基于花粉管通道法的转基因玉米材料的获得、分子检测和表型鉴定

发布时间：2019-03-04 18:53

【摘要】：玉米是世界三大食粮作物之一,同时也是重要的工业原料,其产量和品质对人类有着至关重要的意义。在众多影响农作物产量和品质的逆境因素中,干旱无疑是很常见且影响甚大的一种逆境胁迫,是制约农业生产的重要问题。干旱胁迫对玉米生长发育的影响十分巨大。我国是个缺水严重的农业生产国家,尤其是西北部地区,在没有有效灌溉方法的情况下,农作物长期处于干旱胁迫中。所以利用转基因技术去提高玉米品种的抗旱能力是减少干旱对玉米产量和品质影响的快捷有效的途径。根系是植物的重要器官,主要作用是吸收水分和养料以及合成必需的激素。玉米的根系发达与否和其抗旱能力强弱有直接关系,从而影响玉米的茎叶等地上部位以及最终产量的构成。细胞分裂素与植物长发育有密切联系,其代谢途径中的关键酶是细胞分裂素氧化酶(CKX),它可调节细胞分裂素在植物体内的含量,进而调控植物的各部分组织器官的生长发育情况。本研究选择一种水稻根特异性表达基因EXP18D启动子与CKX基因相连后,对玉米自交系进行转化,从而促进其根系的生长发育,以期获得耐旱玉米品种。本研究采用花粉管通道法将AtCKX3基因导入玉米自交系。经过对T0代、T1代以及T2代进行草甘膦除草剂喷洒的初步筛选,获得130个具有草甘膦抗性的转基因纯合株系。然后接着对T2代纯合株系进行耐旱鉴定,选出耐旱能力较强的10个转基因株系,然后通过分子检测的方法和一些常规根系测量指标去证明抗草甘膦除草剂基因EPSP以及目的基因CKX3已成功的导入C92、2416、572等玉米自交系的基因组中,且能够稳定遗传和表达。测定干旱处理过的转基因株系和C92、2416自交系的株高、叶面积,千粒重等生理指标和产量指标,结果显示,转CKX3基因株系各项指标都明显优于C92等自交系。另外,还对8个自交系572的T4代高代纯合转基因株系和572自交系的苗期根系相关指标进行了测定,结果表明CKX3基因过表达的转基因纯合株系的耐旱性相比自交系而言有所提高。基于微滴式数字PCR平台,以转基因玉米为例,建立了转基因作物外源基因拷贝数分析方法,对待测样品进行了快速鉴定,并从T0代转基因玉米株系中鉴定出多个单拷贝单株。对该方法与实时荧光定量PCR方法在分析结果的准确性方面进行了比较,从试验数据可以看出,两种检测方法的结果比较一致,单拷贝检测结果高度一致;但是ddPCR试验操作更加简便,试验结果可重复性强,试验数据更加准确可靠。研究表明,ddPCR方法是一种更加便捷、快速和准确的外源基因拷贝数分析新方法,基于其在准确性和灵敏度方面的显著优势,将会在转基因作物的外源基因拷贝数分析中得到广泛的应用。
[Abstract]:Corn is one of the three major food crops in the world, and it is also an important industrial raw material. Its yield and quality are of vital importance to human beings. Among the many adversity factors affecting crop yield and quality drought is undoubtedly a common and influential adversity stress which is an important problem restricting agricultural production. The effects of drought stress on the growth and development of maize were very great. China is an agricultural production country with serious water shortage, especially in northwest China. Without effective irrigation methods, crops are in drought stress for a long time. Therefore, using transgenic technology to improve drought resistance of maize varieties is a quick and effective way to reduce the effect of drought on maize yield and quality. Root system is an important organ of plants. Its main function is to absorb water and nutrients and synthesize essential hormones. Whether the root system is developed or not is directly related to the drought resistance ability of maize, which affects the composition of the upper ground parts of maize such as stem and leaf, as well as the final yield. Cytokinin is closely related to plant growth and development. The key enzyme in its metabolic pathway is cytokinin oxidase (CKX), which can regulate the content of cytokinin in plants. And then regulate the growth and development of each part of plant tissues and organs. In this study, a rice root-specific expression gene, EXP18D promoter, was linked to the CKX gene and transformed into maize inbred lines to promote the growth and development of their roots, with a view to obtaining drought-tolerant maize varieties. In this study, AtCKX3 gene was introduced into maize inbred lines by pollen tube pathway. After spraying glyphosate herbicide on T _ 0, T _ 1 and T _ 2 generations, 130 transgenic homozygous lines with glyphosate resistance were obtained. Then, the T _ 2 homozygous lines were identified for drought tolerance, and 10 transgenic lines with strong drought tolerance were selected. Then it was proved that glyphosate-resistant herbicide gene EPSP and target gene CKX3 had been successfully introduced into the genome of maize inbred lines C92, 2416572 by molecular detection and some routine root measurements, and the gene could be inherited and expressed stably. The physiological and yield indexes such as plant height, leaf area, 1000-grain weight and so on in drought-treated transgenic lines and C92, 2416 inbred lines were determined. The results showed that the CKX3 transgenic lines were obviously superior to C92 inbred lines in each index. In addition, eight inbred lines (572) with T _ 4 homozygous transgenic lines and 572 inbred lines at seedling stage were tested. The results showed that the drought tolerance of transgenic homozygous lines with over-expression of CKX3 gene was higher than that of inbred lines. The results showed that the drought tolerance of transgenic homozygous lines with higher T _ 4 generation and 572 inbred lines was higher than that of inbred lines. Based on the micro-drop digital PCR platform, a copy number analysis method of foreign genes in transgenic maize was established, and the samples were identified quickly. Several single copies of transgenic maize lines were identified from T _ 0 generation transgenic maize lines, and a single copy analysis method for transgenic maize plants was established, and several single copy single plants were identified from the T _ 0 generation transgenic maize lines. The accuracy of the method is compared with that of real-time fluorescence quantitative PCR. From the experimental data, it can be seen that the results of the two methods are relatively consistent, and the results of single copy detection are highly consistent. But the ddPCR test operation is more simple, the test results are repeatable, and the test data are more accurate and reliable. Studies have shown that ddPCR is a more convenient, rapid and accurate method for the analysis of foreign gene copy numbers, which is based on its significant advantages in accuracy and sensitivity. It will be widely used in the analysis of foreign gene copy number in transgenic crops.
【学位授予单位】：长江大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S513

【相似文献】