制高抗草甘膦棉花种质系,分析其抗性表现及相关农艺性状,探讨其生产应用前景。主要研究结果如下：1、利用本实验室构建的表达载体pCAMBIA1300-EPSPS-G6,酶切获得目的基因EPSPS-G6.并与空载体pCAMBIA2301连接构建棉花高效表达载体——pCAMBIA2301-EPSPS-G6,去除原载体中的NPII筛选标记基因,将目的基因作为筛选标记。2、对本实验室创制的农杆菌活体转化方法进行优化。结果表明,含50mg/L乙酰丁香酮(acetosyringone,AS)的转化液可将阳性率提高至22.18%,是对照(9.72%)的2.28倍。此外,不同转化时间转化效率差异显著,其中开花当天中午12：00～14：00转化得到阳性率最高(22.18%),下午13：00～15：00次之(18.86%),上午8：00～10：00转化效率最低(11.29%)。3、采用优化的农杆菌活体转化方法,将EPsPS-G6基因导入到陆地棉推广品种——中棉所49,共收获转化种子14.3 kg,T0转化植株经20 mM草甘膦喷施,阳性率达22.18%。经PCR验证,共获得了35株含EPSPS-G6基且抗草甘膦的To转基因植株。4、T1转基因后代的抗性分离比例从1/4-3/4不等。对T1阳性植株进行自交,按单株收获,T2抗性稳定材料占19.8～32.1%,基本符合一对杂合基因分离的规律。5、田间草甘膦抗性水平和莽草酸含量结果表明,转EPSPS-G6基因棉花种质系可抗3倍以上的推荐浓度,其抗性水平明显优于美国抗草甘膦品种和本实验室创制的第一代抗草甘膦棉花种质系,其中EPSPSG6-501种质系抗草甘膦的能力最强。6、转EPSPS-G6基因棉花种质系籽棉产量、皮棉产量、衣分均与中棉所49近似；铃重、籽指略高于对照；株高与对照近似；果枝数略低于对照；单株铃数与对照一致。纤维品质检测结果表明,转EPSPS-G6基因棉花种质系的纤维长度和整齐度略优于对照；马克隆值与对照在同一品级且略好于对照；伸长率略低于对照；断裂比强度与对照相同。说明外源基因对于转基因棉花种质系的产量、农艺性状和纤维品质无明显影响。7、调查了金华地区的杂草发生情况与防控成本,为抗草甘膦棉花品种的应用提供依据。结果表明,金华地区棉田杂草以牛筋草、马齿苋和小飞蓬为主,化学除草可节省植棉除草用工开支约1000元/亩,抗草甘膦棉花品种的生产应用可取得显著的经济效益,具有广阔的生产应用前景。
[Abstract]:Cotton is the only crop that can produce fiber by seed. Cotton production needs more labor force. Due to the transfer of labor force, the labor price of our country is rising, and the comparative benefit and economic benefit of cotton planting are decreased. In recent years, cotton production in China has been declining year after year. The cotton production in China is still dominated by manual work, among which weeding in cotton fields is one of the main contents of cotton planting. Glyphosate of glyphosate is a typical broad-spectrum, endo-absorption-conductive herbicide. In this study, EPSPS-G6, a glyphosate resistant herbicide gene with independent intellectual property rights, was constructed into plant expression vector. Using Agrobacterium tumefaciens transformation method, the target gene was transferred to Zhongmiansuo 49, a upland cotton extension variety, to create germplasm lines with high resistance to glyphosate, and to analyze its resistance and related agronomic characters. The main results are as follows: 1, using the expression vector pCAMBIA1300-EPSPS-G6 constructed in our laboratory. The target gene EPSPS-G6 was digested and ligated with empty vector pCAMBIA2301 to construct pCAMBIA2301-EPSPS-G6. The NPII screening marker gene was removed from the original vector and the target gene was used as the screening marker. 2. The transformation method of Agrobacterium tumefaciens was optimized. The conversion solution containing 50 mg / L acetosyringone AS) could increase the positive rate to 22.18, 2.28 times as much as that of the control group (9.72%). There were significant differences in transformation efficiency among different transformation times, and the highest positive rate was obtained at 12: 00: 14: 00 at noon on the flowering day. At 13: 00 in the afternoon, 15: 00 followed by 18.860.The conversion efficiency of 8: 00 to 10: 00 in the morning was the lowest, 11.29 and 3, and the optimized transformation method of Agrobacterium tumefaciens in vivo was adopted. The EPsPS-G6 gene was introduced into the upland cotton cultivar Zhongmiansuo 49, and the transformed seeds were harvested and transformed by spraying glyphosate with 20mm glyphosate. The positive rate was 22.18%. 35 transgenic plants containing EPSPS-G6 and glyphosate resistant to glyphosate were obtained by PCR. The percentage of resistance segregation of T1 transgenic progenies ranged from 1 / 4 to 3 / 4. For T1 positive plants, 19.832. 1% of the resistant stable materials were harvested per plant. The results of glyphosate resistance level and shikimic acid content showed that transgenic cotton germplasm lines with EPSPS-G6 gene could resist more than 3 times the recommended concentration. Its resistance level was obviously superior to that of the American glyphosate resistant variety and the first generation of glyphosate resistant cotton germplasm developed in our laboratory, and the EPSPSG6-501 germplasm line had the strongest resistance to glyphosate. The yield of seed cotton, lint yield and lint percentage of cotton germplasm lines with EPSPS-G6 gene were similar to those of Zhongmiansuo 49. The boll weight and seed index were slightly higher than those of the control. Plant height was similar to that of control. The number of fruit branches was slightly lower than that of the control. The results of fiber quality test showed that the fiber length and uniformity of cotton germplasm lines with EPSPS-G6 gene were slightly better than that of the control. The micronaire value was in the same grade as the control and was slightly better than that of the control. The elongation was slightly lower than that of the control. The breaking ratio intensity was the same as that of the control. The results showed that exogenous gene had no significant effect on yield, agronomic characters and fiber quality of transgenic cotton germplasm lines. The occurrence and control cost of weeds in Jinhua area were investigated. The results showed that the main weeds in cotton field in Jinhua area were Herba canola, Portulaca oleracea and Prunus vulgaris, and chemical weeding could save about 1000 yuan per mu in the cost of weeding and weeding. The production and application of glyphosate resistant cotton varieties can obtain remarkable economic benefits and have a broad prospect of production and application.
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S562

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