【作者】 高叶；

【导师】 王宝山；

【作者基本信息】 山东师范大学 ， 植物学， 2010， 硕士

【摘要】 盐生植物是一类能够在含盐量相当于200 mM NaCl或更高的盐土上完成生活史的天然植物类群,在与盐土协同演化过程中形成了一系列适应盐生环境的特殊生存策略。因此,利用分子生物学技术研究盐生植物耐盐的遗传及分子机理对于利用基因工程等生物技术培育耐盐经济作物开发利用盐碱地具有重要意义。但是,目前尚为建立真盐生植物的遗传转化体系。盐地碱蓬(Suaeda salsa)是一种肉质化的真盐生植物(euhalophyte),可以在潮间带完成生活史,其愈伤组织也具有较强的抗盐性。因此,不管是在细胞水平还是在整株水平,盐地碱蓬在植物耐盐基因工程的研究利用方面都显示出了广阔的应用前景和巨大的利用价值。目前,农杆菌介导法是植物遗传转化上应用最广泛的方法,本实验将探讨农杆菌介导法转化盐地碱蓬茎尖的影响因素,并对转化效率及转基因在茎尖中的表达情况进行研究。本文主要研究结果如下:1.以盐地碱蓬茎尖为外植体,探讨了不同浓度的潮霉素对茎尖分化能力的影响。结果发现,当Hyg(Hygromycin B,潮霉素)浓度达到10 mg.L-1时茎尖的分化被完全抑制,100%的茎尖发黄死亡。综合考虑,在盐地碱蓬农杆菌介导的遗传转化中,茎尖分化的过程宜选用浓度为500 mg.L-1的哌拉西林钠来抑制农杆菌的生长,Hyg筛选的临界浓度为10 mg.L-1。2.以盐地碱蓬茎尖作为根癌农杆菌(LBA4404)介导转化的受体,通过对GUS基因稳定表达率的分析,研究该转化体系的最佳参数。结果表明预培养时间、共培养时间、农杆菌浓度、感染时间和恢复时间等对转化效率都有一定的影响。最佳转化参数是:切取10 d苗龄的无菌苗茎尖10～20 mm,预培养2 d后于OD600 0.5农杆菌悬浮液中感染l0 min,吸去多余的菌液,将茎尖转移到共培养基中共培养2 d,然后转移到脱菌培养基中进行抑菌培养3天后,将茎尖转到分化培养基中恢复2～3 d,最后再转入筛选培养基上连续筛选3～4代,每代12 d,获得抗性茎尖,转化频率为3%左右。3.对转基因抗性茎尖进行GUS基因组织化学染色和PCR检测,证实了外源基因己经整合到盐地碱蓬茎尖的基因组中,并得到稳定表达。我们正在利用转基因抗性茎尖获得转基因植株。更多还原

【Abstract】 Halophytes are a class of the natural plant taxa which can complete their life cycle in the saline with salt content equivalent of 200 mM NaCl or higher. They developed a series of survival strategy in particular to adapt to the high salinity in the process of co-evolution with the saline soil. Therefore, studies on genetic and molecular mechanism of salt tolerance of halophytes will be very important for cultivating salt-tolerant crops through biothechnology, such as genetic engineering, which could have important ramification in exploitating saline-alkali soil. However, transformation systems of euhalophytes have not yet been established. Suaeda salsa L. is a kind of succulent euhalophyte, which can completed the life cycle in the high saline soil, and even intertidal zone. Agrobacterium-mediated transformation is the most widely used methods for plant molecular research. In the present paper, stem tips of S. salsa L. were used as explant to examine the factors affecting agrobacterium-mediated transformation efficiency.The main results are as follows:1. Stem tips of S. salsa L. were used as explant to study effects of antibiotics on in-vitro callus induction. With the increase of hygromycin concentration, the meristem differentiation rate markedly decreased. Meristem differentiation from shoot explants was completely inhibited in presence of 10 mg.L-1 hygromycin, about 100% shoot died. It was considered that piperalillin sodium (500 mg.L-1) was suitable for Suaeda salsa to elimination of Agrobacterium tumefaciens during transgenic callus induction; hygromycin 10 mg.L-1 was suitable in the selection of transgenic versus false-positive shoot from shoot explants.2. Stem tips of S. salsa L. were used as explant to study agrobacterium tumefaciens-mediated transformation efficiency. Hygromycin-resistant shoot were induced after co-cultivation of shoot with Agrobacterium tumefaciens strain LBA4404 that harbored a binary vector (pBI1301) with the genes for GUS and HPT. Using stable expression frenqueny of GUS gene, the optimal parameters for Agrobacterium tumefaciens-mediated transformation of Suaeda salsa were examined, such as bacterium concentration, preculture time, infection time, co-culture time and recovery time. Histochemical GUS assay showed that the optimal parameters for Agrobacterium tumefaciens-mediated transformation of Suaeda salsa are as follows: one to two millimeters of stem tip of the 10 day-old sterile seedlings, preculture for 2 days, infection for l0 min, co-culture for 2 days, bacterium concentration OD600=0.5.Then the shoot tip to restore in the differentiation medium 2 to 3 days and eventually transferred to selection medium containing 3 or 4 consecutive screening generations, each generation for 12 days, leading to a tansformation frequency of about 3%.3. The described above successful transformation systems was further confirmed by histochemical analysis of GUS activity and PCR in hygromycin-resistant shoot. The results showed that the extra gene Hyg was integrated into Suaeda salsa shoot genomes and GUS gene had stable expression. An efficient plant regeneration protocol from Agrobacterium tumefaciens-mediated transformed shoot is being studied.更多还原