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林业科学 ›› 2007, Vol. 43 ›› Issue (03): 46-50.doi: 10.11707/j.1001-7488.20070308

• 论文及研究报告 • 上一篇    下一篇

不同因素对农杆菌介导的杉木转化效率的影响

席梦利 施季森   

  1. 南京林业大学国家林业局林木遗传和基因工程重点实验室,南京210037
  • 收稿日期:2006-01-10 修回日期:1900-01-01 出版日期:2007-03-25 发布日期:2007-03-25

An Assessment of Factors Influencing the Efficiency of Transformation of Cunninghamia lanceolata Mediated by Agrobacterium tumefaciens

Xi Mengli,Shi Jisen   

  1. Key Laboratory of Forest Tree Genetics and Gene Engineering,State Forestry Administration Nanjing Forestry University Nanjing 210037
  • Received:2006-01-10 Revised:1900-01-01 Online:2007-03-25 Published:2007-03-25

摘要:

以杉木试管苗靠近茎尖的茎段为受体材料,研究6个影响杉木遗传转化效果的因素,初步确定杉木茎段较为合适的转化体系为预培养1~3d,OD600nm为0.1~0.4的菌液浸染10~15min,共培养3~5d,共培养基附加乙酰丁香酮(AS)80μmol·L-1,共培养后延迟3d筛选。在初次筛选培养基上共得到186个卡那霉素(Km)抗性芽,在二次筛选培养基上获得39个Km抗性芽。PCR检测有2个Km抗性芽呈稳定阳性,初步证明外源天麻抗真菌蛋白(GAFP)基因已整合到这2个Km抗性芽的基因组DNA中。

关键词: 杉木, 农杆菌介导, 天麻抗真菌蛋白基因

Abstract:

Using plantlet shoot sections near the shoot tip as explants, after studying the six factors to affect transformation, we have preliminarily determined an effective system for genetic transformation. The protocol was: 1~3 days for pre-culture, OD600 nm =0.1~0.4, 10~15 minutes for infection, 3~5 days for co-culture, and acetosyringone (AS) 80 μmol·L-1 as co-culture medium, and then lasting 3 days before selecting. And then 186 regenerated kanamycin(Km)-resistant buds were obtained from the first selection medium. 39 Km-resistant buds were obtained from the second selection medium. PCR analysis on 39 regenerated Km-resistant buds showed that 2 buds were PCR positive. The result indicated that gastrodia antifungal protein (GAFP) gene was integrated into the genome of the two Km-resistant buds.

Key words: Cunninghamia lanceolata, Agrobacterium tumefaciens mediated transformation, gastrodia antifungal protein gene