【作者】 殷东旭；

【导师】 刘立侠；

【作者基本信息】 东北师范大学 ， 植物学， 2009， 硕士

【摘要】 紫花苜蓿是世界上非常重要的牧草之一。由于近年来土壤盐渍化程度的加重,使苜蓿大幅减产,培育高耐盐性苜蓿新品种就显得至关重要。目前利用转基因技术能够快速有效的获得抗盐苜蓿新品种。SsNHXI基因是来源于盐地碱蓬的一个编码Na+/H+逆向转运蛋白的基因,其可将Na+区隔到液泡中,从而提高植物的耐盐性。近几年研究还发现,解旋酶在植物抗逆性方面也起着至关重要的作用。PDH45是来源于豌豆的一种DNA解旋酶,该基因转化烟草后可大幅提高其抗盐能力。本论文对实验室已构建的质粒pBISsNHXI和pBIDH45进行重组,构建了同时含有SsNHXI和PDH45基因的植物双元表达载体,利用根癌农杆菌介导法将其双元载体转入紫花苜蓿中,以期获得耐盐性好的苜蓿材料。通过卡那霉素初步筛选得到80株抗性植株,PCR技术显示目的基因已经整合到紫花苜蓿基因组中。选择其中20株转基因材料进行耐盐生理功能检测,用250mM NaCl同时处理非转基因型和转基因型植株5周,结果表明,尽管盐胁迫下转基因植株与非转基因植株的生长均受抑制,但转基因植株的生长状况明显好于非转基因型植株。与此同时,转基因植株的脯氨酸含量约是非转基因型植株的20倍,其叶绿素含量约高于非转基因型植株40%,且其细胞膜损伤程度低于非转基因型植株90%。这些实验结果表明过量表达SsNHXI与PDH45基因确实显著提高了紫花苜蓿的耐盐性。本研究为通过转基因技术培育紫花苜蓿耐盐新品种提供了新的材料基础。更多还原

【Abstract】 Alfalfa is one of the most important forage. As more and more saline-sodic soil grately reduced the yeild of Alfalfa, it is much more important to culture new varieties of Alfalfa with higher salt tolerance, while the transgenetic technique provides a quick and effective method to culture the new variety of Alfalfa.SsNHXI, a gene from suaeda salsa, encodes a Na+/H+ antiporter in vacuole membrane,it can pump Na+ into the vacuole to improve the salt tolerance of plants. Recent studies have shown that helicase also has key effects on stress tolerance. PDH45 is a DNA helicase from pea, which has been proved to be able to largely enhance the salt tolerance of transgenic tobacoo.In this study, the pBISsNHXI and pBIDH45 had been recombined to construct a bivalence expression vector with SsNHXI and PDH45 two genes.We transfered the two genes, SsNHXI and PDH45, into Alfalfa by Agrobacterium tumefaciems induced method to obtain a new variety of Alfalfa with higher salt tolerance. 80 kanymycin-resistant transgenic plants were obtained and PCR results proved that the interested gene had been integrated into the genome of Afalfa and then 20 transgenetic plants were selected to do further physiology test. After treated with 250mmol NaCl for 5 weeks, the growth of both nontransgenic and transgenic plants were suppressed, but the latter grew much better than nontransgenic wild type plants. Meanwhile, the experiment of measuring the percentage of the proline and chlorophyll and cell membrane permeability was carried out between the transgenic and nontransgenic wild type plants, and the results showed that the proline content of the transgenic alfalfa was 20 fold of the wild-type plants, the chlorophyll content was 40% higher than the wild plants and the degree of membrane permeability of transgenic Afalfa was 90% lower than the wild plants. Taken togather, these results indicated that over expression of SsNHXI and PDH45 indeed greatly improve the salt tolerance of Alfalfa。This study provided some basis for culturing new salt-resistant varieties of Alfalfa by transgenetic techinique.更多还原