【作者】 梁宏伟；

【导师】 杨传平； 刘桂丰；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2001， 硕士

【摘要】 在渗透胁迫下，有些耐盐的微生物和植物细胞能够主动积累一些小分子有机化合物和蛋白类来维持渗透平衡和体内水分。甘氨酸甜菜碱（glycine betaine，简称甜菜碱）就是这样一种盐胁迫下很好的调渗保护物质，当前被认为是最有希望的植物渗透调节剂之一。但甜菜碱在许多农作物和林木中不能积累，如果利用基因工程手段将其合成途径相关基因转入这些植物中，既可积累甜菜碱，又将可能达到提高植物耐盐性的目的。 高等植物甜菜碱的生物合成包括两步反应：胆碱→甜菜碱醛→甘氨酸甜菜碱，这两步反应分别由胆碱单加氧酶（CMO）和甜菜碱醛脱氢酶（BADH）催化。源于大肠杆菌（Escherichiacoli）的胆碱脱氢酶（CDH）可兼具有CMO和BADH的催化功能，合成甜菜碱。它是由Bet基因家族中的Bet-A基因编码，基因全长1668bp，成熟蛋白质分子量为61.9KD。本研究通过农杆菌介导法将Bet-A导入小黑杨（Populus simonii×P.nigra）中，使其积累甘氨酸甜菜碱，从而期望获得耐盐的杨树新品系。 首先建立小黑杨组培再生体系。设置BA和NAA的不同浓度梯度组合试验，确立小黑杨叶片分化再生最适宜培养基配方为：MS+0.5mg/L BA+0.1mg/L NAA；组培苗生根培养基配方为1/2MS+0.1mg/L NAA。由于目的基因和选择标记基因NPT Ⅱ（卡那霉素抗性基因）嵌合在一起，这样在遗传转化中卡那霉素可作为选择剂。因此需要进行叶片分化和生根对卡那霉素的敏感性测定，以确定合理的选择压。组培系统建立后，通过浓度梯度试验确立叶片分化对卡那霉素的临界浓度为50mg/L，即在进行外源目的基因对小黑杨叶片转化时，50mg/L的卡那霉素为最低选择剂量。芽诱导生根对卡那霉素的临界浓度为40mg/L。 然后以小黑杨组培苗为转化受体材料，通过农杆菌介导法导入Bet-A基因。分别调整工程菌液浓度、浸染时间及共培养时间等影响介导转化的关键因子，确立介导法转化小黑杨叶片的高效遗传转化体系。对获得的11株转化苗进行了PCR扩增检测，其中7株有特异性扩增条带产生，而阴性对照没有任何扩增条带。取其中3株进行了Southern杂交检测，转化苗获得了阳性杂交带，而阴性对照没有杂交带产生。由此可以证明，外源基因已整合进小黑杨的基因组中。更多还原

【Abstract】 Some salt梩olerant microbes and plant cell can maintain osmotic balance and water in the plant through active accumulation of compatible solute under osmotic stress, such as glucide, polyol, osmotin etc. One of the osmoregulatory is glycine betaine which is considered as one of the most important plant osmoprotectant at present, but glycine betaine can not be accumulated in some important corps and trees. If we transform synthetical gene of glycine betaine into plant梒ell, we might obtain high salt梩olerant transgenic plants. In plant, glycine betaine is synthesized by two step oxidation from choline to glycine betaine . The proceeding is choline梑etaine aldehyde梘lycine betaine, which the first step is established by choline monooxygenase(CMO), and the second step is done by betaine aldehyde dehydrogenase(BADH). Choline dehydrogenase gene isolated from Escherichia co]i, is coded by Bet桝 gene which is one of the Bet gene family . The Bet桝 gene is consist of 1 668 nucleotides, that is capable of encoding a protein in 61.9 KD, choline dehydrogenase(CDH). Since CDH has both function of CMO and BADH in catalyzing reaction, it is a necessary enzyme in the way of betaine synthesis. In this study , we have introduced the Bet桝 gene into Popu]us simonhi XP. nigra by agrobacterium ?mediated transformation. lYe hope to see that glycine betaine can be accumulated and enhance salt stress tolerance in transgenic plant in the future. Firstly, the leaves of this hybrid variety is used as explants and the medium is added some plant hormones, such as NAA and BA in different concentration as experiment component. The optimal medium of cell polarization and multiplication is MS + 0. 5mg/L BA + 0. lmg/L NAA. The best medium of rooting is 1/2 MS + 0. lmg/L NAA. Kanamycin is used as selective agent in transformation, because Bet桝 and NPT II genes is connected in the vector. It needs to test the sensitivity of the explants to Kanamycin in order to decide a reasonable selective pressure for transformation. The critical concentration of kanamycin to the cell polarization is 50 mg/L. That is to say, the lowest concentration of kanamycin in cell polarization is 50 mg/L, and 4Omg/L during transplants rooting in Populus simoniiXP. nigra. Secondly, bet桝 gene was introduced into leaves of asepsis plants of poplar by agrobacterium-mediated transformation. The efficient system of transformation via agrobacterium ?mediated is established by regulating the crucial factor, such as the bacterial concentration, the soaking time, the co梒ulture time, respectively. Then, transgenic plants are analysed by PCR and southern blotting. The result shows that Bet桝 gene has been introduced into the genome of Populus simoniiXP. nigra.更多还原