哈茨木霉T2-16的GFP标记及其生防特性
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摘要
优化高效拮抗生防菌哈茨木霉T2-16的转化条件,筛选出与野生型菌株具有相似生防特性的阳性转化子,为生防木霉菌T2-16的定殖动态、分布规律等研究打下基础。通过PCR和分子克隆技术构建具有G418抗性基因的绿色荧光(GFP)表达载体pKN-sGFP,利用PEG-CaCl_2介导的原生质体转化法,获得强荧光表达的哈茨木霉T2-16转化子,并将其与野生型菌株的生物特性进行比较,筛选出与野生型菌株具有相似生防特性的阳性转化子。试验结果显示,哈茨木霉T2-16在20℃培养条件下对1000μg/mL G418敏感,在上述优化条件下,转化获得稳定遗传的阳性转化子TG2-10;进一步比较其与野生型菌株的生物特性发现,两者之间无明显差异,可用于下一步哈茨木霉T2-16生防机理的研究。
Abtract: Optimizing the transformation conditions of highly antagonistic Trichoderma harzianum T2-16 and screening the positive transformants with similar biocontrol characteristics to wild-type strain will lay a foundation for determination of colonization dynamics and distribution of T. harzianum T2-16. In this study, the green fluorescence protein(GFP) expression vector with G418 resistance gene, named pKN-sGFP, was constructed by PCR and molecular cloning technology. T. harzianum T2-16 transformant with high fluorescent expression level was obtained by PEG-CaCl_2-mediated protoplast transformation, and a positive transformant with similar biocontrol properties to wild-type strain was screened out. T. harzianum T2-16 strain was sensitive to 1000 μg/mL G418 at 20 ℃. Under the above optimized conditions, the stable genetic positive transformant TG2-10 was obtained and compared with the wild-type strain. There was no significant difference in biocontrol characteristics between the two strains. Thus, the transformant TG2-10 could be used to further study the biocontrol mechanism of T. harzianum T2-16 in the future.
Abtract: Optimizing the transformation conditions of highly antagonistic Trichoderma harzianum T2-16 and screening the positive transformants with similar biocontrol characteristics to wild-type strain will lay a foundation for determination of colonization dynamics and distribution of T. harzianum T2-16. In this study, the green fluorescence protein(GFP) expression vector with G418 resistance gene, named pKN-sGFP, was constructed by PCR and molecular cloning technology. T. harzianum T2-16 transformant with high fluorescent expression level was obtained by PEG-CaCl_2-mediated protoplast transformation, and a positive transformant with similar biocontrol properties to wild-type strain was screened out. T. harzianum T2-16 strain was sensitive to 1000 μg/mL G418 at 20 ℃. Under the above optimized conditions, the stable genetic positive transformant TG2-10 was obtained and compared with the wild-type strain. There was no significant difference in biocontrol characteristics between the two strains. Thus, the transformant TG2-10 could be used to further study the biocontrol mechanism of T. harzianum T2-16 in the future.
引文
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[30]Mukherjee P K,Latha J,Hadar R,et al.Role of two G-protein alpha subunits,Tga A and Tga B,in the antagonism of plant pathogens by Trichoderma virens[J].Applied and Environmental Microbiology,2004,70(1):542-549.
[2]夏海,吴琼,陆志翔,等.有效霉素A对棘孢木霉的影响及协同防治玉米纹枯病作用[J].微生物学通报,2018,45(1):1-10.
[3]程东美,张志祥,区丽文,等.哈茨木霉T2菌株耐药性的测定及其对几种病原菌的抑制作用研究[J].安徽农业科学,2008,36(10):4170-4172.
[4]梁志怀,魏宝阳,魏林,等.哈茨木霉在水稻体内的定殖及对水稻生理生化特性的影响[J].湖南农业科学,2008(4):51-53.
[5]李松鹏.两株水稻根际木霉菌株生物学特性及生防潜能研究[D].武汉:华中农业大学,2017.
[6]Sghir F,Touati J,Mouria B,et al.Effect of Trichoderma harzianum and endomycorrhizae on growth and Fusarium wilt of tomato and eggplant[J].World Journal of Pharmaceutical and Life Sciences,2016,2(3):69-93.
[7]王丽荣,蒋细良,Estifanos,等.铜胁迫下的哈茨木霉Th-33转录组分析[J].中国生物防治学报,2017,33(1):103-113.
[8]吕黎,许丽媛,罗志威,等.哈茨木霉生物防治研究进展[J].湖南农业科学,2013(17):92-95.
[9]彭可为,李婵.木霉菌的生物防治研究进展[J].安徽农业科学,2010,38(2):780-782.
[10]胡春华,魏岳荣,刘凯,等.几丁质酶基因克隆及其野生蕉转化[J].分子植物育种,2010,8(4):719-724.
[11]黄艳青,庄敬华,高增贵,等.木霉菌诱导甜瓜抗枯萎病相关防御反应酶系的研究[J].沈阳农业大学学报,2005,36(5):546-549.
[12]Enkerli J,Felix G,Boller T.The enzymatic activity of fungal xylanase is not necessary for its elicitor activity[J].Plant Physiology,1999,121(2):391-397.
[13]Elamathi E,Malathi P,Viswanathan R,et al.Expression analysis on mycoparasitism related genes during antagonism of Trichoderma,with Colletotrichum falcatum,causing red rot in sugarcane[J].Journal of Plant Biochemistry and Biotechnology,2018,27(5):1-11.
[14]肖荣凤,刘波,史怀,等.生防菌哈茨木霉FJAT-9040的GFP标记及土壤定殖示踪[J].植物保护学报,2011,38(6):506-512.
[15]徐文,黄媛媛,黄亚丽,等.木霉-植物互作机制的研究进展[J].中国生物防治学报,2017,33(3):408-414.
[16]杜婵娟,付岗,潘连富,等.哈茨木霉gz-2菌株在土壤中的定殖规律研究[J].西南农业学报,2016,29(1):138-142.
[17]贺字典,宋士清,高玉峰,等.棘孢木霉Trichoderma asperellum在土壤中定殖量的荧光定量PCR检测[J].植物保护学报,2016,43(4):552-558.
[18]赵志文,李艳娇,户勋,等.用于植物病原细菌标记的p BB-GFP载体构建及应用[J].生物技术通报,2018,34(3):136-141.
[19]刘芳,杨佳颖,陈红漫.绿色木霉外源基因表达系统的构建[J].湖北农业科学,2013,52(13):3180-3183.
[20]辛鑫.绿色木霉H6-L4在土壤中定殖能力及对香蕉枯萎病原菌的抑制作用[D].海口:海南大学,2013.
[21]Sarrocco S,Mikkelsen L,Vergara M,et al.Histopathological studies of sclerotia of phytopathogenic fungi parasitized by a GFP transformed Trichoderma virens antagonistic strain[J].Mycological Research,2006,110(2):179-187.
[22]王晓利.农杆菌介导的瑞氏木霉转化及绿色荧光蛋白的表达研究[D].济南:山东大学,2008.
[23]吕桂云.西瓜与枯萎病菌互作的组织学和转录组学初步分析[D].北京:中国农业科学院,2010.
[24]黄亚丽.农杆菌介导哈茨木霉转化系统优化及突变体分析[D].北京:中国农业科学院,2008.
[25]赵兴丽,陶刚,赵玳琳,等.钩状木霉ACCC31649的GFP标记及其对辣椒定殖和促生作用[J].菌物学报,2017,23(5):1276-1285.
[26]车建美,蓝江林,刘波.转绿色荧光蛋白基因的青枯雷尔氏菌生物学特性[J].中国农业科学,2 008,41(11):3626-3635.
[27]刘路宁.绿木霉(TRICHODERMA VIRENS)TY009菌株胶霉毒素分离纯化及绿色荧光蛋白标记研究[D].杭州:浙江大学,2008.
[28]Castellanos F,Schmoll M,Martínez P,et al.Crucial factors of the light perception machinery and their impact on growth and cellulase gene transcription in Trichoderma reesei[J].Fungal Genetics and Biology,2010,7(5):468-476.
[29]Casas-Flores S,Rios-Momberg M,Bibbins M,et al.BLR-1 and BLR-2,key regulatory elements of photoconidiation and mycelial growth in Trichoderma atroviride[J].Microbiology,2004,150(11):3561-3569.
[30]Mukherjee P K,Latha J,Hadar R,et al.Role of two G-protein alpha subunits,Tga A and Tga B,in the antagonism of plant pathogens by Trichoderma virens[J].Applied and Environmental Microbiology,2004,70(1):542-549.