番茄中BR相关基因的遗传转化研究

【作者】 於维维；

【导师】 汪俏梅；

【作者基本信息】 浙江大学 ， 蔬菜学， 2005， 硕士

【摘要】 油菜素甾醇类化合物(BRs)是在植物的生长发育中起重要作用的一类植物激素。BR的主要生理作用是促进植物的生长和提高植物对环境逆境的抗性。目前BR信号转导途径已基本清楚,BR信号转导的主要组分包括膜受体BRI1、与哺乳动物糖元合成酶激酶3相似的BIN2激酶、核蛋白BZR1和BZR2/BES1等。最新研究表明BZR1在调节BR信号转导、BR生物合成和生长响应中发挥关键性作用。 本研究选用模式植物番茄作为遗传转化受体,引入BR信号转导重要组分基因——BZR1基因,以期为进一步分析BZR1在调节BR的信号转导途径、BR水平和BR诱导基因的表达中的功能以及探讨能否通过对BZR1的调控进行作物改良奠定基础。因此,我们建立了番茄离体培养植株再生体系,构建了BZR1基因植物表达载体,并将目的基因导入根癌农杆菌LBA4404菌株中,进行了番茄的遗传转化研究,获得了19株番茄Kan~R植株。主要研究结果如下: (1)通过不同的激素配比的筛选,提出了一个番茄离体培养再生频率较高的培养基配方(MS培养基+3%蔗糖+0.7%琼脂+2mg/L 6-BA+0.2mg/L IAA),子叶外植体的再生频率达89%,下胚轴外植体的再生频率达94%。 (2)检测鉴定了已构建的含BZR1基因的植物表达载体质粒35S-BZR1-CFP和含bzrl基因的植物表达载体质粒35S-bzrl-CFP,分别将它们导入农杆菌LBA4404菌株中。 (3)经过对番茄外植体Kan筛选浓度的确定、抑菌剂Amp的敏感性试验、预培养和共培养时间的筛选,建立了一个番茄遗传转化体系。具体操作为:将番茄子叶外植体在预培养基上培养2-3d后,用含目的基因的农杆菌LBA4404菌液侵染15min,在灭菌滤纸上吸干菌液,置于共培养基上培养3d,随后将外植体转入含Kan 20mg/L的选择分化培养基上诱导不定芽,2-3周转瓶一次,当抗性不定芽长至2-3cm时,在愈伤组织处切下幼苗,置于生根培养基上诱导不定根,20d左右后不定根长成,开瓶炼苗,然后移栽至营养土中,正常管理。 (4)按已建立的番茄遗传转化体系,获得了19株卡那霉素抗性苗,对全部的Kan~R转基因番茄植株进行了PCR检测,结果有16株含672bp标记基因(NPTⅡ基因)扩增产物。 目前,番茄转基因植株的进一步分子检测和田间鉴定及遗传特性等研究还在继续中。更多还原

【Abstract】 Brassinosteroid (BRs) are a class of steroid hormones that play important roles in plant growth and development. BR can promote plant growth and enhance the resistance of plants to environmental stresses. The picture of BR signalling is emerging in recent years. The main components of BR signal transduction include the cell-suface receptor kinase BRI1, the glycogen synthase kinase-3-like kinase BIN2, nuclear proteins BZR1 and BZR2/BES1. A recent study defines BZR1 as a central regulator for coordinating BR signalling, BR biosynthesis, and growth responses.In this study, we constructed an efficient tomato genetic transformation system, and transformed BR signal transduction gene (BZR1 gene) into tomato, which we hope to make a strong basis for further investigating the function of BZR1 in regulating BR signalling, the level of BR in plant, and expression of BR-induced genes, and therefore developing strategy for crop improvement by regulation of BZR1. The main results are as follow:(1) We have developed an efficient method for plant regeneration from tomato explant of cotyledon and hypocotyl. MS medium, supplemented 3% sucrose, 0.7% agar, 2mg/L 6-BA and 0.2mg/L IAA was the optimum medium in obtaining high frequency shoot regeneration in tomato. The frequency of shoot regeneration from cotyledon was 89%, while the frequency of shoot regeneration from hypocotyls reached as high as 94% by this means.(2) The plant expressing plasmid vectors 35S-BZR1-CFP and 35S-bzrl-CFP, which contain BZR1 and bzrl gene respectively were constructed, and introduced to Agrobacterium tumefaciens strain LBA4404 by triparental cross.(3) An efficient gene transformation system was established, and the optimum conditions for the transformation of BZR1 or bzrl were as follow: The explants were infected by Agrobacterium tumefaciens strain LBA4404 for 15 minutes, and cocultivated for 3 days after they were precultivated for 2-3 days, Then the explants were inoculated into the regeneration medium containing 20mg/L Kan to obtain the KanR shoot, and the explants were transferred to fresh medium at intervals of 2-3 weeks. The KanR shoots were cut off from the explants and placed in root-inducing medium when they grew to 2-3cm long. Roots were induced in the shoots after 20 days, and then the cover of the container were opened gradually within the first week, after that, the young transgenic plants were transferred to soil in larger pots, and grown in a greenhouse.(4) 19 KanR young plants were obtained in the tomato genetic transformation test. PCR analysis showed that 16 of the 19 plants contain the 672bp (NPT Ⅱ) amplification products.更多还原