昆虫抗冻蛋白基因Mpafp149对棉花内生菌的转化及转化菌回接棉花的耐寒性初探

Transformation of Insect Antifreeze Protein Gene Mpafp149 to an Endophyte of Cotton and Cold Tolerance of Cotton Carrying the Transformed Endophyte

【目的】将转化了小胸鳖甲抗冻蛋白基因的棉花内生菌回接棉花,检测转化菌在棉花的定殖及棉花的耐寒性,探索利用内生菌携带外源抗冻基因快速有效提高棉花耐寒性的方法。【方法】利用穿梭载体PBE2构建抗冻蛋白-绿色荧光蛋白转基因质粒;将重组质粒电转至内生菌,然后将转化菌回接棉花进行荧光检测;低温-1℃处理棉花后测定叶片电导率。【结果】重组质粒pBE2-Mpafp149-gfp转化内生菌M17可显著提高其抗冻性。转化的内生菌回接棉花后,在-1℃处理14 h的叶片相对电导率显著低于对照棉花,表明抗冻蛋白对棉花有一定的抗冻保护作用。荧光检测结果表明绿色荧光蛋白在转化菌回接组和对照组无明显差异。【结论】通过回接转化了外源抗冻基因的内生菌能够使棉花快速获得一定的耐寒性。植物组织自发荧光现象使通过绿色荧光蛋白检测转化菌回接情况的效果不明显。

[ Objective] This study aimed to explore a rapid and effective method for improving cold tolerance of cotton plant by inoculating the endophyte of cotton that was transformed by an insect antifreeze protein gene Mpafp149 from the desert beetle Microdera punetipennis. [ Method] Recombinant plasmid expressing antifreeze protein and greer, fluorescence fusion protein was constructed based on shuttle plasmid pBE2; The recombinant plasmid was electronically transformed into endophyte M17, and then the transformed endophyte was inoculated into cotton seedling; cotton seedlings with and without transformed endophyte inoculation were subject to fluorescence observation and to cold treatment at - 1 ℃ for a period of time. The relative conductivity of cotton leaves ,was determined after 14 h at - 1℃. [ Result] The plasmid pBE2 - Mpafp149 -gfp is transformed into Bacillus subtilis M17 successfully and the transformed endophytes showed increased freeze tolerance. The cotton ：seedlings carrying the transformed M17 showed significant lower relative conductivity compared with the cont：rol cotton, indicating that the antifreeze protein expressed by the transformed endophyte exerts certain level of cryoprotective effect, although there was no obvious difference in the fluorescent intensity between the inoculated and the control cotton. [ Conclusion ] The cold tolerance of cotton plant can be improved rapidly by inoculating endophyte carrying an insect antifreeze protein gene. The localization of the inoculated enophyte in cotton was not distinguishable by observing the green fluorescence due to the endogenous fluoresce of cotton plant.