纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化

doi:10.13560/j.cnki.biotech.bull.1985.2016.09.030

生物技术通报 ›› 2016, Vol. 32 ›› Issue (9): 225-231.doi: 10.13560/j.cnki.biotech.bull.1985.2016.09.030

纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化

林艳梅, 李瑞杰, 张慧杰, 秦秀林, 冯家勋

广西大学生命科学与技术学院亚热带农业生物资源保护与利用国家重点实验室,南宁 530004

收稿日期:2016-01-03 出版日期:2016-09-25 发布日期:2016-10-10
作者简介:林艳梅,女,硕士,研究方向：微生物学;E-mail：930231670@qq.com
基金资助:
国家自然科学基金项目（31300076）,广西自然科学基金项目（2013GXNSFBA019096）

Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase

LIN Yan-mei, LI Rui-jie, ZHANG Hui-jie, QIN Xiu-lin, FENG Jia-xun

College of Life Science and Technology,State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,Guangxi University,Nanning 530004

Received:2016-01-03 Published:2016-09-25 Online:2016-10-10

摘要/Abstract

摘要： 旨在优化深绿木霉（Trichoderma atroviride）菌株HP35-3原生质体制备和转化条件,便于对该菌株进行遗传操作以提高其纤维素酶产量。分别对制备深绿木霉原生质体的菌龄、酶解时间、酶组分及比例和转化条件进行优化。结果显示,利用3 mg/mL蜗牛酶、3 mg/mL溶菌酶和3 mg/mL裂解酶酶组分酶解菌龄10 h的菌丝2 h,获得的原生质浓度达到3.5×10⁷个/mL以上,原生质体再生率为61%。利用原生质体进行PEG介导转化,当原生质体浓度为1×10⁸个/mL、外源DNA为5 μg时,转化率达到35个转化子/μg DNA。建立的高效原生质体制备及转化体系可用于深绿木霉的遗传转化及菌株改造。

关键词: 深绿木霉, 丝状真菌, 原生质体制备, 原生质体转化

Abstract: The purpose of this work is to optimize the conditions of formatting and transforming the protoplast of Trichoderma atroviride HP35-3,aiming at genetically manipulating the strain for increasing the yield of cellulase. The individual condition for formatting the protoplast of Trichoderma atroviride,i.e.,mycelium age,hydrolysis time of enzyme,constituents and ratios of enzymes,as well as transformation conditions were optimized respectively. Results showed that the combination of 3 mg/mL snailase,3 mg/mL lysing enzyme,and 3 mg/mL lysozyme enzyme was effective in releasing protoplasts from T. atroviride HP35-3 mycelium（at the age of 10 h）after digestion for 2 h,the concentration was over 3.5×10⁷ protoplasts/mL,and the regeneration rate was 61%. The protoplasts were mediated and transformed by PEG,and the transformation rate was 35 transformants/μg DNA when 1×10⁸ protoplasts were employed with 5 μg DNA. In conclusion,establishing efficient formation and transformation of protoplasts may be applied in strain improvement and gene transformation of T. atroviride in biotech industries and laboratories.

Key words: Trichoderma atroviride, filamentous fungi, formation of protoplast, transformation of protoplast

林艳梅, 李瑞杰, 张慧杰, 秦秀林, 冯家勋. 纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化[J]. 生物技术通报, 2016, 32(9): 225-231.

LIN Yan-mei, LI Rui-jie, ZHANG Hui-jie, QIN Xiu-lin, FENG Jia-xun. Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase[J]. Biotechnology Bulletin, 2016, 32(9): 225-231.

参考文献

[1] 刘士旺, 王政逸, 郭泽建. 绿色木霉的原生质体制备与转化条件[J]. 农业生物技术学报, 2004, 12（1）：96-101.
[2] Prabavathy VR, Mathivanan N, Sagadevan E, et al. Self-fusion of protoplasts enhances chitinase production and biocontrol activity in Trichoderma harzianum[J]. Bioresource Technology, 2006, 97（18）：2330-2334.
[3] Dillon AJP, Camassola M, Henriques JAP, et al. Generation of recombinants strains to cellulases production by protoplast fusion between Penicillium echinulatum and Trichoderma harzianum[J]. Enzyme and Microbial Technology, 2008, 43（6）：403-409.
[4] Lee KM, Yu J, Son M, et al. Transmission of Fusarium boothii mycovirus via protoplast fusion causes hypovirulence in other phytopathogenic fungi[J]. PLoS One, 2011, 6（6）：e21629.
[5] Jiang D, Zhu W, Wang Y, et al. Molecular tools for functional genomics in filamentous fungi：recent advances and new strategies[J]. Biotechnology Advances, 2013, 31（8）：1562-1574.
[6] De BC, Wiebenga A, Aguilar G, et al. An enzyme cocktail for efficient protoplast formation in Aspergillus niger[J]. Journal of Microbiological Methods, 2009, 76（3）：305-306.
[7] Liu TH, Lin MJ, Ko WH. Factors affecting protoplast formation by Rhizoctonia solani[J]. New Biotechnology, 2010, 27（1）：64-69.
[8] Patil NS, Jadhav JP. Penicillium ochrochloron MTCC 517 chitinase：An effective tool in commercial enzyme cocktail for production and regeneration of protoplasts from various fungi[J]. Saudi Journal of Biological Sciences, 2015, 22（2）：232-236.
[9] Feng HT, Sun ZG, Li HJ, et al. Preparation, purification and regene- ration optimizing research of protoplasts from Rhizoctonia solani [J]. African Journal of Microbiology Research, 2012, 6（13）：3222-3230.
[10] 任志红, 苏彩云, 闫敬, 等. 霉酚酸产生菌原生质体转基因方法的建立[J]. 微生物学, 2013, 53（11）：1226-1232.
[11] 徐新丽, 谢必峰. 深黄被孢霉3. 3410原生质体的制备和再生研究[J]. 生物技术, 2009, 19（2）：46-48.
[12] Tschen SM, Li IF. Optimization of formation and regeneration of protoplasts from biocontrol agents of Trichoderma species[J]. Mycoscience, 1994, 35（3）：257-263.
[13] Balasubramanian N, Lalithakumari D. Characteristics of protoplast inter, intra-fusant and regeneration of antagonistic fungi Trichoderma harzianum and Trichoderma viride[J]. African Journal of Biotechnology, 2008, 7（18）：3235-3243.
[14] You YR, Liu SW, Wu YF, et al. The Transformation in Trichoderma viride protoplasts by restriction enzyme mediated integration（RE-MI）[J]. Advanced Materials Research, 2012, 518-523：545-548.
[15] 黄玉茜, 刘根, 程根武, 等. 木霉菌T23菌株原生质体制备及再生研究[J]. 上海交通大学学报：农业科学报, 2005, 23（3）：303-307.
[16] Zhang Z, Liu JL, Lan JY, et al. Predominance of Trichoderma and Penicillium in cellulolytic aerobic filamentous fungi from subtropical and tropical forests in China, and their use in finding highly efficient beta-glucosidase[J]. Biotechnology for Biofuels, 2014, 7（1）：1-14.
[17] Churchill ACL, Ciuffetti LM, Hansen DR, et al. Transformation of the fungal pathogen Cryphonectria parasitica with a variety of heterologous plasmids[J]. Current Genetics, 1989, 17（1）：25-31.
[18] 吕天晓, 徐凤花, 李世贵, 等. 生防木霉菌原生质体的制备及再生研究[J]. 生物技术通报, 2009（4）：130-134.
[19] Cartwright GM, Tanaka A, Eaton CJ, et al. Formation of arthrocon-idia during regeneration and selection of transformed Epichloe festucae protoplasts[J]. Fungal Biology, 2014, 118（5-6）：462-471.
[20] 卢明, 张月杰. 丝状真菌AL18的原生质体制备和再生条件的优化[J]. 生物技术, 2012, 22（3）：86-89.
[21] Balasubramanian N, Juliet GA, Srikalaivani P, et al. Release and regeneration of protoplasts from the fungus Trichothecium roseum[J]. Canadian Journal Microbiology, 2003, 49（4）：263-268.
[22] Savitha S, Sadhasivam S, Swaminathan K. Regeneration and mole- cular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion [J]. Biotechnology Advances, 2010, 28（3）：285-292.
[23] 张晓立, 郑小梅, 满云, 等. 黑曲霉柠檬酸工业菌株原生质体制备与转化[J]. 生物技术通报, 2015, 31（3）：171-177.
[24] Villanueva JR, Acha G. Chapter XXIV Production and use for fungal protoplast[J]. Methods in Microbiology, 1971, 4：666-718.
[25] Tamova G, Betina V, Farkas V. An efficient method for the preparation of protoplasts from Trichoderma viride[J]. Folia Microbiologica, 1993, 38（3）：214-218.
[26] Mrinalini C, Lalithakumari D. Integration of enhanced biocontrol efficacy and fungicide tolerance in Trichoderma spp. by electrofusion[J]. Journal of Plant Diseases and Protection, 1998, 105（1）：34-40.

纤维素酶高产菌株Trichoderma atroviride HP35-3原生质体制备及转化

Formation and Transformation of Protoplast of Trichoderma atroviride HP35-3 Highly Yielding Cellulase

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