花粉管通道法介导的铁皮石斛转基因技术

Transgene by pollen-tube pathway of Dendrobium officinale

关键词:  铁皮石斛, 花粉管通道法, 质粒, 农杆菌, 转基因

Key words:  Dendrobium officinale, pollen-tube pathway, plasmid, agrobacterium, transgene

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OD₆₀₀=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:

The transgenic technology of Dendrobium officinale was studied by using pollen-tube pathway with plasmid and agrobacterium vectors as target genes delivery. The main results were summarized as follows:(1)The minimum lethal concentration of kanamycin to seed germination and protocorm growth were 90 and 150 mg·L^-1 respectively. Further study showed that 100 and 150 mg·L^-1 kanamycin could be added for selecting transgenic seeds and protocorms during selective culture in vitro respectively.(2)Plasmid and agrobacterium contained GFP or GUS gene was transferred into D. officinale via pollen-tube pathway. The technology and method were summarized as follows: Collected target plasmid(pSuper1300 and pBI121)and agrobacterium which contained GFP or GUS gene; resuspended the plasmid pSuper1300 and pBI121 to concentration of 100 ng·μL^-1 with thrice distilled water, while resuspended the agrobacteria which carried plasmid pSuper1300 and pBI121 to OD₆₀₀=0.7-0.8 with the solution of 2% sucrose+1/2MS+0.1% silwet-77+0.1% AS or 5% sucrose+0.1% silwet-77+0.1 mmol·L^-1 AS respectively; during 0.5-2.5 h after artificial pollination, used stigma dripping method to transfer into plasmids and agrobacteria which contained target genes; collect the mature seeds; selective culture and PCR certificated that the resistant materials of almost all treatments were integrated GFP and GUS genes. Moreover, compared with agrobacterium as target gene delivery, the plasmid gene delivery could harvest more fruits.