导　　师： 裴炎

学科专业： G1010

授予学位： 硕士

作　　者： ;

机构地区： 西南农业大学

摘　　要： 紫花苜蓿(medicago sativa l.)是一种温带地区重要的豆科牧草,被誉为'牧草之王'.在全世界约有3500万公顷的种植面积.其蛋白质和必需氨基酸在单位面积上的产量几乎比任何一种作物都要高.由于它有很高的饲用价值,人们试图将其从温带冷凉的北方地区引种到中国南方.在中国黄河流域和西北地区是最适合苜蓿种植和生产区域,而长江流域及以南等广大地区由于存在温热多雨和酸性土壤等不适于苜蓿生长的环境,紫花苜蓿分布较少.酸性土壤最主要的问题是铝毒害问题,因而提高紫花苜蓿的耐铝性是解决紫花苜蓿顺利引种到中国南方的关键之一.该实验就是要通过基因工程手段提高植物的耐铝性.建立了紫花苜蓿高效的遗传转化体系,并将棉花铝诱导蛋白ghalin基因转入紫花苜蓿,以提高其耐铝性,为将紫花苜蓿顺利引种到中国南方做有益的探索.主要的研究结果如下:1、建立了紫花苜蓿高频再生体系;2、建立了紫花苜蓿高效的遗传转化体系;3、将耐铝相关基因转入紫花苜蓿,并进行了转基因植株的分子鉴定;4、进行了转基因植株的实验,验证了所转耐铝基因的功能. Alfalfa /(Medicago saliva L/), known as 'king of forage', is a highly valued forage legume that is cultivated on more than 35million hectares in the warm temperate and cool subtropical regions all over the world. Alfalfa most likely produces a greater yield of protein and essential amino acid per hectare than any other crop. The most fining area to the growth of Alfalfa in china'is the Yellow River regions and northwest of china. Alfalfa is not adapted to south of china because of the rainy weather and acidic soil. Many people want to migrate Alfalfa to south of china from the cool subtropical region, because of its important feeding values to animals. Aluminum toxicity is the main problem in acidic soil. So it is very important for Alfalfa to improve its aluminum tolerance ability in order that it can grow better in south of china. It has been achieved in aboard to improve plant aluminum tolerance ability by gene engineering technique. In this experiment, we want to improve Alfalfa aluminum tolerance ability via introducing aluminum simulated protein gene into Alfalfa. First we established a highly effective regeneration and transformation system and introduced one gene associated to aluminum resistant to improve the Alfalfa aluminum tolerance ability, which will be benefit to immigrate Alfalfa to south of china. The main results are as follows: 1 .Establishing the highly effective regeneration system The highly regenerative genotype was screened from the four Alfalfa varieties, SHEGNSH1804, CUF101. BADDING and WL52540. The effective regeneration protocol was established. BAODING has the highest regenerative ability of the four varieties. The cotyledon of BAODING was incubated on UM media to induce embryogenic callus, and then the embrogenic callus were transferred to UM liquid media to suspend 1-2 weeks, embryogenic callus suspension were transferred to SHE embryo developmental medium, the mature embryoies were selected and incubated on the 1//2SHGN medium to recover into plantlet. The whole regeneration progress can be achieved within 3-4 months. 2.Establishing the highly effective transformation system. The genotypes apt to transformation were screened from the six Alfalfa varieties. BAODING, CLIF 101, WL52540 are the genotypes apt to transformation. The factors affecting transformation rate, including the genotype of explant, the state of the explant, the state of the Agrobacterium tumefaciens , co-cultural time, were investigated using GUS transient expression technique. In this way, the Agrobacterium-mediated transformation system was established taken leaves as acceptor via vacuum infiltration. And a highly effective transformation system was also established taken embryogenic callus as acceptor. The transient transformation rates can be achieved almost 50/%, via transforming embryogenic callus suspension mediated by Agrobacterium tumefaciens. The transgenic plant can be obtained for about four month. 3.Introducing one aluminum tolerance associated gene into Alfalfa and doing molecular analysis to the transgenic plant The GhAlin gene, which was cloned from cotton, was introduced into Alfalfa mediated by Agrobacterium tumefaciens. The leaves and roots of transgenic plant can be stained into blue employing GUS historical stain technique. The same size fragment of interested gene can be emplicated from the transgenic plant genome by PCR analysis, which shows the target gene has transferred into plant genome. 4.Doing the aluminum tolerance experiment to analyze the role of the gene The two-week-old cuttings were placed in hydroponic chamber filled with 3L of 0.5 mM CaCl2 in deionized water, pH4.3. After the roots reached 3~5cm, the solution was replaced with 3L of preconditioning solution containing 0, 25, 50, 100 umol//L AlCl3 and 0.5 mM CaCl2, at pH.4.3. Root length was measured before and after 1d and 7d of AICl3 treatment, respectively. We used the relative root growth as a parameter to elicit the aluminum tolerance ability of transgenic plant. It shows the transgenic plant roots treated with

关 键 词： 紫花苜蓿 棉花铝诱导基因 遗传转化 耐铝

分 类 号： [S51.7.035]

领　　域： [农业科学]