【作者】 李金华；

【导师】 李平；

【作者基本信息】 四川农业大学 ， 生物化学与分子生物学， 2003， 硕士

【摘要】 盐碱地在我国分布广泛，随着人们生产活动的加剧，盐渍化还在进一步恶化。培育耐盐品种是解决盐碱地粮食生产的关键技术，然而大多数农作物对盐碱的耐受性较差，传统育种方法很难选育出真正的耐盐品种。植物抗逆机理研究的进展和相关基因的克隆为我们利用基因工程技术培育高产、抗逆、优质新品种提供了一条有效的途径。 盐碱对植物造成的主要伤害之一是渗透胁迫。高等植物尤其是抗性较强的植物，通过积累一些小分子渗调物质来抵御渗透胁迫造成的危害，其中甜菜碱(betaine)是最重要的一种。甜菜碱在多种生物体内积累，但在一些重要的农作物中并不积累，如水稻、马铃薯、番茄和烟草等。通过基因工程技术将甜菜碱合成的全套机制转移到这些重要的农作物中，使其具有合成甜菜碱的能力而提高其耐盐性。本研究利用来源于藜科盐生植物山菠菜的甜菜碱合成的两个相关基因CMO(胆碱单氧化酶)基因和BADH(甜菜碱醛脱氢酶)基因为供体，以粳稻和杂交籼稻恢复系、保持系为受体，通过基因枪或农杆菌转化法进行遗传转化，获得了转化植株，并经分子验证，目的基因已整合到水稻基因组中，表现出较好的耐盐能力。取得的主要研究结果如下： 1．分别以不同水稻品种的幼穗、幼胚和成熟胚作为外植体，建立了高效的转基因受体系统，并对其进行了优化。采取幼胚低温预处理、愈伤组织的干燥培养和抗性愈伤组织的预分化处理等措施，分别提高了愈伤组织诱导率、抗性愈伤获得率和出苗率。 2．获得了一批转CMO基因和BADH基因的潮霉素和G418抗性植株，PCR检测结果表明大多数抗性植株其选择标记基因与外源目的基因共整合，个别植株只整合了选择标记基因而丢失了目的基因。对随机抽取的PCR检测双阳性植株进行Southern blotting杂交检测分析，表明CMO和BADH基因均已整合到转基因水稻的基因组中。 3．潮霉素和G418抗性植株经室内初步盐胁迫诱导筛选后，在温室盐池中再次进行耐盐性鉴定，得到耐盐植株。发现有少数PCR阳性反应植株在盐胁迫过程中没有耐盐表现而死亡，少数阴性植株则有耐盐表现而存活的现象。 4．对转基因植株进行的室内和田间耐盐性鉴定中，转基因植株的耐盐性均比对照好，但个体间耐盐表现有差异。 5．对转基因水稻植株后代进行的遗传分析表明，大多数符合孟德尔单基因遗传规律，目的基因能稳定地遗传和表达。更多还原

【Abstract】 Salina spread widely in China. With human activity pricking up, such problem will become more and more serious. Breeding salt-tolerance variety is a key technology to settle the foodstuff problem of Salina area. However, most of the crops we planted can not tolerate the salted soil, and it is almost impossible to get the genuine salt-tolerance variety by traditional breeding methods (Ashraf M et al). The research progress of plant adversity resistance mechanism and the cloning of correlative gene provide us an effective approach to breed new variety with high production, adversity resistance and good quality by use of genetic engineering.One of the major damages caused by salt is osmotic stress. Advanced plants, especially those which have perfect tolerance, can resist the harm caused by osmotic stress through accumulating some low molecule weight Osmoprotectants, among which, glycine betaine is one of the most important substance. Glycine Betaine can be accumulated in many kinds of organism, but not in some important crops such as rice, potato, tomato and tobacco (Weretilnyk E A et al, 1999). We can transform the whole bataine synthesis mechanism into these important crops providing them the ability of synthesizing bataine to increase their salt-tolerance by genetic engineering. The research using two glycine betaine synthesis related genes namely Choline dehydrogenase (CMO) and Betain-aldehyde dehydrogenase (BADH) from Atriplex hortensis as donor, the japonica rice and some indica hybrid rice restorer or maintainer lines as receptor, gotsome transgenic plants by using micro-particle bombardment or agri-bacterium, and proved that the target gene have been integrated into the rice genome after molecule testing. And these gene modified rice express better salt tolerance. From the experiment, such results below were got1. Effective gene transferring system was set up with young panicles, immature embryos and mature embryos of different rice varieties. Cold pretreatment of immature embryos, desiccation culture and predifferentiation of resistence calli could increase the callus inducing ratio, resistance calli and plant regeneration ratio, respectively.2. Some hygromicine and G418 resistant’plants containing CMO and BADH were obtained in mis study, and PCR testing indicated that most of the resistant plants contained the selective gene and the foreign target gene, but some plants just integrated selective gene and lost the targetgene. Southern Blot with positive plants of PCR showed that CMO and BADH have been integrated into the rice genome.3. The salt tolerance was tested indoors with all the hygromicine and G418 resistant plants and confirmed in salt pool in greenhouse, some salt tolerance plants were screened. A few of the positive plants could not express salt tolerance and died, while some negative plants survived in salty condition.4. Both in field testing and indoors testing, all the transgenic plants show better tolerance to salt than the control, but the expression degree are different in different plants.5. Genetic analysis of the transgenic plant offspring shows that the separating ratio is in accordance with Mendel’s single gene inheritance law, and the target gene can entail to the offspring and express stably.更多还原