尖孢镰刀菌Six1d启动子的克隆及其在真菌分泌表达载体构建中的应用

Cloning of Six1d Promoter of Fusarium oxysporum and Its Application in the Construction of Fungal Secretory Expression Vector

前期研究中构建的真菌分泌表达载体p74HSP-EGFP可在真菌中表达目标蛋白,在真菌侵染并定殖植物的过程中分泌目标蛋白到植物中,从而验证目的基因的功能,但p74HSP-EGFP载体中驱动目的基因表达的毒素基因(toxin A,ToxA)启动子不是尖孢镰刀菌古巴专化型(Fusarium oxysporum f. sp. cubense, Foc)自身的启动子。为能在Foc中更有效地表达其自身基因,本研究从尖孢镰刀菌1号生理小种(Foc1)中克隆到高丰度表达的木质部分泌蛋白基因(secreted in xylem 1d, Six1d)的启动子,来替换p74HSP-EGFP载体中的ToxA启动子,并将新构建的真菌分泌表达载体命名为pSix1d74HSPG。为比较2个分泌载体的表达效率,本研究进一步将p74HSP-EGFP和pSix1d74HSPG载体转化尖孢镰刀菌热带4号生理小种(Foc TR4),获得的转化菌株分别在钾盐液体培养基(KK)中振荡培养5 d后,检测分泌到胞外的EGFP荧光强度和EGFP蛋白的表达量。结果显示,pSix1d74HSPG中Six1d启动子驱动的EGFP表达强度能够达到p74HSP-EGFP中ToxA启动子驱动的1.6~1.7倍,说明pSix1d74HSPG载体可代替p74HSP-EGFP载体在Foc中更高效分泌表达目的蛋白,为香蕉枯萎病抗感病分子机理研究提供重要的实验工具。

The fungal secretory expression vector p74HSP-EGFP constructed in a previous study can express the target gene in fungal pathogens and secrete the target protein into plants during fungal infection and colonization,thus verify-ing the function of the target gene.However,the promoter of toxin A gene(ToxA)driving the target gene in p74HSP-EGFP is not a endogenous promoter of Fusarium oxysporum f.sp.cubense(Foc).For more efficient expression of target genes in Foc,the promoter of secreted in xylem 1d(Six1d),which is highly expressed in Foc,was cloned using the genomic DNA of Foc race 1(Foc1)as a template,replacing the ToxA promoter in p74HSP-EGFP,and the newly constructed fungal secretory vector was named pSix1d74HSPG.Foc tropical race 4(Foc TR4)was transformed by p74HSP-EGFP and pSix1d74HSPG.The transformed strains were cultured in potassium salt liquid medium(KK)con-taining 100μg/ml hygromycin for 5 days,respectively.The fluorescence intensity and the abundance of EGFP protein secreted outside the cell were detected. The results showed that the expression intensity of EGFP driven by Six1d pro-moter in pSix1d74HSPG was 1.6-1.7 times of that driven by ToxA promoter in p74HSP-EGFP, which could replace the p74HSP-EGFP vector for more efficient expression and secretion of target proteins in Foc for functional characteriza-tion. Our work provides an important experimental tool for the study of the molecular mechanism of resistance and susceptibility to banana fusarium wilt.