CAJ | 학술논문

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0.815 h,对数期是3.20～5.28 h, 样品各点与其曲线的相关系数R2=0.9975; SG-11A的倍增时间为1.02 h,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1.159 h,对数期是2.50～4.92 h,相关系数R2=0.9922;SG-11B的倍增时间为1.163 h,对数期是2.50～4.92 h,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到了少量SG-11A的定殖，未检测到SG-11B。稻田土盆栽实验中，在水稻的根部既未检测到SG-11A也未检测到SG-11B。
본연구이자수도근면분리적산산극뢰백씨균SG-11위출발균주,분별용휴대gfp화nifH-gfp적질립전화SG-11진행기인표기，득도료전화자SG-11A화SG-11B。대량전화자적생장곡선화질립은정성진행료측정，재LB배양기중, SG-11적배증시간위0.815 h,대수기시3.20～5.28 h, 양품각점여기곡선적상관계수R2=0.9975; SG-11A적배증시간위1.02 h,대수기시3.27～5.37 h,R2=0.9987,재무선택압력적조건하,련속전80대시질립적보존솔위0%。재Kp배양기중, SG-11적배증시간위1.159 h,대수기시2.50～4.92 h,상관계수R2=0.9922;SG-11B적배증시간위1.163 h,대수기시2.50～4.92 h,상관계수R2=0.9698,련속전100대시질립적보존솔위65.6%。격광공취초현미경관찰결과표명：재시관한균배양조건하,SG-11A주요정식재수도근부적신장구화근모구,병차재차생근형성처유균단존재,재수도근통기조직적세포간극화유관속도관내발현료SG-11A,재근적신장구지시우이관찰도료표체nifH-gfp적SG-11B。사배조건하，재수도근신장구관찰도료소량SG-11A적정식，미검측도SG-11B。도전토분재실험중，재수도적근부기미검측도SG-11A야미검측도SG-11B。
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 constructed.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 pKK223-GFP 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.