抗玉米螟、抗草甘膦、抗旱的转基因复合性状玉米种质的创制

发布时间：2018-11-26 20:44

【摘要】：玉米是世界第一大粮食作物,被广泛用于食品、饲料、工业加工、新能源开发等方面,玉米产量的波动会在世界范围内带来一系列负面效应。保证玉米高产优质和稳产,首先需要加强病虫害和自然灾害的防治。近20年来,人们已经利用基因工程手段成功培育出了具有抗虫、抗病、抗逆以及耐除草剂等优良性状的玉米新种质,抗虫和耐除草剂的转基因玉米更是被广泛种植,有效提高了玉米产量和降低了生产成本。当前严重影响玉米产量的因素有虫害、草害和干旱,培育具有抗虫、耐除草剂、抗旱性状的转基因玉米品种是一项具有战略意义的工作。利用多基因转化技术培育具有复合性状的转基因玉米具有明显竞争优势,是值得探索的课题。本工作采用了对玉米螟具有强杀毒活性的crylAc-M基因以防治对我国玉米危害严重的亚洲玉米螟。crylAc-M基因是在保证cry1Ac氨基酸序列不变的前提下进行密码子及编码框长度优化的产物,使其在单子叶植物中能高效表达。为了提高玉米对除草剂草甘膦的耐受性,我们导入epsps和GAT两个基因。epsps基因是从草甘膦极度污染的土壤中分离的可变盐单胞菌(Halomonas Variabilis)中克隆的,拥有我国自主知识产权。GAT基因能使进入植物体的草甘膦乙酰化而解除毒性。两个耐除草剂基因同时发挥作用,希望能有效提高玉米植株抗草甘膦的能力。ZmPIS基因是本实验室从玉米中克隆得到的,该基因的表达强度受多种逆境胁迫诱导,且过表达该基因可明显提高玉米植株的抗旱能力。这4个基因构建到同一植物表达载体中用于遗传转化。本工作通过农杆菌介导的玉米茎尖遗传转化法将上述4个基因(crylAc-M、 epsps、GAT、ZmPIS)分别转入玉米自交系9801和齐319(Q319),获得了转基因植株。通过PCR检测确定了目标基因在转化植株中存在,利用RT-PCR检测了4个基因的转录强度,肯定它们在转基因植株中稳定表达。此外,利用Real-time RT-PCR和Western blot方法检测了cryl Ac基因的表达丰度和cryl Ac蛋白的表达水平,肯定了cryl Ac基因在玉米植株中稳定表达。通过逐代除草剂筛选、PCR检测及抗虫性鉴定,从大量转基因株系中优选出6个稳定遗传且抗玉米螟、抗除草剂草甘膦和耐旱性良好的转基因玉米株系,其中株系L1-L3来自骨干自交系9801,Q1-Q3来自骨干自交系Q319。在此基础上,以非转基因自交系9801、Q319为对照,在严格控制条件下对6个转基因株系进行抗虫、抗除草剂和抗旱检测试验。转基因植株的抗虫性分析由于不同生长时期玉米对玉米螟危害的敏感程度有差异,本工作检测了生长不同阶段的转基因玉米的抗虫性,分别进行了田间和室内玉米螟接种试验。植株抗虫性观察结果显示,这些高抗株系的营养生长期植株仅在最初接虫的叶片上出现少量的幼虫取食小孔,没有受到进一步危害的迹象,与对照植株相比差异达到极显著差异水平。取灌浆期玉米的籽粒和苞叶进行饲喂玉米螟幼虫实验,得出喂食转基因玉米籽粒或苞叶的玉米螟死亡率显著高于饲喂非转基因的。这些结果表明转基因玉米可有效防治玉米螟危害,达到实用标准。转基因植株的草甘膦抗性分析在营养生长期,玉米叶片数与植株对草甘膦抗性成正相关,即较大植株对草甘膦抗性也较强。在玉米三叶期喷洒1.05kg ae ha-1草甘膦的水溶液筛选抗草甘膦株系。在田间草甘膦抗性测试中,对六叶期玉米植株喷洒0.84kg ae ha-1(即大田除草应用剂量)的草甘膦溶液,转基因株系表现出了良好抗性,即达到生产中应用水平。在进行高浓度除草剂喷洒试验中,以两倍应用剂量的草甘膦溶液喷施植株,发现转基因植株较未转基因对照植株受除草剂伤害程度轻,受害症状出现晚,而对照植株几乎全部死亡。转基因植株的抗旱性分析对10叶期玉米植株的干旱胁迫试验,观察转基因植株的抗旱能力。干旱处理期每天监测并控制土壤相对含水量在17-18%左右,持续处理7天,然后恢复正常浇水。在干旱控水期间,相比于对照植株,转基因株系叶片较伸展,萎焉程度较轻,恢复浇水后,也能够较快恢复正常生长。光合作用和叶绿素荧光测定结果显示：转基因植株维持较强的光合能力和光系统II活性,对干旱胁迫的抗性显著高于对照植株。综上所述,将crylAc-M、epsps、GAT、ZmPIS基因一同转入玉米,赋予了植株抗玉米螟、抗除草剂草甘膦特性,提高了植株的抗旱性。本工作培育出了具有优良复合性状的转基因玉米新材料,为培育多性状叠加的转基因新品种奠定了基础。
[Abstract]:Corn is the first major food crop in the world, and is widely used in food, feed, industrial processing, new energy development and so on, and the fluctuation of corn output will bring a series of negative effects in the world. the high-yield and stable yield of the corn is ensured, and the prevention and control of the diseases and insect pests and the natural disasters are first needed. In the past 20 years, the genetic engineering method has been used to successfully cultivate new corn germplasm, anti-insect and herbicide resistant transgenic corn having excellent characters such as insect resistance, disease resistance, stress resistance and herbicide resistance, and is widely planted, thereby effectively improving the yield of the corn and reducing the production cost. The factors that have seriously affected the yield of the corn are insect, grass and drought, and the cultivation of the transgenic corn variety with the resistance to insects, herbicide and drought resistance is a strategic task. The use of multi-gene transformation technology to cultivate transgenic corn with compound character has obvious competitive advantage, and it is a topic to be explored. The invention adopts the cryl Ac-M gene with strong anti-virus activity on the corn silk to prevent and treat the Asian jade which has serious harm to the corn in China The m. crylAc-M gene is a product of codon and coding frame length optimization under the premise of ensuring that the amino acid sequence of the cry1Ac is unchanged, so that it can be highly effective in monocotyledonous plants. In order to improve the tolerance of the corn to the herbicide, we have introduced epsps and GAT The gene. epsps is cloned from the highly polluted soil of the grass and has the autonomous knowledge of China. Property rights. The GAT gene can be released from the plant, and can be released from the plant. The two herbicide-resistant genes play a role at the same time, hoping to effectively improve the resistance of the corn plants to the grass The ZmPIS gene is cloned from the corn, and the expression strength of the gene is induced by a plurality of adversity stress, and the overexpression of the gene can obviously improve the drought resistance of the maize plant. Ability. The four genes were built into the same plant expression vector for inheritance The above four genes (crylAc-M, epsps, GAT, ZmPIS) were transferred to the maize inbred lines 9801 and Q319 (Q319) by Agrobacterium-mediated maize stem-tip genetic transformation. The presence of the target gene in the transformed plant was determined by PCR, and the transcription level of the 4 genes was detected by RT-PCR, and they were positive in the transgenic plants. In addition, the expression level of crylAc gene and the expression level of crylAc were detected by Real-time RT-PCR and Western blot. 6 stable and anti-maize, anti-herbicide, and well-resistant transgenic corn lines are preferably selected from a large number of transgenic lines by screening, PCR and insect-resistant identification on a per-generation herbicide, wherein the lines L1-L3 are from the backbone inbred lines 9801, Q1-Q3 from the backbone selfing Based on the above, the non-transgenic inbred lines 9801 and Q319 were used as control, and 6 transgenic lines were anti-insect, anti-herbicide and anti-herbicide under the control of strict control. drought test The insect-resistance of the plants is different from the sensitivity of the corn to the corn in different growth stages, and the insect-resistance of the transgenic corn in different stages of growth is detected, and the field is respectively carried out. The results of the observation on the insect-resistance of the plants showed that the vegetative growth period of these high-resistant strains only showed a small number of larvae feeding the small holes on the leaves of the first worm, and there were no further signs of further harm, compared with the control plants. The difference reached a very significant level of difference. The grain and bract leaf of the corn in the grouting period were fed with the experiment of the corn and larvae, and the mortality rate of the corn in the grain or bract of the transgenic corn was obtained. The results show that the transgenic corn can effectively prevent and treat the jade. The hazard of the rice has reached the practical standard. The resistance of the plant of the transgenic plant was analyzed in the vegetative period, the number of the leaves of the corn and the resistance of the plant to the plant were positive. The results showed that the resistance of the larger plants to the plant was stronger than that of the larger plants.-1. The anti-plant strain was selected from the aqueous solution of the grass and the root of the plant. In the field of the resistance test of the plant in the field, a total of 0. 84kg ae ha-1 (i.e., the application dose of the field weeding) was sprayed on the maize plants in the six-leaf period, and the expression of the transgenic lines In the high-concentration herbicide spraying test, the plant was sprayed with a two-fold application dose of the grass-calamine solution, and the transgenic plants were found to be less harm to the herbicide than the non-transgenic control plants. The symptoms were late, and the control plants Almost all of the deaths. The drought resistance of the transgenic plants is about 10 leaf. The drought stress test of the rice plant was carried out to observe the drought resistance of the transgenic plants. The relative water content of the soil was monitored and controlled every day in the drought treatment period. 7-18%, continuous treatment for 7 days, and then normal watering. During drought-controlled water, the transgenic line leaves were spread, withering, compared to the control plants. The results of photosynthesis and chlorophyll fluorescence showed that the transgenic plants maintained strong photosynthetic capacity and light. In conclusion, cryl Ac-M, epsps, GAT and ZmPIS gene were transferred to the corn, and the resistance to drought stress was significantly higher than that of control plants. The plant has the characteristics of resistance to the corn and the herbicide, and the drought resistance of the plant is improved.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：S513

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