用绿色荧光蛋白基因(gfp)标记产酸克雷伯氏菌SG-11研究其在水稻苗期根部的定殖  

Klebsiella oxytoca SG-11 Labeling by Green Fluorescent Protein Gene (gfp) and its Colonization in Rice Seedling Roots

作　　者：吕泽勋[1] 李久蒂[1] 朱至清[1] 

机构地区：[1]中国科学院植物研究所,北京100093

出　　处：《农业生物技术学报》2001年第1期13-18,共6页Journal of Agricultural Biotechnology

摘　　要：本研究以自水稻根面分离的产酸克雷伯氏菌 ＳＧ－１１为出发菌株，分别用携带ｇｆｐ和ｎｉｆＨ－ｇｆｐ的质粒转化 ＳＧ－１１进行基因标记，得到了转化子ＳＧ－１１Ａ和ＳＧ－１１Ｂ。对两转化子的生长曲线和质粒稳定性进行了测定，在ＬＢ培养基中，ＳＧ－１１的倍增时间为０．８１５ｈ，对数期是３．２０～５．２８ｈ，样品各点与其曲线的相关系数Ｒ２＝０．９９７５；ＳＧ－１１Ａ的倍增时间为１．０２ｈ，对数期是 ３．２７～５．３７ ｈ， Ｒ２＝０．９９８７，在无选择压力的条件下，连续传 ８０代时质粒的保存率为 ０％。在Ｋｐ培养基中， ＳＧ－１１的倍增时间为１．１５９ｈ，对数期是２．５０～４．９２ｈ，相关系数Ｒ２＝０．９９２２；ＳＧ－１１Ｂ的倍增时间为１．１６３ｈ，对数期是２．５０～４．９２ｈ，相关系数Ｒ２＝０．９６９８，连续转１００代时质粒的保存率为６５．６％。激光共聚焦显微镜观察结果表明：在试管限菌培养条件下，ＳＧ－１１Ａ主要定殖在水稻根部的伸长区和根毛区，并且在次生根形成处有菌团存在，在水稻根通气组织的细胞间隙和维管束导管内发现了ＳＧ－１１Ａ，在根的伸长区只是偶尔观察到了表达ｎｉｆＨ－ｇｆｐ的ＳＧ－１１Ｂ。砂培条件下，在水稻根伸长区观察?The Klebsiella oxytoca SG-11 isolated from rice rhizosphere was used to conduct microbial colonization in roots of rice seedlings, which were respectively transformed by plasmids pMGFP2. 1 carrying nifH-gfp and PKK223-GFP carrying gfp newly constricted. The transformants of SG-11A with pKK223-GFP and SG-11B with pMGFP2.1 were obtained. The detection of growth curve and plasmid persistence of SG-11, SG-11A in LB medium, and SG-11, SG-11B in Kp medium were established respectively. The results showed that the doubling time of SG-11 is 0.815 h, logarithmic phase is 3.20~5.28 h, coefficient of correlation is R2=0.9975; the doubling time of SG-11A is 1.02 h, logarithmic phase is 3.27~5.37 h, coefficient of correlation is R2=0.9987. The plasmid pKK223GFP in SG-11A was completely loosed after 80 generation of continuous cultivation. For SG-11in KP, the doubling time is 1.159 h, logarithmic phase is 2.50~4.92 h, coefficient of correlation is R2=0.9922; For SG-11B, the doubling time is 1 .163 h, logarithmic phase is 2.50~4.92 h, coefficient of correlation is R2=0.9698. The plasmid persistence rate in SG-11B is 65.6% after 100 generation of continuous cultivation. The researches of spatial distribution of SG-11A and SG-11B by laser scanning cofocal microscope revealed that SG-11A was predominantly localized in the zones of elongation and hairy formation, sites of lateral formation with larger cell aggregates and the zones of elongation with less SG-11B gnotobiotically. Much interestedly, the SG-11A was visualized in epidermal cells, intercellular layers and vascular system of roots. When the rice seedlings were grown in sterilized sand, only SG-11A was found in the zone of elongation. Nether SG-11A nor SG-11B was detected under field soil-grown rice seedlings.

关 键 词：产酸克雷伯氏菌SG-11 绿色荧光蛋白 激光共聚焦显微镜 水稻 苗期 根部 定殖 

分 类 号：S511.01[农业科学—作物学]