| 引用本文: | 冼康华, 付传明, 何金祥, 龚庆芳, 苏 江, 黄宁珍.花粉管通道法介导的铁皮石斛转基因技术[J].广西植物,2017,37(9):1101-1110.[点击复制] |
| XIAN Kang-Hua, FU Chuan-Ming, HE Jin-Xiang, GONG Qing-Fang,
SU Jiang, HUANG Ning-Zhen.Transgene by pollen-tube pathway of Dendrobium officinale[J].Guihaia,2017,37(9):1101-1110.[点击复制] |
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| 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。 |
| 关键词: 铁皮石斛, 花粉管通道法, 质粒, 农杆菌, 转基因 |
| DOI：10.11931/guihaia.gxzw201608034 |
| 分类号:Q943.2, Q785 |
| 文章编号:1000-3142(2017)09-1101-10 |
| 基金项目: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)[Supported by Key Special Fund of Science and Technology of Guangxi(1355001-3-1,14124002-6,14124002-7); Key Program of Science and Technology of Guangxi(1346008-5); Program of Forestry Science and Technology of Guangxi([2013]No. 15)]。 |
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| Transgene by pollen-tube pathway of Dendrobium officinale |
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XIAN Kang-Hua, FU Chuan-Ming, HE Jin-Xiang, GONG Qing-Fang,
SU Jiang, HUANG Ning-Zhen*
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Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany,
Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, Guangxi, China
Guangxi Key Laboratory of Plant Conservation and Restoration Ecology in Karst Terrain, Guangxi Institute of Botany,
Guangxi Zhuang Autonomous Region and Chinese Academy of Sciences, Guilin 541006, Guangxi, China
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| 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. |
| Key words: Dendrobium officinale, pollen-tube pathway, plasmid, agrobacterium, transgene |
