【作者】 侯德英；

【导师】 祖元刚； 安利佳；

【作者基本信息】 东北林业大学 ， 植物学， 2003， 硕士

【摘要】 甜菜碱是已研究过的最好的渗透调节剂之一，且合成途径简单，遗传操作方便。在高等植物中，甜菜碱由胆碱经两个酶催化得到，催化第一步反应的酶是胆碱单加氧酶(CMO)，催化第二步反应的酶是甜菜碱醛脱氢酶(BADH)。已见转CMO和BADH基因且耐盐和耐旱性提高的转基因烟草，但尚未见关于耐旱性生理研究的详细报道。杨树是重要的速生经济树种，通过基因工程研究进行遗传改良相对成熟，已获得抗除草剂、抗虫等转基因杨树，但是未见转CMO、BADH基因的报道。因此，本文对转甜菜碱合成酶基因烟草的耐旱性进行了系统分析，并对山杨进行了甜菜碱合成酶基因的遗传转化研究，建立了根癌农杆菌介导的山杨遗传转化体系并获得了PCR及Southern dot检测阳性的转基因植株。对干旱胁迫下转甜菜碱合成酶基因的烟草的SOD酶和POD酶的活性、电解质的渗透率、叶片水势、失水速率及含水量的变化的分析结果表明，转基因烟草的SOD酶及POD酶活性、叶片水势、叶片相对含水量均高于对照，而电解质的渗透率和失水速率低于对照。说明CMO及BADH基因对提高转基因植物的耐旱性有一定的作用。通过根癌农杆菌介导的叶片转化法，进行了甜菜碱合成酶基因的遗传转化研究，获得了48株转CMO基因及6株转BADH基因的抗生素抗性植株。其中部分植株的PCR及Southern dot检测为阳性，初步证明外源基因已转移进山杨基因组。更多还原

【Abstract】 Betaine is one of the best osmopretectant studied, and its synthetic pathway is simple. In higher plants, betaine is synthesized in two-steps: the fist one is catalyzed by choline monooxygenase (CMO) and the second one is catalyzed by betaine aldehyde dehydrogenase(BADH). Several studies on drought-resistance and salt-resistance improved transgenic tobacco expressing CMO and BADH gene were reported, but there is no systematic physiological study on their drought-resistance characters. Populus is an important fast-grow economical forest tree species to which genetic manipulation system is well established and transgenic Populus with improved insect-resistance, herbicide-resistance and other characters were obtained, but no report is related to CMO and BADH transformation. We reported here systematic physiological studies on drought-resistant characters of transgenic tobacco expressing betaine synthetase genes. At the same time, we established Agrobacterium-tumefacience mediated gene transformation system of Aspen (Populus davidiana Dove) and obtained PCR and Southern dot positive plants transformed with CMO and BADH gene.In stress conditions, superoxide dismutase(SOD) activity, peroxidase (POD)activity and water potential of transgenic plants were generally higher than that of the control, while leaf relative leakage electrolytes and lose rate of transgenic plants were generally lower. These results indicated that betaine synthetase gene transformation contributed to improved drought resistance of tobacco.Populus is an importantly economical forest tree species, and have small genomes and are easy for tissue culture that make them well study as model plants of tree genetic engineering. At the same time, the traditional tree breeding limiting the improving the resistance of the populus. Engineering has quickly been developed and widely applied to improved the quality of populus. In this paper, the rapid and stable regeneration system was established that is suit to use for gene-transformation mediate by Agrobacterium tumefaciens.Leaf explants were infected with A.t. strain LBA4404 carrying antibiotic-resistance gene and betaine synthetase gene. 48 Km resistant shoots transformed with CMO gene and 6 hygromycin resistant shoots transformed with BADH gene were obtained. PCR and Southern dot analysis of some resistant shoots preliminarily indicated foreign genes had integrated into the genome of Aspen.Studies on factors influencing Aspen leaf disc transformation implied that 500mg/L Cef., 20mg/LKm or 3-5mg/L Hyg, 2-3 days preculture, about OD6oo 0.5 of Agrobacterium culture, 5min infection and 2~3days co-culture were optimized conditions.更多还原