番茄遗传转化筛选及其子代选育体系的优化研究  

Optimization of tomato genetic transformation, kanamycin-resistant screening and seed selection

作　　者：应彩云[1] 黄小琴[1] 郭瑜琪[1] 钟莉莉[1] 刘艳[1] 黎事伦[1] 顾晓敏[1] 周晓红[1] 

机构地区：[1]南方医科大学公共卫生与热带医学学院病原生物学系,广东广州510515

出　　处：《南方医科大学学报》2008年第7期1117-1122,共6页Journal of Southern Medical University

基　　金：国家自然科学基金(30080024、30400367);广东省自然科学基金(001052、5004747);广东省科技计划项目(2003c104041、73007)~~

摘　　要：目的以弓形虫多表位抗原基因(MAG)和截短的速殖子主要表面抗原1(tSAG1)为插入子建立优化的番茄遗传转化筛选及其子代选育体系。方法从培养基激素配方、番茄外植体选择等方面进行番茄再生体系的优化。自番茄品种、外植体选材、培养温度、培养基激素配方以及在共培养时是否加入乙酰丁香酮(AS)等进行以农杆菌介导的番茄基因组稳定转化体系的优化。采用不同浓度卡那霉素抗性压力对转基因小苗进行根筛选诱导的比对。转基因番茄幼苗练苗移栽开花结果后收集种子,以无菌播种卡那霉素抗性筛选法进行番茄转基因植株子代的选育。结果子叶较下胚轴具有更强的再生能力,在优化的番茄再生培养基ZB3中,番茄子叶可达到98%(59/60)的高频再生芽萌发率。番茄的转化体系优化,培养基激素配方、培养温度是番茄转化芽诱导过程中的关键影响因素,(23±1)℃能显著提高转化芽的萌发率,AS的施加亦能显著提高番茄转化芽萌发率,Zhongshu No.5番茄子叶于ZB2培养基和(23±1)℃的条件下转化芽萌发率为35%(28/81)。80mg/L卡那霉素抗性进行HK种番茄转化小苗根的筛选诱导比较适宜,生根率为48%(31/65)。抗性苗移栽成活,正常开花结果的转化植株117株。无菌播种,卡那霉素浓度≥100mg/L时能显著抑制非转基因番茄种子的发育尤其是根的萌发与植株的伸长,根出现紫化且无侧根生长;将转基因番茄种子置于150mg/L卡那霉素抗性浓度下培养,能有效进行转基因植株后代遗传分离的选育。结论成功建立番茄高频再生体系以及优化的番茄转化、抗性筛选及其转基因植株子代的选育体系。Objective To optimize the process of tomato genetic transformation, screening and seed selection using multiepitope antigenic gene （MAG） and tnmcated major surface antigen 1 （tSAG1） of Toxoplasma gondii as the target insert genes. Methods Tomato high-frenquency regeneration system was optimized with different choices of media and explants. The genetic transformation procedure was optimized using different tomato cultivars, explants, culture temperatures, media and acetosyringone （AS） supplements. Three concentrations ofkanamycin were utilized for resistant selection of the transgenic candidate roots. The selected lines were trained, transplanted to soil and grown in a greenhouse till maturity. Sterile seeding using kanamycin-incorporated medium was conducted for screening transgenic tomato generations. Results and Conclusion Cotyledons were better than hypocotyls as the regeneration explants. The regeneration rate of cotyledons reached 98%（59/60） using the optimized regeneration medium ZB3. The culture medium and temperature were the key factors for tomato transgenic shoot induction. The number oftransgenic buds increased significantly at the appropriate temperature condition （23±1）℃, and AS of 100 μmol/L in the medium before inoculation also significantly raised transformation rate. The budding rate of Zhongshu No.5 cotyledons was 35%（28/81 ）using the medium ZB2 under （23±1）℃. Kanamycin at 80 mg/L was optimal for transgenic plantlet rooting selection with the rooting rate of 48% （31/65）. 117 transgenic lines were obtained. Non-transgenic tomato plant growth, especially the root and elongation, was inhibited obviously with kanamycin at 100 mg/L or above, and the roots became purple and lacked lateral roots. The transgenic tomato seeds could be selected effectively with kanamycin at 150 mg/L.

关 键 词：转基因番茄 弓形虫 遗传转化 

分 类 号：Q943.2[生物学—植物学]