康氏木霉与酿酒酵母原生质体融合构建产酒精新菌株
|
摘要
原生质体融合技术是一种微生物育种方法,该方法简便、易行。利用该方法选育目的菌株的过程中仍然存在许多需要解决的问题。本试验以康氏木霉和酿酒酵母为出发菌株,对原生质体融合技术进行了研究,确定了纤维素酶高产菌株康氏木霉原生质体制备及再生的适宜条件,为该菌细胞融合和诱变育种提供适宜的真菌状态。同时对融合株的一些遗传性状进行了相关研究。该研究为微生物育种工作奠定了基础。
本试验对影响康氏木霉原生质体形成和再生的各种因素(菌龄,蜗牛酶浓度,酶解温度和时间,渗透压稳定剂的成份和浓度)进行试验比较。结果表明,康氏木霉原生质体制备的最佳条件:菌龄为22 h,蜗牛酶浓度为2.0%,酶解温度为30℃,酶解时间为2.5 h,蜗牛酶溶液中的渗透压稳定剂为0.6 mol/L NaCl,在制备康氏木霉原生质体时观察到原生质体的释放方式主要有两种:顶端释放和段端位释放。本试验原生质体制备方法可以获得细胞融合和细胞突变诱导所需要的原生质体数量。
本试验对康氏木霉原生质体的再生培养基进行了研究,确定了康氏木霉原生质体的最佳再生培养基为0.8 mol/L蔗糖YPD再生培养基,康氏木霉原生质体在0.8 mol/L蔗糖YPD再生培养基上平均再生菌落数为4.13×105个/mL。同时观察到了康氏木霉原生质体的一种再生方式。
本试验采用抗药性遗传标记和热灭活两种方法作为融合菌株的筛选标记,确定了制霉菌素的标记浓度为750 U/mL,无水乙醇的标记浓度为23%。康氏木霉的热灭活温度为67℃,时间为10 min。本试验采用康氏木霉原生质体单亲热灭活(酿酒酵母采用抗药性遗传标记)和双亲均采用抗药性遗传标记两种方法共筛出246株融合菌株。
本试验初筛采用TTC作为显色剂筛选出发酵葡萄糖能够产酒精的融合株,然后将此融合菌株在羧甲基纤维素钠培养基上进行复筛,筛选出分解葡萄糖产水解圈较大的融合株,TTC和羧甲基纤维素钠培养基能够筛选出既产酒精又产纤维素酶的融合株。将筛选出的融合株进行遗传稳定性试验最终得到4株稳定融合菌株。
本试验最后对遗传性稳定的4株融合菌株进行了个体形态、生化特性、特定基因序列和可溶性蛋白等方面的遗传性质研究,研究发现:融合菌株B1、B2、B3确实进行了基因重组,但其酶活和产酒精能力都比亲本低。但是这些研究为原生质体融合育种工作提供了有益的参考。
The protoplast fusion technology is a way of microbial breeding.This method is simple and easy.But there are still many questions about selecting objective bacterial strain to be solved by this method.This paper is focused on Trichoderma koningii and Saccharomyces cerevisiae,protoplast fusion techonology and inherited characteristics of fusant are analyzed.To provide a suitable fungal material for protoplast-fusion and mutation-breeding of Trichoderma koningii.This research work for microbial breeding laid a foundation.
Conditions for Trichoderma koningii protoplast preparation and regeneration including mycelium growth time,the content of cellulose,temperature and time for reaction,various compounding of osmotic stabilizers were examined.The optimal conditions for protoplast preparation of Trichoderma koningii were as follows:22h mycelium growth time,the content of cellulase was 2.0%,2.5 h reaction at 30℃,the optimal osmotic stabilizer of cellulase was 0.6 mol/L NaCl.Microscopical observation indicated that the protoplasts of Trichoderma koningii were largely released from the apex and hypha segment.A satisfied concentration of Trichoderma koningii protoplasts could be achieved for the cell fusion and the cell mutation induced in this research.
The regeneration culture medium was researched , determining the optimal regeneration culture medium for Trichoderma koningii protoplast was 0.8 mol/L sucrose YPD.The research showed that 4.13×105 regeneration colonies were in this regeneration culture medium . One modes forming irregular outshoot was found to regulate regeneration of Trichoderma koningii.
Fusants were got by two methods.Drug resistance genetic marker and the single heated died parent were used for selection marker in this experiment.The nystatin concentration was 750 U/mL,The alcohol concentration was 23%,the single heated died parent of Trichoderma koningii protoplast was heated up to 67℃,10 min.The 246 fusants were selectd by the two methods.
The fusants through the preliminary selection for fermenting glucose to the alcohol were selected by TTC.Then the fusants through continuous selection for breakdown of glucose were selected by sodium carboxymethyl cellulose culture medium.The fusants have the strong ability for production alcohol.The fusants have the highest enzymic activity strain by TTC and sodium carboxymethyl cellulose culture medium.The selected fusants were to be tested for genetic stability.The results were that the four fusants were the genetic stability fusants.
Individual figure、biochemical characteristic、specific genetic sequence and soluble protein were studied by this experiment.The results were that the fusants for B1、B2、B3 and B4 proceeded gene recombination,but the ability for enzymic activity and production alcohol were lower than the parents.But this experiment provided the foundation for protoplast fusion breeding.
本试验对影响康氏木霉原生质体形成和再生的各种因素(菌龄,蜗牛酶浓度,酶解温度和时间,渗透压稳定剂的成份和浓度)进行试验比较。结果表明,康氏木霉原生质体制备的最佳条件:菌龄为22 h,蜗牛酶浓度为2.0%,酶解温度为30℃,酶解时间为2.5 h,蜗牛酶溶液中的渗透压稳定剂为0.6 mol/L NaCl,在制备康氏木霉原生质体时观察到原生质体的释放方式主要有两种:顶端释放和段端位释放。本试验原生质体制备方法可以获得细胞融合和细胞突变诱导所需要的原生质体数量。
本试验对康氏木霉原生质体的再生培养基进行了研究,确定了康氏木霉原生质体的最佳再生培养基为0.8 mol/L蔗糖YPD再生培养基,康氏木霉原生质体在0.8 mol/L蔗糖YPD再生培养基上平均再生菌落数为4.13×105个/mL。同时观察到了康氏木霉原生质体的一种再生方式。
本试验采用抗药性遗传标记和热灭活两种方法作为融合菌株的筛选标记,确定了制霉菌素的标记浓度为750 U/mL,无水乙醇的标记浓度为23%。康氏木霉的热灭活温度为67℃,时间为10 min。本试验采用康氏木霉原生质体单亲热灭活(酿酒酵母采用抗药性遗传标记)和双亲均采用抗药性遗传标记两种方法共筛出246株融合菌株。
本试验初筛采用TTC作为显色剂筛选出发酵葡萄糖能够产酒精的融合株,然后将此融合菌株在羧甲基纤维素钠培养基上进行复筛,筛选出分解葡萄糖产水解圈较大的融合株,TTC和羧甲基纤维素钠培养基能够筛选出既产酒精又产纤维素酶的融合株。将筛选出的融合株进行遗传稳定性试验最终得到4株稳定融合菌株。
本试验最后对遗传性稳定的4株融合菌株进行了个体形态、生化特性、特定基因序列和可溶性蛋白等方面的遗传性质研究,研究发现:融合菌株B1、B2、B3确实进行了基因重组,但其酶活和产酒精能力都比亲本低。但是这些研究为原生质体融合育种工作提供了有益的参考。
The protoplast fusion technology is a way of microbial breeding.This method is simple and easy.But there are still many questions about selecting objective bacterial strain to be solved by this method.This paper is focused on Trichoderma koningii and Saccharomyces cerevisiae,protoplast fusion techonology and inherited characteristics of fusant are analyzed.To provide a suitable fungal material for protoplast-fusion and mutation-breeding of Trichoderma koningii.This research work for microbial breeding laid a foundation.
Conditions for Trichoderma koningii protoplast preparation and regeneration including mycelium growth time,the content of cellulose,temperature and time for reaction,various compounding of osmotic stabilizers were examined.The optimal conditions for protoplast preparation of Trichoderma koningii were as follows:22h mycelium growth time,the content of cellulase was 2.0%,2.5 h reaction at 30℃,the optimal osmotic stabilizer of cellulase was 0.6 mol/L NaCl.Microscopical observation indicated that the protoplasts of Trichoderma koningii were largely released from the apex and hypha segment.A satisfied concentration of Trichoderma koningii protoplasts could be achieved for the cell fusion and the cell mutation induced in this research.
The regeneration culture medium was researched , determining the optimal regeneration culture medium for Trichoderma koningii protoplast was 0.8 mol/L sucrose YPD.The research showed that 4.13×105 regeneration colonies were in this regeneration culture medium . One modes forming irregular outshoot was found to regulate regeneration of Trichoderma koningii.
Fusants were got by two methods.Drug resistance genetic marker and the single heated died parent were used for selection marker in this experiment.The nystatin concentration was 750 U/mL,The alcohol concentration was 23%,the single heated died parent of Trichoderma koningii protoplast was heated up to 67℃,10 min.The 246 fusants were selectd by the two methods.
The fusants through the preliminary selection for fermenting glucose to the alcohol were selected by TTC.Then the fusants through continuous selection for breakdown of glucose were selected by sodium carboxymethyl cellulose culture medium.The fusants have the strong ability for production alcohol.The fusants have the highest enzymic activity strain by TTC and sodium carboxymethyl cellulose culture medium.The selected fusants were to be tested for genetic stability.The results were that the four fusants were the genetic stability fusants.
Individual figure、biochemical characteristic、specific genetic sequence and soluble protein were studied by this experiment.The results were that the fusants for B1、B2、B3 and B4 proceeded gene recombination,but the ability for enzymic activity and production alcohol were lower than the parents.But this experiment provided the foundation for protoplast fusion breeding.
引文
[1]张继泉,王瑞明,孙玉英.利用木质纤维素生产燃料酒精的研究进展[J].酿酒科技,2003, 115(1):39-41.
[2]黄治玲.燃料乙醇的生产与利用[J].中小企业科技,2003,1(12):25.
[3]孙逸,贺稚非.纤维素发酵生产酒精的研究进展[J].农产品加工·创新版,2009,170(04):70-71.
[4]张小希,张丽春.纤维素乙醇可持续发展道路的探讨[J].现代化工,2008,28(02):26-27.
[5] Habib G,Hassan M F,Siddiqui M M,Degradation characteristics of straw different wheat genotypes and their response tourea-ammoniation treatment[J] . Animal Feed Science and Technology,1998,72:373-386.
[6]赵彩云,王异静,关东明.纤维素乙醇研究进展[J].酿酒科技,2009,184(10):87-89.
[7]张作阳,田沈,孟繁艳,等.基因组改组及其在木质纤维素水解液乙醇发酵菌种选育中的应用展望[J].微生物学通报,2009,36(06):900-904.
[8]高阳,刘四新.天然纤维素原料生产生物燃料的应用现状[J].广州农业科学,2009,1(07):205.
[9]阴春梅,刘忠,齐宏升.生物质发酵生产乙醇的研究进展[J].酿酒科技,2007,1(151):87-90.
[10]欧阳嘉,李鑫,王向明,等.纤维素结合域的研究进展[J].2008,6(02):10-11.
[11]曲音波.纤维素乙醇产业化—进展与展望[R].山东济南:山东大学,2007.
[12]吴创之,马隆龙.生物质能现代化利用技术[M].北京:化学工业出版社,2003.
[13]段钢,孙长平.酶在生物质转化为燃料酒精中的应用[J].食品与发酵工业,2005,31(5):73-77.
[14]宋新南,宋爽.纤维素生物燃料酒精技术的现状及发展方向[J].酿酒科技,2007,9(159):89-90.
[15]王敏辉.饲用纤维素酶的研究进展[J].添加剂世界,2009,1(11):27.
[16]文少白,李勤奋,侯宪文,等.微生物降解纤维素的研究概况[J].中国农学通报,2010,26(01):231-236.
[17]杨友坤,朱凤香,王卫平,等.纤维素酶及其应用现状[J].安徽农业通报,2009,15(13):59-60.
[18]PALMAROLA-ADRADOS B,CHOTEBORSKA P,GALBE M.etal.Ethanol production from non-starch carbohydrates of wheat bran [J].Biores Tech,2005(96):843-856.
[19]张继泉,孙玉英.发酵戊糖产酒精酵母菌株的选育[J].生物技术通讯,2002,13(4):275-277.
[20]蒋加拉,刘素纯,关美玲.纤维素降解的菌株筛选及其应用[J].现代生物医学进展,2009,9(18):3451.
[21]成亚利,珠宝成,李亮亮,等.金针菇原生质体紫外诱变选育[J].食用菌学报,1995,2(3):61-64.
[22]李德舜,刘正学.平菇山大1号紫外诱变育种研究[J].食用菌,2002,24(3):9-10.
[23]陈五岭,姚胜利.He-Ne激光在香菇速生高产菌株选育中的应用(I)(J).光子学报,1997,26(11):972-976.
[24]李耀维,冯文新,张素梅.He-Ne激光对金针菇SOD高产株的诱变效应[J].激光生物学报,2002,11(4):283-286.
[25]夏志兰,艾幸.60Co射线对杏鲍菇菌丝的诱变效应[J].激光生物学报,2004,13(4):298-301.
[26]李卫旗,何国庆.60Co射线诱变选育热凝胶多糖高产菌株的研究[J].核农学报,2003,17(5):343-346.
[27]曲音波,高培基,王祖农.青霉的纤维素酶抗降解物阻遏突变株的选育[J].真菌学报,1984,3(4):238-242.
[28]陈宏军.蛋白酶高产菌株的紫外诱变选育[J].安徽农业科学,2010,38(3):1110-1111.
[29]崔国艳,陈五岭.He-Ne激光诱变选育高温乳酸菌[J].中外医疗,2009,1(11):15.
[30]程艳,张克诚,赵明富,等.应用Co60γ射线诱变选育武夷菌素高产菌的研究[J].中国农学通报,2010,26(2):234-237.
[31]武秀琴.纤维素酶高产菌株的诱变选育[J].中国酿造,2009,3(204):84-84.
[32]傅力,涂正东,叶凯,等.里氏木霉高产纤维素酶菌株的选育及产酶培养基的优化[J].食品与机械,2009,25(3):10.
[33]戴群,戴传超,顾敏.花生四烯酸高产菌株的诱变和筛选研究[J].食品科技,2010,35(1):19.
[34]Lilly M,Lambrechts M G,Pretorius I S.Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates[J].Applied and Environmental Microbiology,2000,66(2):744-753.
[35]Laura-Leena,Kiiskinen,Markku Saloheimo.Molecular Cloning and Expression in Saccharomyces cereviside of a Laccase Gene from the Ascomycete Melanocarpus albomyces[J].Applied and Environmental Microbiology,2004,70(1):137-144.
[36]Nina Jarvinen,Minna Maki,Jarkko Rabina,etal.Cloning and expression of Helicobacter pylori GDP-L-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae[J].Eur.J.Biochem,2001,268:6458-6464.
[37]TOSHIO FUJII,NAOSHI NAGASAWA,AKIHIRO IWAMATSU,etal.Molecular Cloning,Sequence Analysis,and Expression of the Yeast Alcohol Acetyltransferase Gene[J].Applied and Environmental Microbiology,1994,60(8):2786-2792.
[38]TSUNG TSNA WANG,LONG LIU LIN,WEN HWEI HSU,Cloning and Expression of a Schwanniomyces occidentalis OL-Amylase Gene in Saccharomyces cerevisiae[J].Applied and Environmental Microbiology,1989,55(12):167-3172.
[39]杨宁,王惠娟,郭利健.工业微生物育种综述[J].湖北农机化,2008,1(3):29.
[40]孟甜,李玉锋.现代工业微生物育种技术研究进展[J].2009,7(12):5.
[41] Salusj?rvi L, Kankainen M, Soliymani R, Pitk?nen J-P, Penttil? M, Ruohonen L. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae. Microbial Cell Factories, 2008, 7: 18. doi:10 .1186/1475-2859-1187-1118
[42]何明雄,祝其丽,潘科,等.利用木质纤维素类生物质发酵生产乙醇重组菌株研究进展[J].应与环境生物学报,2009,15(4):579-584.
[43] Masahiro Nogawa,Masahiro Goto,Hirofumi Okada,Yasushi-Morikawa.L-Sorbose induces cellulose gene transcription in the cellulolytic fungus Trichodema reesei[J].Curr Genet,2001,38:329-334.
[44]刘泽寰,台艳,唐根云,等.绿色木霉EGⅢ基因的克隆及其在工业酿酒酵母中的整合表达[J].中山大学学报(自然科学版),2009,48(6):83.
[45]王利英,刘一,杨登峰,等.绿色木霉葡聚糖内切酶cDNA基因的克隆及其在酿酒酵母中的表达[J].2007,14(3):315-319.
[46] SONDEREGGER M,SCHUMPERLI M,SAUER U.Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae[J].Appl. Environ .Microbiol.,2004,70(5):2892-2897.
[47]刘建军,赵祥颖,姜鲁燕,等.高产酒精絮凝酵母SY-130菌株的选育[J].山东食品发酵,2004,135(4):8.
[48]黄玉茜,梁春浩,陈捷.绿色木霉菌T23原生质体的制备和再生[J].吉林农业大学学报,2007,29(1):24-27.
[49]高榕.高生产效率纤维素酶菌株的初筛方法的研究[J].纤维素科学与技术,2004,912 (3):23-27.
[50]滕利荣,等.生物学基础实验教程(第一版)[M].吉林科学出版社,1999.316-490.
[51]王蕾,等.DNS法测定海藻糖的研究[J].食品科技,2004,2(2):82-84.
[52]苏畅,等.氨基葡萄糖和乙酰氨基葡萄糖的测定方法[J].食品工业科技,2004,1(6):74-76.
[53]靖德兵,李培军,王力华,等.康氏木霉固体发酵生产纤维素酶优化研究—培养基用料与工艺条件优化[J].食品科学,2004,25(5):82-83.
[54]刘海波,王义,陈介南,等.一株高产纤维素酶菌的筛选与鉴定[J].生物学杂志,2008,25(3):16-17.
[55]苏艳秋,朱卫华,吴鹏,等.耐高温、耐酸产酒精酵母的筛选与鉴定[J].微生物学杂志,2009,29(2):44-45.
[56]蒋加拉,刘素纯,关美玲,等.纤维素降解的菌株筛选及其运用[J].现代生物医学进展,2009,9(18):3451-3453.
[57]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994.
[58]张怀东,刘作易,周礼红,等.高耐性高产酒精酵母的分离和筛选[J].酿酒科技,2009,177(3):40-41.
[59]Kurtzman CP,Robnett CJ.Identification of clinically important ascomycetous yeasts based on nucleotide divergence in 5′end of the large-subunit(26S) ribosomal DNA gene[J]..J Clin Microbiol,1997,35(5):1216-1223.
[60]姜明国,何小丹,裴氏秋河,等.板栗疫病菌致病性机理的双向凝胶电泳法的研究[J].广西植物,2008,28(1):117-120.
[61]邱雁临,曾莹,胡赛阳,等.原生质体融合子代的筛选和鉴定[J].生物技术,2003,13(6):17-18.
[62]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994.
[63]赵乐辉,李颖,吕淑霞.木霉T21和T22原生质体制备和再生研究初报[J].沈阳农业大学学报,2005,36(1):96-98.
[64]王景林,殷勤燕,陈宗泽,等.康氏木霉B-7和黑曲霉X-15原生质体的形成和再生[J].微生物学杂志,1999,1(19):20-21.
[65]周庆新,陈静,于恺,等.疏绵状嗜热丝孢菌原生质体的制备与再生[J].菌物研究,2006,4(2):1-5.
[66]朱艳,薛正莲,张相美,等.林肯链霉菌原生质体制备条件的研究[J].四川食品与发酵,2008,44(3):13-14.
[67]包莹玲,潘力.茂原链霉菌原生质体制备条件的优化[J].现代食品科技,2008,24(2):138-140.
[68]金卫根,孙丽萍.茶新菇原生质体制备条件的分析[J].江西农业学报,2009,21(11):147-148.
[69]祝子坪,李娜,贺建东.渗透压稳定剂对桑黄菌原生质体分离与再生的影响[J].食用菌,2009 (5):25-26.
[70]张拥华,李晶晶,彭志刚,等.粉红粘帚霉67-1菌株原生质体的形成和再生[J].中国生物防治,2006,22(4):303-307.
[71]梁平彦.产黄青霉两株营养缺陷型原生质体的形成和再生的因素的研究[J].植物生理学报,1981,7(1):1-9.
[72]邢来君,张军.米曲霉两株营养缺陷型原生质体的形成和再生的条件实验[J].真菌学报,!987,6(4):242-247.
[73]王立洪,杨土风,杨涛,等.平菇原生质体的高效制备及再生优化条件[J].应用与环境生物学报,2008,14(3):351-355.
[74]王慧,王玉华,段宁宁,等.出芽短梗霉原生质体制备及再生的研究[J].食品科技,2008,2(5):1-3.
[75]朱藴兰,周卫东,陈安徽,等.冬虫夏草无性型原生质体再生条件研究[J].安徽农业科学,2009,37(30):14629-14631.
[76]任选.漆酶高活性菌株的选育及其融合子F49的初步鉴定[D].山东:山东农业大学,2008.
[77]阎晓星.绿粘帚霉和球孢白僵菌原生质体制备、再生和融合条件的研究[D].四川:四川农业大学,2007.
[78]董博宇,陈叶福,岳瑞雪,等.TTC在发酵木糖高产乙醇的休哈塔假丝酵母选育中的应用[J].酿酒科技,2008,10(172):40-42.
[79]岳思君,李学斌,李爱华,等.高酶活纤维素分解菌分离筛选的研究[J].安徽农业科学,2009,37(1):11-12,15.
[80]沈雪亮,夏黎明.产纤维素酶细菌的筛选及酶学特性研究[J].林产化学与工业,2002,2(22):47-51..
[81]张晓晖,郭春华,江晓霞,等.康氏木霉基因组DNA提取方法的比较研究[J].生物技术通报,2007(5):129-130.
[82]李艾.耐高温高产酒精酵母的分离鉴定及其应用研究[D].河北农业大学,2007.
[83]王庆国,刘天明.酵母菌分类学方法研究进展[J].微生物学杂志,2007,27(3):97-98.
[84]赵丽丽,陈存社,郭凤莲.26S rDNA序列分析法鉴定酵母菌[J].中国酿造,2008,192(15):49-51.
[85]卢乡怀,明霞.裂殖酵母的酶及可溶性蛋白质聚丙烯酰胺凝胶电泳[J].真菌学报,1990,1(1):50-55.
[86]梁平彦,陈开英.曲霉和青霉内杂种和亲株的蛋白质电泳图谱比较[J].真菌学报,1991,2(2):149-151.
[87]邱雁临,曾莹,胡赛阳,等.原生质体融合子代的筛选和鉴定[J].生物技术,2003,13(6):17-18.
[88]单志萍,姜文侯,孟妤.丝状真菌三孢布拉霉原生质体制备及再生条件的研究[J].江苏食品与发酵,1999,4(99):1-3.
[89]薛平海,宫正,谢鲲鹏,等.灰树花原生质体制备与再生条件的研究[J].食用菌,2004,1(1):13-14.
[90]刘玲,叶博,刘长江.原生质体融合亲本菌株抗药性标记的筛选[J].江苏农业科学,2007,4(3):226-227.
[2]黄治玲.燃料乙醇的生产与利用[J].中小企业科技,2003,1(12):25.
[3]孙逸,贺稚非.纤维素发酵生产酒精的研究进展[J].农产品加工·创新版,2009,170(04):70-71.
[4]张小希,张丽春.纤维素乙醇可持续发展道路的探讨[J].现代化工,2008,28(02):26-27.
[5] Habib G,Hassan M F,Siddiqui M M,Degradation characteristics of straw different wheat genotypes and their response tourea-ammoniation treatment[J] . Animal Feed Science and Technology,1998,72:373-386.
[6]赵彩云,王异静,关东明.纤维素乙醇研究进展[J].酿酒科技,2009,184(10):87-89.
[7]张作阳,田沈,孟繁艳,等.基因组改组及其在木质纤维素水解液乙醇发酵菌种选育中的应用展望[J].微生物学通报,2009,36(06):900-904.
[8]高阳,刘四新.天然纤维素原料生产生物燃料的应用现状[J].广州农业科学,2009,1(07):205.
[9]阴春梅,刘忠,齐宏升.生物质发酵生产乙醇的研究进展[J].酿酒科技,2007,1(151):87-90.
[10]欧阳嘉,李鑫,王向明,等.纤维素结合域的研究进展[J].2008,6(02):10-11.
[11]曲音波.纤维素乙醇产业化—进展与展望[R].山东济南:山东大学,2007.
[12]吴创之,马隆龙.生物质能现代化利用技术[M].北京:化学工业出版社,2003.
[13]段钢,孙长平.酶在生物质转化为燃料酒精中的应用[J].食品与发酵工业,2005,31(5):73-77.
[14]宋新南,宋爽.纤维素生物燃料酒精技术的现状及发展方向[J].酿酒科技,2007,9(159):89-90.
[15]王敏辉.饲用纤维素酶的研究进展[J].添加剂世界,2009,1(11):27.
[16]文少白,李勤奋,侯宪文,等.微生物降解纤维素的研究概况[J].中国农学通报,2010,26(01):231-236.
[17]杨友坤,朱凤香,王卫平,等.纤维素酶及其应用现状[J].安徽农业通报,2009,15(13):59-60.
[18]PALMAROLA-ADRADOS B,CHOTEBORSKA P,GALBE M.etal.Ethanol production from non-starch carbohydrates of wheat bran [J].Biores Tech,2005(96):843-856.
[19]张继泉,孙玉英.发酵戊糖产酒精酵母菌株的选育[J].生物技术通讯,2002,13(4):275-277.
[20]蒋加拉,刘素纯,关美玲.纤维素降解的菌株筛选及其应用[J].现代生物医学进展,2009,9(18):3451.
[21]成亚利,珠宝成,李亮亮,等.金针菇原生质体紫外诱变选育[J].食用菌学报,1995,2(3):61-64.
[22]李德舜,刘正学.平菇山大1号紫外诱变育种研究[J].食用菌,2002,24(3):9-10.
[23]陈五岭,姚胜利.He-Ne激光在香菇速生高产菌株选育中的应用(I)(J).光子学报,1997,26(11):972-976.
[24]李耀维,冯文新,张素梅.He-Ne激光对金针菇SOD高产株的诱变效应[J].激光生物学报,2002,11(4):283-286.
[25]夏志兰,艾幸.60Co射线对杏鲍菇菌丝的诱变效应[J].激光生物学报,2004,13(4):298-301.
[26]李卫旗,何国庆.60Co射线诱变选育热凝胶多糖高产菌株的研究[J].核农学报,2003,17(5):343-346.
[27]曲音波,高培基,王祖农.青霉的纤维素酶抗降解物阻遏突变株的选育[J].真菌学报,1984,3(4):238-242.
[28]陈宏军.蛋白酶高产菌株的紫外诱变选育[J].安徽农业科学,2010,38(3):1110-1111.
[29]崔国艳,陈五岭.He-Ne激光诱变选育高温乳酸菌[J].中外医疗,2009,1(11):15.
[30]程艳,张克诚,赵明富,等.应用Co60γ射线诱变选育武夷菌素高产菌的研究[J].中国农学通报,2010,26(2):234-237.
[31]武秀琴.纤维素酶高产菌株的诱变选育[J].中国酿造,2009,3(204):84-84.
[32]傅力,涂正东,叶凯,等.里氏木霉高产纤维素酶菌株的选育及产酶培养基的优化[J].食品与机械,2009,25(3):10.
[33]戴群,戴传超,顾敏.花生四烯酸高产菌株的诱变和筛选研究[J].食品科技,2010,35(1):19.
[34]Lilly M,Lambrechts M G,Pretorius I S.Effect of Increased Yeast Alcohol Acetyltransferase Activity on Flavor Profiles of Wine and Distillates[J].Applied and Environmental Microbiology,2000,66(2):744-753.
[35]Laura-Leena,Kiiskinen,Markku Saloheimo.Molecular Cloning and Expression in Saccharomyces cereviside of a Laccase Gene from the Ascomycete Melanocarpus albomyces[J].Applied and Environmental Microbiology,2004,70(1):137-144.
[36]Nina Jarvinen,Minna Maki,Jarkko Rabina,etal.Cloning and expression of Helicobacter pylori GDP-L-fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae[J].Eur.J.Biochem,2001,268:6458-6464.
[37]TOSHIO FUJII,NAOSHI NAGASAWA,AKIHIRO IWAMATSU,etal.Molecular Cloning,Sequence Analysis,and Expression of the Yeast Alcohol Acetyltransferase Gene[J].Applied and Environmental Microbiology,1994,60(8):2786-2792.
[38]TSUNG TSNA WANG,LONG LIU LIN,WEN HWEI HSU,Cloning and Expression of a Schwanniomyces occidentalis OL-Amylase Gene in Saccharomyces cerevisiae[J].Applied and Environmental Microbiology,1989,55(12):167-3172.
[39]杨宁,王惠娟,郭利健.工业微生物育种综述[J].湖北农机化,2008,1(3):29.
[40]孟甜,李玉锋.现代工业微生物育种技术研究进展[J].2009,7(12):5.
[41] Salusj?rvi L, Kankainen M, Soliymani R, Pitk?nen J-P, Penttil? M, Ruohonen L. Regulation of xylose metabolism in recombinant Saccharomyces cerevisiae. Microbial Cell Factories, 2008, 7: 18. doi:10 .1186/1475-2859-1187-1118
[42]何明雄,祝其丽,潘科,等.利用木质纤维素类生物质发酵生产乙醇重组菌株研究进展[J].应与环境生物学报,2009,15(4):579-584.
[43] Masahiro Nogawa,Masahiro Goto,Hirofumi Okada,Yasushi-Morikawa.L-Sorbose induces cellulose gene transcription in the cellulolytic fungus Trichodema reesei[J].Curr Genet,2001,38:329-334.
[44]刘泽寰,台艳,唐根云,等.绿色木霉EGⅢ基因的克隆及其在工业酿酒酵母中的整合表达[J].中山大学学报(自然科学版),2009,48(6):83.
[45]王利英,刘一,杨登峰,等.绿色木霉葡聚糖内切酶cDNA基因的克隆及其在酿酒酵母中的表达[J].2007,14(3):315-319.
[46] SONDEREGGER M,SCHUMPERLI M,SAUER U.Metabolic engineering of a phosphoketolase pathway for pentose catabolism in Saccharomyces cerevisiae[J].Appl. Environ .Microbiol.,2004,70(5):2892-2897.
[47]刘建军,赵祥颖,姜鲁燕,等.高产酒精絮凝酵母SY-130菌株的选育[J].山东食品发酵,2004,135(4):8.
[48]黄玉茜,梁春浩,陈捷.绿色木霉菌T23原生质体的制备和再生[J].吉林农业大学学报,2007,29(1):24-27.
[49]高榕.高生产效率纤维素酶菌株的初筛方法的研究[J].纤维素科学与技术,2004,912 (3):23-27.
[50]滕利荣,等.生物学基础实验教程(第一版)[M].吉林科学出版社,1999.316-490.
[51]王蕾,等.DNS法测定海藻糖的研究[J].食品科技,2004,2(2):82-84.
[52]苏畅,等.氨基葡萄糖和乙酰氨基葡萄糖的测定方法[J].食品工业科技,2004,1(6):74-76.
[53]靖德兵,李培军,王力华,等.康氏木霉固体发酵生产纤维素酶优化研究—培养基用料与工艺条件优化[J].食品科学,2004,25(5):82-83.
[54]刘海波,王义,陈介南,等.一株高产纤维素酶菌的筛选与鉴定[J].生物学杂志,2008,25(3):16-17.
[55]苏艳秋,朱卫华,吴鹏,等.耐高温、耐酸产酒精酵母的筛选与鉴定[J].微生物学杂志,2009,29(2):44-45.
[56]蒋加拉,刘素纯,关美玲,等.纤维素降解的菌株筛选及其运用[J].现代生物医学进展,2009,9(18):3451-3453.
[57]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994.
[58]张怀东,刘作易,周礼红,等.高耐性高产酒精酵母的分离和筛选[J].酿酒科技,2009,177(3):40-41.
[59]Kurtzman CP,Robnett CJ.Identification of clinically important ascomycetous yeasts based on nucleotide divergence in 5′end of the large-subunit(26S) ribosomal DNA gene[J]..J Clin Microbiol,1997,35(5):1216-1223.
[60]姜明国,何小丹,裴氏秋河,等.板栗疫病菌致病性机理的双向凝胶电泳法的研究[J].广西植物,2008,28(1):117-120.
[61]邱雁临,曾莹,胡赛阳,等.原生质体融合子代的筛选和鉴定[J].生物技术,2003,13(6):17-18.
[62]诸葛健,王正祥.工业微生物实验技术手册[M].北京:中国轻工业出版社,1994.
[63]赵乐辉,李颖,吕淑霞.木霉T21和T22原生质体制备和再生研究初报[J].沈阳农业大学学报,2005,36(1):96-98.
[64]王景林,殷勤燕,陈宗泽,等.康氏木霉B-7和黑曲霉X-15原生质体的形成和再生[J].微生物学杂志,1999,1(19):20-21.
[65]周庆新,陈静,于恺,等.疏绵状嗜热丝孢菌原生质体的制备与再生[J].菌物研究,2006,4(2):1-5.
[66]朱艳,薛正莲,张相美,等.林肯链霉菌原生质体制备条件的研究[J].四川食品与发酵,2008,44(3):13-14.
[67]包莹玲,潘力.茂原链霉菌原生质体制备条件的优化[J].现代食品科技,2008,24(2):138-140.
[68]金卫根,孙丽萍.茶新菇原生质体制备条件的分析[J].江西农业学报,2009,21(11):147-148.
[69]祝子坪,李娜,贺建东.渗透压稳定剂对桑黄菌原生质体分离与再生的影响[J].食用菌,2009 (5):25-26.
[70]张拥华,李晶晶,彭志刚,等.粉红粘帚霉67-1菌株原生质体的形成和再生[J].中国生物防治,2006,22(4):303-307.
[71]梁平彦.产黄青霉两株营养缺陷型原生质体的形成和再生的因素的研究[J].植物生理学报,1981,7(1):1-9.
[72]邢来君,张军.米曲霉两株营养缺陷型原生质体的形成和再生的条件实验[J].真菌学报,!987,6(4):242-247.
[73]王立洪,杨土风,杨涛,等.平菇原生质体的高效制备及再生优化条件[J].应用与环境生物学报,2008,14(3):351-355.
[74]王慧,王玉华,段宁宁,等.出芽短梗霉原生质体制备及再生的研究[J].食品科技,2008,2(5):1-3.
[75]朱藴兰,周卫东,陈安徽,等.冬虫夏草无性型原生质体再生条件研究[J].安徽农业科学,2009,37(30):14629-14631.
[76]任选.漆酶高活性菌株的选育及其融合子F49的初步鉴定[D].山东:山东农业大学,2008.
[77]阎晓星.绿粘帚霉和球孢白僵菌原生质体制备、再生和融合条件的研究[D].四川:四川农业大学,2007.
[78]董博宇,陈叶福,岳瑞雪,等.TTC在发酵木糖高产乙醇的休哈塔假丝酵母选育中的应用[J].酿酒科技,2008,10(172):40-42.
[79]岳思君,李学斌,李爱华,等.高酶活纤维素分解菌分离筛选的研究[J].安徽农业科学,2009,37(1):11-12,15.
[80]沈雪亮,夏黎明.产纤维素酶细菌的筛选及酶学特性研究[J].林产化学与工业,2002,2(22):47-51..
[81]张晓晖,郭春华,江晓霞,等.康氏木霉基因组DNA提取方法的比较研究[J].生物技术通报,2007(5):129-130.
[82]李艾.耐高温高产酒精酵母的分离鉴定及其应用研究[D].河北农业大学,2007.
[83]王庆国,刘天明.酵母菌分类学方法研究进展[J].微生物学杂志,2007,27(3):97-98.
[84]赵丽丽,陈存社,郭凤莲.26S rDNA序列分析法鉴定酵母菌[J].中国酿造,2008,192(15):49-51.
[85]卢乡怀,明霞.裂殖酵母的酶及可溶性蛋白质聚丙烯酰胺凝胶电泳[J].真菌学报,1990,1(1):50-55.
[86]梁平彦,陈开英.曲霉和青霉内杂种和亲株的蛋白质电泳图谱比较[J].真菌学报,1991,2(2):149-151.
[87]邱雁临,曾莹,胡赛阳,等.原生质体融合子代的筛选和鉴定[J].生物技术,2003,13(6):17-18.
[88]单志萍,姜文侯,孟妤.丝状真菌三孢布拉霉原生质体制备及再生条件的研究[J].江苏食品与发酵,1999,4(99):1-3.
[89]薛平海,宫正,谢鲲鹏,等.灰树花原生质体制备与再生条件的研究[J].食用菌,2004,1(1):13-14.
[90]刘玲,叶博,刘长江.原生质体融合亲本菌株抗药性标记的筛选[J].江苏农业科学,2007,4(3):226-227.