花粉管通道法介导的铁皮石斛转基因技术

发布时间：2018-08-25 16:17

【摘要】：该研究以含有GFP和GUS基因的质粒和农杆菌为载体,采用花粉管通道法对铁皮石斛进行转基因技术研究。结果表明:(1)铁皮石斛种子萌发和原球茎生长对卡那霉素的最低致死浓度分别为90和150 mg·L~(-1)。进一步研究证实,在筛选转化种子和原球茎时,可分别向培养基中添加100和150 mg·L~(-1)的卡那霉素进行选择培养。(2)以携带GFP和GUS基因的质粒(pSuper1300和pBI121)和农杆菌为载体,用无菌去离子水重悬质粒pSuper1300和pBI121至浓度为100 ng·μL~(-1),用2%蔗糖+1/2MS+0.1%silwet-77+0.1%AS或5%蔗糖+0.1%silwet-77+0.1 mmol·L~(-1)AS重悬携带质粒pSuper1300和pBI121的农杆菌至菌液浓度为OD_(600)=0.7~0.8;在授粉后0.5~2.5 h内使用柱头滴加法导入携带外源基因的质粒或农杆菌溶液,收集成熟的转化种子,经选择培养及PCR检测发现,几乎所有处理的转化材料均能检测出外源GFP和GUS基因片段。另外,与农杆菌相比,以质粒为载体进行转化,可获得更高的结实率。该研究结果为铁皮石斛的基因工程育种提供了参考。
[Abstract]:In this study, the plasmid containing GFP and GUS genes and Agrobacterium tumefaciens were used as vectors to study the transgenic technology of Dendrobium candidum by pollen tube channel method. The results showed that: (1) the lowest lethal concentrations of kanamycin for seed germination and protocorm growth of Dendrobium candidum were 90 and 150 mg / L ~ (-1), respectively. Further studies confirmed that kanamycin containing 100 and 150 mg L ~ (-1) was added to the medium for selective culture when the transformed seeds and protocorms were screened. (2) plasmids carrying GFP and GUS genes (pSuper1300 and pBI121) and Agrobacterium tumefaciens were used as vectors. The concentration of Agrobacterium tumefaciens suspended with aseptic deionized water heavy suspension plasmids pSuper1300 and pBI121 was 100 ng 渭 L ~ (-1) with 2% sucrose 1/2MS 0.1%silwet-77 0.1%AS or 5% sucrose 0.1%silwet-77 0.1 mmol L ~ (-1) AS to carry plasmids pSuper1300 and pBI121. Introduction of a plasmid carrying a foreign gene or Agrobacterium tumefaciens solution, The mature transformed seeds were collected. After selective culture and PCR detection, it was found that almost all the transformed materials could detect exogenous GFP and GUS gene fragments. In addition, compared with Agrobacterium tumefaciens, plasmids were used as vectors to obtain higher seed setting rate. The results provide a reference for genetic engineering breeding of Dendrobium candidum.
【作者单位】：广西喀斯特植物保育与恢复生态学重点实验室广西壮族自治区中国科学院广西植物研究所;
【基金】：广西科技重大专项(桂科重1355001-3-1,桂科重14124002-6,桂科重14124002-7) 广西科技攻关计划项目(桂科攻1346008-5) 广西林业科技项目(桂林科字[2013]第15号)~~
【分类号】：Q943.2;S567.239

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