GFP -labeled Transformation of Trichoderma harzianum T2-16 and Its Biocontrol Characteristics

doi:10.16409/j.cnki.2095-039x.2019.02.007

Abstract

Abstract: 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-CaCl₂-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.

Key words: Trichoderma harzianum, green fluorescent protein, vector construction, biological control

CLC Number:

S476

TIAN Cheng, ZHANG Yi, XIAO Jiling, ZHU Feiying, TANG Yanying, WEI Lin, LIANG Zhihuai. GFP -labeled Transformation of Trichoderma harzianum T2-16 and Its Biocontrol Characteristics[J]. journal1, 2019, 35(2): 247-254.

References

[1] 梁志怀, 魏林, 陈玉荣, 等. 哈茨木霉在水稻体内的定殖及其对水稻纹枯病抗性的影响[J]. 中国生物防治学报, 2009, 25(2):143-147.
[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pBB-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,2008, 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, TgaA and TgaB, in the antagonism of plant pathogens by Trichoderma virens[J]. Applied and Environmental Microbiology, 2004, 70(1):542-549.