【作者】 韩凯；

【导师】 薛吉全；

【作者基本信息】 西北农林科技大学 ， 种子工程， 2011， 硕士

【摘要】 自世界上首例转基因烟草问世以来,植物转基因技术得到广泛应用,特别是在作物育种方面。尽管在过去的几年中玉米的转化技术取得了较大进展,但对研究者来说遗传转化技术仍然是玉米转基因研究的一个关键限制因素。目前常用的方法是基因枪法和农杆菌介导法。因此需开发一种新的遗传转化方法,建立稳定、便捷和高效的遗传转化,加速作物新品种培育的步伐和功能基因组学研究的发展。应用细胞穿透多肽(cell penetrate peptides,CPPs)为介导对花粉进行的转基因研究,无需大量准备工作,从收集花粉到完成遗传转化可在几小时内完成。本文就超声处理参数、植物表达载体构建、转基因植株草甘膦筛选浓度及转基因检测等方面做了相关研究。主要研究结果如下:1.以玉米自交系昌7-2为材料,利用TTC染色法和花粉离体萌发的方法研究了不同培养基组分和不同超声参数对花粉活力的影响。结果表明:花粉在液体中的活力急剧下降;随着超声处理强度的加大,花粉的活力明显降低;相同超声强度下,超声处理时间越长,花粉的活力越低。SAS软件分析表明在四种不同的培养基中,PLM1和PLM3对花粉活力的影响相对小一些。不同超声处理对花粉活力的影响:N1<1<3<4<2<5<6。为以花粉作为受体进行转基因的研究提供超声参数参考。2.利用In-FusionTM Advantage PCR Cloning Kit(Clontech Laboratories, Takara)试剂盒,将玉米谷氨酰胺合成酶基因(GS1)插入pCUbi1390载体的Ubi启动子后面PstI酶切位点处,然后在此基础之上,再将选择标记基因5-烯醇丙酮莽草酸-3-磷酸合成酶基因( CaMV35S + EPSPS )插入HindIII位点,构建植物表达载体pCUbi1390-Ubi-GS1-35S-EPSPS。结果表明In-Fusion法正确构建了设计的转化载体,简化了载体构建的过程,也适用于任何一个基于单酶切位点把外源DNA重组插入目标序列,具有应用与推广价值。3.以玉米自交系18-599(红)为材料,当幼苗4-5叶时,分别喷施不同浓度的草甘膦溶液,确定转基因植株草甘膦筛选浓度。幼苗在喷施0.2%的草甘膦一周后死亡77株,受抑制163株,正常9株,96.4%的非转基因玉米苗对0.2%的草甘膦没有抗性。因此,选用0.2%的草甘膦药液作为筛选转基因植株的最适浓度。4.以玉美头168为材料,5%的蔗糖溶液为超声处理缓冲液进行花粉介导的遗传转化实验。收获种子后,对T0代进行PCR检测、Elisa检测试验。利用不同超声波处理参数处理花粉,授粉后总共得到种子467粒,将种子种到温室里共出苗432株,经过三轮的PCR检测,共有23株幼苗为转基因植株。PCR检测转基因效率约为5.32%。对可以正常结实的18株PCR检测阳性的转基因植株进行Elisa检测,18个单株全部呈现阳性。进一步转基因检测得到的转基因效率为4.17%。更多还原

【Abstract】 Transgenic technology has been widely used since its inception of the first transgenic plant--transgenic tobacco, especially in the area of crop breeding. Although the maize genetic transformation has been made great progresses in the recent years, it still the vital limitation. Particle bombardment and agrobacterium-mediated techniques are commonly used these days. It’s necessary to develop new transformation methods and built stable, rapid and high-efficient transformation system for breeding new crop variety and the study of functional genomics. With the help of cell penetrating peptides, pollen-mediated transformation need not lots of preparatory work. And the complete genetic transformation can be completed in a few hours. In this paper, ultrasonic processing parameters, construction of plant expression vector, transgenic plants glyphosate screening concentration and transgenic detection were studied.The results were as follows:1. We used the maize inbred line Chang 7-2 to study the viability of maize pollen in different media components and different ultrasonic parameters with the methods TTC staining and pollen germination in vitro. The results showed that: The viability of pollen was sharply decreased in liquid. With the increasing intensity of ultrasound treatment, the viability of pollen gradually decreased; and also the time of ultrasound treatment. Analysis of SAS confirmed that PLM1 and PLM3 had less effect on pollen viability among the four media; and the influence of different ultrasound treatment was: N1<1<3<4<5<6<2. This work can provide ultrasound parameters reference for pollen-mediated transgenic research.2. We inserted two genes: maize glutamine synthase gene (GS1) and 5-enolpyruvlshimimate-3-phosphate synthase gene (CaMV35S + EPSPS ) into the Pst I and Hind III sites of vector pCUbi1390 separately to construct plant expression vector pCUbi1390-Ubi-GS1-35S-EPSPS. The results showed the expecting pCUbi1390-Ubi-GS1-35S-EPSPS was successfully constructed. The procedures were largely simplified and the method could be widely used in any recombination of insertion of any DNA sequence not very long at single restriction site of any DNA sequence.3. Used maize inbred line 18-599(red) as the material, we sprayed 4-5 leaf seedling glyphosate solution of different concentrations to determine the transgenic plants, glyphosate screening concentration. Seedlings of spraying 0.2% glyphosate had 77 death, 163 inhibition and 9 normal. That said 96.4% of seedling had no resistance to 0.2% glyphosate solution. So we choose 0.2% as the optimal concentration to screen Transgenic plants.4. The pollen-mediated genetic transformation was carried out using Yumeitou 168 as material, 5% sucrose solution for ultrasound processing buffer. After harvest, we did PCR amplification analysis and Elisa test on T0 generation seedlings to detect the transgenic efficiency of this method. Sowed the seeds we harvested (467 in total), 432 seeds emerged. 23 seedlings were positive after PCR of three pair of primers, and the transgenic efficiency were approximately 5.32%. 18 of PCR positive plants which were fertile were assayed again with Elisa test. They all tested positive. Further transgenic detected of transgenic efficiency was 4.17%.更多还原