GFP标记短小芽孢杆菌LYMC-3在马尾松体内的定殖

Colonization of GFP-tagged Bacillus pumilus strain LYMC-3 in masson pine

为研究松材线虫拮抗细菌短小芽孢杆菌LYMC-3的内生性及其在马尾松体内的定殖规律,采用扫描电子显微镜(SEM)观察菌株在马尾松组培苗体内的定殖,并采用高渗透法将质粒pGFP78转入菌株LYMC-3,对其进行绿色荧光蛋白(GFP)标记,同时测定标记菌株的遗传稳定性。在此基础上,以GFP标记和抗性标记作为示踪手段,将标记菌株接种到2年生马尾松盆栽实生苗的根部,借助激光扫描共聚焦显微镜(LSCM)和稀释涂板的方法,对马尾松根部、茎部的标记菌株进行定期回收检测。结果显示,通过SEM成功观察到菌株LYMC-3在马尾松体内的定殖,并成功获得LYMC-3菌株荧光表达强烈且遗传稳定的转化子,经过连续8次的稀释培养,其遗传稳定性为96.8%。GFP标记菌株在接种马尾松根部后的第4天,根部、茎部都能回收到大量标记菌株的存在,随后呈持续下降的趋势,且根部的下降速度快于茎部。接种40d后,根部回收标记菌株数量为0.3×102cfu/g,茎部回收标记菌株数量为0.8×102cfu/g。以上结果表明短小芽孢杆菌LYMC-3具有内生性,能在马尾松体内良好地定殖和传导。

SEM （scanning electronic microscopy） ,LSCM （laser scanning confocal microscope） and GFP （green fluorescent protein） techniques were used to study the endogeneity and colonization rules of Bacillus pumilus strain LYMC-3 ,which has high nematicidal activity.The shuttle vector pGFP78 carrying gfpmutea gene,was transformed into B. pumilus strain LYMC-3 for fluorescence-tagging by nature transformation and expressed successfully.The plasmid stability was then determined. After inoculating the bacteria in root surface of masson pine,the colonization of LYMC-3 tagged with GFP was studied by combining antibiotics plate recovery and LSCM.GFP-tagged T.YMC-3 with bright green fluorescence was observed under 481 nm wavelength and the genetic stability was 96.8% after 8 continuous dilution culture. On 4th day after inoculation in masson pine root,a large number of labeled strains were recovered, followed by a downward trend,and the number decreased faster in the root than in the stem.On 40th day after inoculation,the number of the labeled strain was 0.3×10^2cfu/g in the root,and 0.8×10^2cfu/g in the stem.These results demonstrated that the tagged strain could successfully colonize and transmit in masson pine,which provide a scientific basis for the exploitation and utilization of strain LYMC-3.