用gfp基因标记法研究金龟子绿僵菌在玉米根际的定殖动态

利用草胺磷抗性基因bar为筛选标记,将绿色荧光蛋白基因（gfp）转入载体pbarGPE1,得到了组成性表达载体pGPE-GFP,并转入金龟子绿僵菌RCEF3377,获成功表达。用含有gfp基因的金龟子绿僵菌孢子粉配制种衣剂,对玉米种子包衣。于玉米苗期对根际定殖情况进行定期取样观测。结果表明,与非根际土壤相比,在根际土壤中被标记的绿僵菌带菌量不仅最大,而且在播种后10d数量达最大,20～30d内数量即不再显著变动。相比之下,非根际土壤中的带菌量不仅极显著少于根际土壤,且随时间的延长明显降低;在表层土中的带菌量很低,亦随时间的延长显著降低。表明应用该种衣剂剂型,金龟子绿僵菌孢子在玉米根际有较高活力和定殖效果。

Colonization of Metarhizium anisopliae in Corn Rhizosphere as Studied with gfp Marker Gene

A plasmid,pGPE-GFP,carrying green fluorescent protein gene（gfp） and phosphinothricin resistance gene bar was constructed and transformed into Metarhizium anisopliae.The transformants grew normally and GFP fluorescence was detected.Corn seeds were coated with a seed-coating preparation of M.anisopliae containing gfp and were sowed.At seedling stage,soil samples were regularly taken from rhizosphere,non-rhizosphere and soil surface for detection and monitoring of the transformant.Results showed that the inoculum of the gfp-marked M.anisopliae was not only the highest in the rhizosphere soil,but also did not fluctuate significantly from the 20th to the 30th day.Comparatively,the inoculum in the non-rhizosphere soil was not only lower than in the rhizosphere soil,but also declined significantly with time.In surface soil,the inoculum was the lowest and also declined significantly.All the evidences suggested that the conidia of M.anisopliae from the seed-coating agent were very viable with good colonization effect in rhizosphere soil of corn,demonstrating that the seed-coating agent is of potential against root insect pests.