枸杞内生真菌NQ8GⅡ4菌株GFP标签蛋白表达载体的筛选  

Expression vector screening for GFP-labeled endophytic fungus Fusarium nematophilum NQ8GⅡ4 from Lycium barbarum

作　　者：闫思远 任苗苗 杜娟 李金 杨富龙 李嘉泓 顾沛雯[1] YAN Siyuan;REN Miaomiao;DU Juan;LI Jin;YANG Fulong;LI Jiahong;GU Peiwen(Agricultural College,Ningxia University,Yinchuan,Ningxia 750021,China)

机构地区：[1]宁夏大学农学院,宁夏银川750021

出　　处：《西北农林科技大学学报（自然科学版）》2021年第4期123-132,共10页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家自然科学基金项目(31460484)。

摘　　要：[目的]筛选适合枸杞内生真菌(Fusarium nematophilum)NQ8GⅡ4菌株GFP标签蛋白的表达载体,为后期研究该菌株在宿主植物中的侵染行为和定殖规律奠定基础。[方法]采用PEG介导原生质体转化法,分别将GFP标签蛋白表达载体pFL2、pDL2、r-KNT和KNTG转入NQ8GⅡ4菌株,对比4种NQ8GⅡ4转化子的荧光强度、遗传稳定性、拮抗活性和致病性,筛选GFP标签蛋白的最佳表达载体。[结果]枸杞内生真菌NQ8GⅡ4菌株对潮霉素B(HmB)的耐受质量浓度为20 mg/L,对遗传霉素(G418)的耐受质量浓度为80 mg/L。通过PEG介导原生质体转化,获得pFL2转化子272株、pDL2转化子57株、r-KNT转化子73株和KNTG转化子226株,其转化效率分别为13.60,2.85,3.65和11.30株/μg。荧光显微观察发现,表达载体pDL2和KNTG的转化子所产生的荧光强度明显强于表达载体r-KNT和pFL2的转化子。连续培养5代后,pDL2转化子的遗传稳定率高达89.47%,pFL2、r-KNT和KNTG转化子的遗传稳定率分别仅为67.28%,68.49%和58.85%。4种转化子对枸杞胶孢炭疽菌(Colletotrichum gloeosporioides)的拮抗活性以及对枸杞离体叶片的致病性均与野生型菌株无明显差异。[结论]pDL2更适合用作NQ8GⅡ4菌株GFP标签蛋白的表达载体。[Objective] This study screened expression vector for GFP-labeled endophytic fungus Fusarium nematophilum NQ8 GⅡ4 from Lycium barbarum to provide basis for understanding determination dynamics and distribution of NQ8 GⅡ4.[Method] The expression vectors pFL2,pDL2,r-KNT and KNTG transformed by PEG-mediated protoplast transformation were transformed into protoplast of NQ8 GⅡ4.The best expression vector was obtained by comparing fluorescence observation, mitotic stability, antagonistic role against Colletotrichum gloeosporioides and pathogenicity on L. barbarum leaves of the four NQ8 GⅡ4 transformants.[Result] The tolerable concentration of NQ8 GⅡ4 to hygromycin B was 20 mg/L,and that to G418 was 80 mg/L.The expression vectors pFL2,pDL2,r-KNT and KNTG were successfully transformed into the protoplast of NQ8 GⅡ4.A total of 272 pFL2 transformants, 57 pDL2 transformants, 73 r-KNT transformants and 226 KNTG transformants were obtained, and the transformation frequencies of NQ8 GⅡ4 were 13.60,2.85,3.65 and 11.30 transformants per microgram.Under fluorescence microscopy, the pDL2 and KNTG transformants could produce stronger fluorescence than pFL2 and r-KNT transformants.After five generations, 89.47% pDL2 transformants were mitotically stable, while the ratios for pFL2 transformants, r-KNT transformants and KNTG transformants were 67.28%,68.49% and 58.85%.There were no significant differences in antagonistic activity and pathogenicity among the four transformants and wild strain.[Conclusion] The expression vector pDL2 was more suitable for GFP-labeled NQ8 GⅡ4 strain.

关 键 词：内生真菌 NQ8GⅡ4菌株 PEG介导原生质体转化 表达载体 枸杞 

分 类 号：S567.1[农业科学—中草药栽培]