灵芝尿嘧啶营养缺陷型菌株的筛选和分子鉴定
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摘要
【背景】营养缺陷型是一种应用广泛的分子标记,但是目前在灵芝中还未有研究和应用报道。【目的】为灵芝遗传转化研究、杂交育种和菌种鉴别提供亲本材料和技术支持。【方法】采用紫外光诱变、单单杂交、孢子单核化的方法从灵芝单核体菌株出发得到尿嘧啶营养缺陷型双核体菌株。【结果】获得8株稳定的尿嘧啶营养缺陷型单核体突变菌株和7株尿嘧啶营养缺陷型双核体菌株。【结论】灵芝尿嘧啶营养缺陷型菌株在添加外源营养物的基础上可恢复正常生长,可以为灵芝遗传转化体系的构建和灵芝育种提供材料。
[Background] Auxotrophy is a widely applied biomarker in production and scientific research, but there is no report on Ganoderma lucidum. [Objective] This study was conducted to provide parent strains and technique supports for genetics research, cross breeding and spawn identification of Ganoderma lucidum. [Methods] The uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. The randomly selected uracil auxotrophic monokaryon strains were used as parents, and crossed with each other by Monkaryon-monkaryon mating to establish uracil auxotrophic dikaryons. [Results] Eight uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. Seven uracil auxotrophic dikaryon strains were obtained by Monkaryon-monkaryon crossing. [Conclusion] The testing results showed these auxotrophic strains restored their growth on PDA medium with uracil. The uracil auxotrophic dikaryon strains would be used as a tool for genetic transformation study and breeding.
[Background] Auxotrophy is a widely applied biomarker in production and scientific research, but there is no report on Ganoderma lucidum. [Objective] This study was conducted to provide parent strains and technique supports for genetics research, cross breeding and spawn identification of Ganoderma lucidum. [Methods] The uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. The randomly selected uracil auxotrophic monokaryon strains were used as parents, and crossed with each other by Monkaryon-monkaryon mating to establish uracil auxotrophic dikaryons. [Results] Eight uracil auxotrophic mutant strains were obtained by UV mutagenesis of protoplast. Seven uracil auxotrophic dikaryon strains were obtained by Monkaryon-monkaryon crossing. [Conclusion] The testing results showed these auxotrophic strains restored their growth on PDA medium with uracil. The uracil auxotrophic dikaryon strains would be used as a tool for genetic transformation study and breeding.
引文
[1] Zhang XY, Yang CQ. The chemical composition and pharmacological action of Ganoderma lucidum[J]. World Phytomedicines, 2006, 21(4):152-155(in Chinese)张晓云,杨春清.灵芝的化学成分和药理作用[J].国外医药·植物药分册, 2006, 21(4):152-155
[2] Liu YP, Zhang ZS, Li YM, et al. The research progress of the hypoglycemic mechanism of Ganoderma lucidum polysaccharide[J]. Food Research and Development, 2018,39(2):215-218(in Chinese)刘亚萍,张泽生,李雨蒙,等.灵芝多糖降血糖机制的研究进展[J].食品研究与开发, 2018, 39(2):215-218
[3] Zhang HB, Shi SS, Chen CM. Research progress in anti-tumor immune mechanism of Ganoderma lucidum polysaccharides[J].Medical Recapitulate, 2016, 22(2):259-262(in Chinese)张华斌,石松生,陈春美.灵芝多糖抗肿瘤免疫机制的研究进展[J].医学综述, 2016, 22(2):259-262
[4] Lv CT, Yao XY, Sun C. Progress of researches on main active substances and pharmacology of Ganoderma lucidum[J]. Anhui Agricultural Science Bulletin, 2011, 17(1):50-51,94(in Chinese)吕超田,姚向阳,孙程.灵芝主要活性物质及其药理作用研究进展[J].安徽农学通报, 2011, 17(1):50-51,94
[5] Ye PF, Zhang MP, Wang KY, et al. Progress of researches on main constituent and pharmacology of Ganoderma lucidum[J].Edible and Medicinal Mushrooms, 2013, 21(3):158-161(in Chinese)叶鹏飞,张美萍,王康宇,等.灵芝主要成分及其药理作用的研究进展综述[J].食药用菌, 2013, 21(3):158-161
[6] Wang WH, Jia W, Zhang JS, et al. Advances in studies on mechanisms of anti-prostate cancer property of Ganoderma lucidum[J]. Acta Edulis Fungi, 2018, 25(1):107-112(in Chinese)汪雯翰,贾薇,张劲松,等.灵芝对前列腺癌的作用机理[J].食用菌学报, 2018, 25(1):107-112
[7] Wang HY, Wang L, Xie KP, et al. The antimicrobial and antioxidant activity of the powder of Ganoderma lucidum[J].Chinese Journal of Biochemical Pharmaceutics, 2017, 37(3):52-54(in Chinese)王红岩,汪璐,谢鲲鹏,等.灵芝全粉的抑菌作用和抗氧化作用[J].中国生化药物杂志, 2017, 37(3):52-54
[8] Kim S, Song J, Choi HT. Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration[J]. FEMS Microbiology Letters,2004, 233(2):201-204
[9] Shi L, Fang X, Li MJ, et al. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum[J]. World Journal of Microbiology and Biotechnology, 2012, 28(1):283-291
[10] Li G, Li RX, Liu QY, et al. A highly efficient polyethylene glycol-mediated transformation method for mushrooms[J].FEMS Microbiology Letters, 2006, 256(2):203-208
[11] Xin YH, Xiao ZY, You LF, et al. Heterologous expression of taxadiene synthase gene in Ganoderma lucidum[J]. Scientia Agricultura Sinica, 2014, 47(3):546-552(in Chinese)辛燕花,肖招燕,尤琳烽,等.紫杉二烯合酶基因在灵芝中的表达[J].中国农业科学, 2014, 47(3):546-552
[12] Sun MK, Zhang M, Tian J, et al. Construction of an overexpression vector containing LZ-8 gene and transformation of Ganoderma lingzhi protoplasts[J]. Mycosystema, 2017,36(12):1625-1631(in Chinese)孙墨可,张曼,田娟,等.真菌免疫调节蛋白基因LZ-8过表达载体的构建及转化灵芝原生质体的研究[J].菌物学报,2017, 36(12):1625-1631
[13] Zhang XT, Han C, Wu LY, et al. Preparation and regeneration of the protoplasts from Ganoderma lucidum and their transformation[J]. Hereditas(Beijing), 2001, 23(5):458-462(in Chinese)张新涛,韩春,吴立宇,等.灵芝原生质体的分离及其转化[J].遗传, 2001, 23(5):458-462
[14] Sun L, Cai HQ, Xu WH, et al. Efficient transformation of the medicinal mushroom Ganoderma lucidum[J]. Plant Molecular Biology Reporter, 2001, 19(4):383-384
[15] Lei W, Liu X. Selectable marker-free technique and food safety of transgenic plants[J]. Journal of Chinese Institute of Food Science and Technology, 2009, 9(5):157-164(in Chinese)雷桅,刘昕.无选择标记技术与转基因植物食品安全性[J].中国食品学报, 2009, 9(5):157-164
[16] Li WF, Ji J, Wang G, et al. Strategies on the safety of selectable marker genes in transgenic plant[J]. Scientia Agricultura Sinica,2010, 43(9):1761-1770(in Chinese)李文凤,季静,王罡,等.提高转基因植物标记基因安全性策略的研究进展[J].中国农业科学, 2010, 43(9):1761-1770
[17] Tian Y, Chai WW, Bao AK, et al. Research progress of biosafety selectable marker genes[J]. Plant Physiology Journal, 2017,53(3):352-362(in Chinese)田野,柴薇薇,包爱科,等.生物安全型选择标记基因及其应用研究进展[J].植物生理学报, 2017, 53(3):352-362
[18] Hu LQ, Pan YJ. The research situation of molecular transformation of edible fungi[J]. Edible Fungi of China, 2006,25(4):7-10(in Chinese)胡乐琴,潘迎捷.食用菌分子转化研究状况[J].中国食用菌,2006, 25(4):7-10
[19] O’Donovan GA, Neuhard J. Pyrimidine metabolism in microorganisms[J]. Bacteriological Reviews, 1970, 34(3):278-343
[20] Chen SL, Xu J, Liu C, et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum[J]. Nature Communications, 2012, 3:913
[21] Wang LH, Mao WJ, Bao DP. Selected and molecular verification for uracil auxotrophic mutants of Lentinula edodes[J]. Acta Agriculturae Shanghai, 2014, 30(3):6-9(in Chinese)王连会,茅文俊,鲍大鹏.香菇尿嘧啶营养缺陷型菌株的筛选与分子验证[J].上海农业学报, 2014, 30(3):6-9
[22] Lu XZ, Wan JN, Mao WJ, et al. Screening and molecular verification of uracil auxotrophic mutants of Flammulina velutipes[J]. Acta Horticulturae Sinica, 2017, 44(3):495-503(in Chinese)卢绪志,万佳宁,茅文俊,等.金针菇尿嘧啶营养缺陷型菌株的筛选与分子鉴定[J].园艺学报, 2017, 44(3):495-503
[23] Zhang H, Qin LH, Tan Q, et al. Extract genomic DNA from Lentinula edodes using CTAB method[J]. Journal of Shanghai University(Natural Science), 2006, 12(5):547-550(in Chinese)张红,秦莲花,谭琦,等.用改进的CTAB法提取香菇基因组[J].上海大学学报:自然科学版, 2006, 12(5):547-550
[24] Cao H, Huang RZ. Cross-breeding of polyspora in Flammulina velutipes[J]. Edible Fungi, 1993, 15(6):6-7(in Chinese)曹晖,黄瑞贞.金针菇担孢子多孢混合杂交育种[J].食用菌,1993, 15(6):6-7
[25] Chen XL, Fan MZ, Li ZZ. Isolation of auxotrophic mutants of G106, a strain of Paecilomyces gunnii var. minor, the anamorph of Cordyceps gunnii var. minor[J]. Journal of Anhui Agricultural University, 2002, 29(2):166-168(in Chinese)陈晓琳,樊美珍,李增智.一种虫草无性型营养缺陷型的诱变筛选[J].安徽农业大学学报, 2002, 29(2):166-168
[26] Tripathi SA, Olson DG, Argyros DA, et al. Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant[J].Applied and Environmental Microbiology, 2010, 76(19):6591-6599
[27] Lu H, Li TM, Liu JL, et al. Construction of a marker-free deletion system based on the uracil phosphoribosyl transferase gene as a negative selection marker in Ketogulonigenium vulgare[J]. Food Science, 2017, 38(4):39-44(in Chinese)陆浩,李天明,刘金雷,等.基于尿嘧啶磷酸核糖转移酶为负向筛选标记的生酮古龙酸杆菌基因组无痕修饰系统的构建[J].食品科学, 2017, 38(4):39-44
[28] Zhang DC, Xu YL, Xin X, et al. Pichia pastoris X-33 with OCH1 gene deletion and its expression of glycoprotein GM-CSF[J]. Acta Microbiologica Sinica, 2011, 51(5):622-629(in Chinese)张大成,许永利,辛鑫,等.毕赤酵母X-33 OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF[J].微生物学报, 2011, 51(5):622-629
[29] Chen BX, Wei T, Ye ZW, et al. Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris[J]. Frontiers in Microbiology, 2018, 9:1157
[30] Zhou CL, Xi LP, Mao WJ, et al. Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding[J]. Breeding Science, 2017, 67(2):135-139
[2] Liu YP, Zhang ZS, Li YM, et al. The research progress of the hypoglycemic mechanism of Ganoderma lucidum polysaccharide[J]. Food Research and Development, 2018,39(2):215-218(in Chinese)刘亚萍,张泽生,李雨蒙,等.灵芝多糖降血糖机制的研究进展[J].食品研究与开发, 2018, 39(2):215-218
[3] Zhang HB, Shi SS, Chen CM. Research progress in anti-tumor immune mechanism of Ganoderma lucidum polysaccharides[J].Medical Recapitulate, 2016, 22(2):259-262(in Chinese)张华斌,石松生,陈春美.灵芝多糖抗肿瘤免疫机制的研究进展[J].医学综述, 2016, 22(2):259-262
[4] Lv CT, Yao XY, Sun C. Progress of researches on main active substances and pharmacology of Ganoderma lucidum[J]. Anhui Agricultural Science Bulletin, 2011, 17(1):50-51,94(in Chinese)吕超田,姚向阳,孙程.灵芝主要活性物质及其药理作用研究进展[J].安徽农学通报, 2011, 17(1):50-51,94
[5] Ye PF, Zhang MP, Wang KY, et al. Progress of researches on main constituent and pharmacology of Ganoderma lucidum[J].Edible and Medicinal Mushrooms, 2013, 21(3):158-161(in Chinese)叶鹏飞,张美萍,王康宇,等.灵芝主要成分及其药理作用的研究进展综述[J].食药用菌, 2013, 21(3):158-161
[6] Wang WH, Jia W, Zhang JS, et al. Advances in studies on mechanisms of anti-prostate cancer property of Ganoderma lucidum[J]. Acta Edulis Fungi, 2018, 25(1):107-112(in Chinese)汪雯翰,贾薇,张劲松,等.灵芝对前列腺癌的作用机理[J].食用菌学报, 2018, 25(1):107-112
[7] Wang HY, Wang L, Xie KP, et al. The antimicrobial and antioxidant activity of the powder of Ganoderma lucidum[J].Chinese Journal of Biochemical Pharmaceutics, 2017, 37(3):52-54(in Chinese)王红岩,汪璐,谢鲲鹏,等.灵芝全粉的抑菌作用和抗氧化作用[J].中国生化药物杂志, 2017, 37(3):52-54
[8] Kim S, Song J, Choi HT. Genetic transformation and mutant isolation in Ganoderma lucidum by restriction enzyme-mediated integration[J]. FEMS Microbiology Letters,2004, 233(2):201-204
[9] Shi L, Fang X, Li MJ, et al. Development of a simple and efficient transformation system for the basidiomycetous medicinal fungus Ganoderma lucidum[J]. World Journal of Microbiology and Biotechnology, 2012, 28(1):283-291
[10] Li G, Li RX, Liu QY, et al. A highly efficient polyethylene glycol-mediated transformation method for mushrooms[J].FEMS Microbiology Letters, 2006, 256(2):203-208
[11] Xin YH, Xiao ZY, You LF, et al. Heterologous expression of taxadiene synthase gene in Ganoderma lucidum[J]. Scientia Agricultura Sinica, 2014, 47(3):546-552(in Chinese)辛燕花,肖招燕,尤琳烽,等.紫杉二烯合酶基因在灵芝中的表达[J].中国农业科学, 2014, 47(3):546-552
[12] Sun MK, Zhang M, Tian J, et al. Construction of an overexpression vector containing LZ-8 gene and transformation of Ganoderma lingzhi protoplasts[J]. Mycosystema, 2017,36(12):1625-1631(in Chinese)孙墨可,张曼,田娟,等.真菌免疫调节蛋白基因LZ-8过表达载体的构建及转化灵芝原生质体的研究[J].菌物学报,2017, 36(12):1625-1631
[13] Zhang XT, Han C, Wu LY, et al. Preparation and regeneration of the protoplasts from Ganoderma lucidum and their transformation[J]. Hereditas(Beijing), 2001, 23(5):458-462(in Chinese)张新涛,韩春,吴立宇,等.灵芝原生质体的分离及其转化[J].遗传, 2001, 23(5):458-462
[14] Sun L, Cai HQ, Xu WH, et al. Efficient transformation of the medicinal mushroom Ganoderma lucidum[J]. Plant Molecular Biology Reporter, 2001, 19(4):383-384
[15] Lei W, Liu X. Selectable marker-free technique and food safety of transgenic plants[J]. Journal of Chinese Institute of Food Science and Technology, 2009, 9(5):157-164(in Chinese)雷桅,刘昕.无选择标记技术与转基因植物食品安全性[J].中国食品学报, 2009, 9(5):157-164
[16] Li WF, Ji J, Wang G, et al. Strategies on the safety of selectable marker genes in transgenic plant[J]. Scientia Agricultura Sinica,2010, 43(9):1761-1770(in Chinese)李文凤,季静,王罡,等.提高转基因植物标记基因安全性策略的研究进展[J].中国农业科学, 2010, 43(9):1761-1770
[17] Tian Y, Chai WW, Bao AK, et al. Research progress of biosafety selectable marker genes[J]. Plant Physiology Journal, 2017,53(3):352-362(in Chinese)田野,柴薇薇,包爱科,等.生物安全型选择标记基因及其应用研究进展[J].植物生理学报, 2017, 53(3):352-362
[18] Hu LQ, Pan YJ. The research situation of molecular transformation of edible fungi[J]. Edible Fungi of China, 2006,25(4):7-10(in Chinese)胡乐琴,潘迎捷.食用菌分子转化研究状况[J].中国食用菌,2006, 25(4):7-10
[19] O’Donovan GA, Neuhard J. Pyrimidine metabolism in microorganisms[J]. Bacteriological Reviews, 1970, 34(3):278-343
[20] Chen SL, Xu J, Liu C, et al. Genome sequence of the model medicinal mushroom Ganoderma lucidum[J]. Nature Communications, 2012, 3:913
[21] Wang LH, Mao WJ, Bao DP. Selected and molecular verification for uracil auxotrophic mutants of Lentinula edodes[J]. Acta Agriculturae Shanghai, 2014, 30(3):6-9(in Chinese)王连会,茅文俊,鲍大鹏.香菇尿嘧啶营养缺陷型菌株的筛选与分子验证[J].上海农业学报, 2014, 30(3):6-9
[22] Lu XZ, Wan JN, Mao WJ, et al. Screening and molecular verification of uracil auxotrophic mutants of Flammulina velutipes[J]. Acta Horticulturae Sinica, 2017, 44(3):495-503(in Chinese)卢绪志,万佳宁,茅文俊,等.金针菇尿嘧啶营养缺陷型菌株的筛选与分子鉴定[J].园艺学报, 2017, 44(3):495-503
[23] Zhang H, Qin LH, Tan Q, et al. Extract genomic DNA from Lentinula edodes using CTAB method[J]. Journal of Shanghai University(Natural Science), 2006, 12(5):547-550(in Chinese)张红,秦莲花,谭琦,等.用改进的CTAB法提取香菇基因组[J].上海大学学报:自然科学版, 2006, 12(5):547-550
[24] Cao H, Huang RZ. Cross-breeding of polyspora in Flammulina velutipes[J]. Edible Fungi, 1993, 15(6):6-7(in Chinese)曹晖,黄瑞贞.金针菇担孢子多孢混合杂交育种[J].食用菌,1993, 15(6):6-7
[25] Chen XL, Fan MZ, Li ZZ. Isolation of auxotrophic mutants of G106, a strain of Paecilomyces gunnii var. minor, the anamorph of Cordyceps gunnii var. minor[J]. Journal of Anhui Agricultural University, 2002, 29(2):166-168(in Chinese)陈晓琳,樊美珍,李增智.一种虫草无性型营养缺陷型的诱变筛选[J].安徽农业大学学报, 2002, 29(2):166-168
[26] Tripathi SA, Olson DG, Argyros DA, et al. Development of pyrF-based genetic system for targeted gene deletion in Clostridium thermocellum and creation of a pta mutant[J].Applied and Environmental Microbiology, 2010, 76(19):6591-6599
[27] Lu H, Li TM, Liu JL, et al. Construction of a marker-free deletion system based on the uracil phosphoribosyl transferase gene as a negative selection marker in Ketogulonigenium vulgare[J]. Food Science, 2017, 38(4):39-44(in Chinese)陆浩,李天明,刘金雷,等.基于尿嘧啶磷酸核糖转移酶为负向筛选标记的生酮古龙酸杆菌基因组无痕修饰系统的构建[J].食品科学, 2017, 38(4):39-44
[28] Zhang DC, Xu YL, Xin X, et al. Pichia pastoris X-33 with OCH1 gene deletion and its expression of glycoprotein GM-CSF[J]. Acta Microbiologica Sinica, 2011, 51(5):622-629(in Chinese)张大成,许永利,辛鑫,等.毕赤酵母X-33 OCH1基因敲除菌株的构建及其表达低糖基化蛋白GM-CSF[J].微生物学报, 2011, 51(5):622-629
[29] Chen BX, Wei T, Ye ZW, et al. Efficient CRISPR-Cas9 gene disruption system in edible-medicinal mushroom Cordyceps militaris[J]. Frontiers in Microbiology, 2018, 9:1157
[30] Zhou CL, Xi LP, Mao WJ, et al. Establishment of uracil auxotrophic dikaryotic strains of Lentinula edodes by crossbreeding[J]. Breeding Science, 2017, 67(2):135-139
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