一株水稻促生长内生真菌的绿色荧光蛋白基因标记与示踪

Labeling and Tracing of Green Fluorescent Protein in Fungal Endophyte with Growth-promoting Activity to Rice Seedlings

将绿色荧光蛋白基因(gfp)转入到碱蓬内生真菌JP4-1中并检测菌株在水稻幼苗中的定殖情况。采用PEG-Ca Cl2介导的原生质体转化方法将携带gfp基因的p CT74质粒与菌株基因组整合获得转化子,用转化子侵染水稻幼苗,荧光显微镜下示踪JP4-1菌株及其侵染特性。转化子经连续传代6次仍能发出绿色荧光且荧光强度良好,能够稳定遗传;经PCR验证gfp基因已成功转入JP4-1菌株和水稻幼苗植株内并表达。转化可获得稳定表达GFP的JP4-1转化子,JP4-1菌株可定殖于水稻幼苗的根、茎、叶,定殖位置为细胞间隙,其促生作用与野生型菌株无明显差别。

This work is to transform the gone of green fluorescem protein （ ffp ） into the fungal endophyte JP4-1 of Suaeda salsa, and to detect the colonization of the fungi in rice seedlings. The plasmid pCT74 with gfp was transformed into the protoplasts with PEG-CaCl2 mediated method and then integrated with the strain for transformants. Further, the transformants was used to infect rice seedlings, and finally the tracer to JP4-1 and its infection characteristics were detected with fluorescence microscope. Results showed that the transformants were quite stable with promising green fluorescence intensity after six successive subcuhures. PCR amplification showed that the gfp was successfully transformed into strain JP4-1 and was expressed stably in rice seedlings. In conclusion, the JP4-1 transformants with stably-expressed gfp was acquired via transformation, the JP4-1 was colonized in the roots, shoots and leaves of rice seedlings, the location of colonization was intercellular, and the growth-promoting activity showed no difference with the wild type.