褐色双孢蘑菇原生质体制备、再生条件的研究
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摘要
为了提高褐色双孢蘑菇原生质体产量和再生率,本研究以褐色双孢蘑菇棕秀一号为材料,利用Design-Expert 8.0.6软件进行二次回归分析对褐色双孢蘑菇制备原生质体的条件进行优化。结果表明,原生质体最佳制备条件为溶壁酶1.35%、蜗牛酶7.01%、酶解温度30.4℃、褐色双孢蘑菇菌丝体菌龄8.36 d、酶解时间3.00 h,在此条件下原生质体产量高达4.39×10~7个·mL~(-1);当溶壁酶含量为1.32%、蜗牛酶含量为6.96%、褐色双孢蘑菇菌丝体菌龄9.00 d、酶解时间2.97 h、酶解温度31.0℃时,原生质体的再生率最高(15.1%)。利用紫外诱变和化学诱变技术对褐色双孢蘑菇原生质体进行生长特性诱变,筛选获得了18株生长速率提高的突变菌株。本研究为褐色双孢蘑菇在遗传转化、基因组重排、基因编辑技术等后续分子遗传学研究提供了理论基础。
In order to improve the yield and regeneration rate of Agaricus bisporus Portabello protoplast. Mushroom strain was ZONGXIU No.1 was selected, and two regression analysis with Design-Expert 8.0.6 software were used to optimize the preparation conditions of protoplast. The results showed that the yield of protoplast was arrived at the highest with 4.39×10~7·mL~(-1) in the condition of lywallzyme concentration1.35%, snailase concentration 7.01%, enzymolysis time 3.00 h, mycelium age 8.36 days and enzymolysis temperature 30.4℃. The highest protoplast regeneration rate with 15.1% was found in the condition of lywallzyme concentration 1.32%, snailase concentration 6.96%, enzymolysis time 2.97 h, mycelium age 9.00 days and enzymolysis temperature 31.0℃. The ultraviolet mutagenesis and chemical mutagenesis were used to mutate the protoplast growth characteristic, and eighteen strains with improved growth characteristics were obtained. The results could provide theoretical basis for protoplasts transformation, genome rearrangement and genome targeted editing for Agaricus bisporus Portobello.
In order to improve the yield and regeneration rate of Agaricus bisporus Portabello protoplast. Mushroom strain was ZONGXIU No.1 was selected, and two regression analysis with Design-Expert 8.0.6 software were used to optimize the preparation conditions of protoplast. The results showed that the yield of protoplast was arrived at the highest with 4.39×10~7·mL~(-1) in the condition of lywallzyme concentration1.35%, snailase concentration 7.01%, enzymolysis time 3.00 h, mycelium age 8.36 days and enzymolysis temperature 30.4℃. The highest protoplast regeneration rate with 15.1% was found in the condition of lywallzyme concentration 1.32%, snailase concentration 6.96%, enzymolysis time 2.97 h, mycelium age 9.00 days and enzymolysis temperature 31.0℃. The ultraviolet mutagenesis and chemical mutagenesis were used to mutate the protoplast growth characteristic, and eighteen strains with improved growth characteristics were obtained. The results could provide theoretical basis for protoplasts transformation, genome rearrangement and genome targeted editing for Agaricus bisporus Portobello.
引文
[1] 蔡为明, 方菊莲, 金群力, 冯伟林, 范丽军. 蘑菇“棕秀一号”的特性及栽培技术[J]. 食用菌, 2003, 25(4): 10
[2] 王泽生, 廖剑华, 陈美元, 蔡志欣. 双孢蘑菇遗传育种和产业发展[J]. 食用菌学报, 2012, 19(3): 1-14
[3] Sinden J W. Strain adaptability[J]. Mushroom Journal, 1981, 101(10): 153-165
[4] Fritsche G. On the use of monospores in breeding selected strains of cultivated mushroom[J]. Theoretical and Applied Genetics, 1972, 42(2): 62-64
[5] Kneebone L R, Patton T G, Schultz P G. Improvement of the brown variety of Agaricus bisporus by single spore selection[J]. Mushroom Science, 1976, 9: 237-243
[6] Nazrul M Ⅰ, bian Y B. Efficiency of RAPD and ISSR markers in differentiation of homo- and hetero-karyoticprotoclones of Agaricus bisporus[J]. Journal of Microbiology and Biotechnology, 2010, 20(4):683-692
[7] Nazrul M I, Bian Y B. ISSR as new markers for the identification of homokaryoticprotoclones of Agaricus bisporus[J]. Current Microbiology, 2009, 60(2):92-98
[8] Raper C A. Sexuality and the life cycle of the edible, wild Agaricus bitorquis[J]. Journal of General Microbiology, 1976, 95(1): 54-66
[9] Wang Z S, Wang H C. Study on the genetic variation of Agaricus bisporus[C]//Proceedings of the International Symposium on Mushroom Biotechnology, Nanjing: Mushroom Biotechnology, 1989: 329-338
[10] Wang Z S, Liao J H. Identification of field-collected isolates of Agaricus bisporus[J]. Micologia Neotrpicla Aplicada, 1993, 3(6): 127-136
[11] Bueno F S, Romuc A, Wac H M, Araujo W L, Azevedo L. Variability in commercial and wild isolates of Agaricus species in Brazil[J]. Mushroom Science, 2008, 17(1): 135-148
[12] Morin E, Kohler A, Baker A R, Foulongne O M, Lombard Ⅴ, Nagy L G, Ohm R A, Patyshakuliyeva A, Brun A, Aerts A L, Bailey A M, Billette C, Coutinho P M, Deakin G, Doddapaneni H, Floudas D, Grimwood J, Hildén K, Kües U, Labutti K M, Lapidus A, Lindquist E A, Lucas S M, Murat C, Riley R W, Salamov A A, Schmutz J, Subramanian Ⅴ, Wösten H A B, Xu J. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(43): 17501-17506
[13] 谭周进, 杨海君, 林曙, 吴铁. 利用原生质体融合技术选育微生物菌种[J]. 核农学报, 2005, 19(1): 75-79
[14] 余波, 吕向云, 董风晴, 王永红. 凝结芽孢杆菌原生质体的制备、再生及灭活[J]. 食品科技, 2017, 42(4): 2-7
[15] 魏洪美, 任锡毅, 杨兵, 黄青青, 刘永祥, 乙引. 竹黄菌原生质体的制备与再生分析[J]. 基因组学与应用生物学, 2016, 35(4): 918-923
[16] 李小六, 张运峰, 张淑红, 李少凤, 方永山, 刘海英. 原生质体EMS诱变选育杏鲍菇高产菌丝多糖菌株[J]. 核农学报, 2017, 31(8): 1463-1468
[17] 李艳丽, 金周雨, 李玉. 原生质体紫外诱变筛选刺芹侧耳高EPS含量菌株及其鉴定[J]. 中国农机化学报, 2016, 37(12): 98-102
[18] 吴思梦, 刘冰, 蒋军喜, 刘英, 周珍. 生防链霉菌ML27和柑橘内生放线菌P3Y2原生质体融合研究[J]. 中国农业科技导报, 2017, 19(3): 31-36
[19] 曾柏全, 李淼, 冯金儒. 双亲灭活青霉菌与枯草芽孢杆菌原生质体融合[J]. 中国食品学报, 2015, 15(6): 45-50
[20] 孙墨可, 张曼, 田娟, 李晓微, 李海燕, 李玉. 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
[21] 朱文静. SQS基因转化日本灵芝原生质体的研究[D]. 吉林: 吉林农业大学, 2014
[22] 陈建中. 基因组重排技术在草菇耐低温菌株选育上的应用[D]. 上海: 上海海洋大学, 2013
[23] 张素敏, 王国增, 叶秀云, 郭耀湘,林娟. 基因组重排技术选育纤维素酶高产菌株[J]. 中国食品学报, 2016, 16(11): 105-111
[24] 罗润, 林俊芳, 郭丽琼, 叶志伟, 郭天芬, 云帆. 基于CRISPR/Cas9系统的金针菇基因组编辑载体构建[J]. 食品工业科技, 2016, 37(20): 230-234
[25] 贺建超,贺聪莹,卢美欢,马英辉,王玛丽. 双孢蘑菇和姬松茸灭活原生质体融合育种研究[J]. 中国食用菌, 2014, 33(1):9-10
[26] 黎雪, 杨涛, 刘慧美, 陈琴, 龙章富. 双孢蘑菇原生质体融合子的特性与分子鉴定[J]. 四川大学学报(自然科学版), 2015, 52(4): 884-888
[27] 王波, 唐利民, 熊鹰, 姜邻. 双孢蘑菇原生质体分离与再生初探[J]. 西南农业学报, 2004, 17(2): 215-216
[28] 孙溪, 郭成金. 双孢蘑菇原生质体制备条件的优化[J]. 天津师范大学学报(自然科学版), 2006, 26(4): 32-35
[29] 王泽生, 廖剑华, 王贤樵. 双孢蘑菇原生质体融合及融合子变异研究[J]. 食用菌,1991, 1(5):10
[30] 韩业君, 曹晖, 陈明杰, 潘迎捷. 草菇原生质体制备与再生及诱变效应研究[J]. 食用菌学报, 2004, 11(2): 1-6
[31] 赖谱富, 陈君琛, 杨艺龙, 翁敏劼, 李怡彬, 沈恒胜. 超声波-内部沸腾法提取杏鲍菇多糖的工艺优化[J]. 核农学报, 2016, 30(12): 2382-2390
[32] 赖谱富, 方汝涛, 陈君琛, 杨艺龙. 超声波-酶法提取大杯蕈菇柄多糖及流变性研究[J]. 核农学报, 2015, 29(10): 1944-1953
[33] 孙姣, 陈振斌, 刘军, 刘赛武, 王小琛. 葡萄糖水溶液乳化柴油制备工艺参数优化[J]. 农业工程学报, 2015, 31(1): 228-235
[34] 马跃腾, 杜双田, 丁建, 纪晓朋, 鲍蕊, 李珍. 秦巴蛹虫草原生质体的制备及再生条件研究[J]. 西北农林科技大学学报(自然科学版), 2015, 43(9):196-202
[35] 田鸿, 张小平, 余桂荣, 李前红. 双孢蘑菇担孢子60Co-γ射线诱变效应初步研究[J]. 四川大学学报(自然科学版), 2016, 53(6): 1423-1428
[2] 王泽生, 廖剑华, 陈美元, 蔡志欣. 双孢蘑菇遗传育种和产业发展[J]. 食用菌学报, 2012, 19(3): 1-14
[3] Sinden J W. Strain adaptability[J]. Mushroom Journal, 1981, 101(10): 153-165
[4] Fritsche G. On the use of monospores in breeding selected strains of cultivated mushroom[J]. Theoretical and Applied Genetics, 1972, 42(2): 62-64
[5] Kneebone L R, Patton T G, Schultz P G. Improvement of the brown variety of Agaricus bisporus by single spore selection[J]. Mushroom Science, 1976, 9: 237-243
[6] Nazrul M Ⅰ, bian Y B. Efficiency of RAPD and ISSR markers in differentiation of homo- and hetero-karyoticprotoclones of Agaricus bisporus[J]. Journal of Microbiology and Biotechnology, 2010, 20(4):683-692
[7] Nazrul M I, Bian Y B. ISSR as new markers for the identification of homokaryoticprotoclones of Agaricus bisporus[J]. Current Microbiology, 2009, 60(2):92-98
[8] Raper C A. Sexuality and the life cycle of the edible, wild Agaricus bitorquis[J]. Journal of General Microbiology, 1976, 95(1): 54-66
[9] Wang Z S, Wang H C. Study on the genetic variation of Agaricus bisporus[C]//Proceedings of the International Symposium on Mushroom Biotechnology, Nanjing: Mushroom Biotechnology, 1989: 329-338
[10] Wang Z S, Liao J H. Identification of field-collected isolates of Agaricus bisporus[J]. Micologia Neotrpicla Aplicada, 1993, 3(6): 127-136
[11] Bueno F S, Romuc A, Wac H M, Araujo W L, Azevedo L. Variability in commercial and wild isolates of Agaricus species in Brazil[J]. Mushroom Science, 2008, 17(1): 135-148
[12] Morin E, Kohler A, Baker A R, Foulongne O M, Lombard Ⅴ, Nagy L G, Ohm R A, Patyshakuliyeva A, Brun A, Aerts A L, Bailey A M, Billette C, Coutinho P M, Deakin G, Doddapaneni H, Floudas D, Grimwood J, Hildén K, Kües U, Labutti K M, Lapidus A, Lindquist E A, Lucas S M, Murat C, Riley R W, Salamov A A, Schmutz J, Subramanian Ⅴ, Wösten H A B, Xu J. Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche[J]. Proceedings of the National Academy of Sciences of the United States of America, 2012, 109(43): 17501-17506
[13] 谭周进, 杨海君, 林曙, 吴铁. 利用原生质体融合技术选育微生物菌种[J]. 核农学报, 2005, 19(1): 75-79
[14] 余波, 吕向云, 董风晴, 王永红. 凝结芽孢杆菌原生质体的制备、再生及灭活[J]. 食品科技, 2017, 42(4): 2-7
[15] 魏洪美, 任锡毅, 杨兵, 黄青青, 刘永祥, 乙引. 竹黄菌原生质体的制备与再生分析[J]. 基因组学与应用生物学, 2016, 35(4): 918-923
[16] 李小六, 张运峰, 张淑红, 李少凤, 方永山, 刘海英. 原生质体EMS诱变选育杏鲍菇高产菌丝多糖菌株[J]. 核农学报, 2017, 31(8): 1463-1468
[17] 李艳丽, 金周雨, 李玉. 原生质体紫外诱变筛选刺芹侧耳高EPS含量菌株及其鉴定[J]. 中国农机化学报, 2016, 37(12): 98-102
[18] 吴思梦, 刘冰, 蒋军喜, 刘英, 周珍. 生防链霉菌ML27和柑橘内生放线菌P3Y2原生质体融合研究[J]. 中国农业科技导报, 2017, 19(3): 31-36
[19] 曾柏全, 李淼, 冯金儒. 双亲灭活青霉菌与枯草芽孢杆菌原生质体融合[J]. 中国食品学报, 2015, 15(6): 45-50
[20] 孙墨可, 张曼, 田娟, 李晓微, 李海燕, 李玉. 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
[21] 朱文静. SQS基因转化日本灵芝原生质体的研究[D]. 吉林: 吉林农业大学, 2014
[22] 陈建中. 基因组重排技术在草菇耐低温菌株选育上的应用[D]. 上海: 上海海洋大学, 2013
[23] 张素敏, 王国增, 叶秀云, 郭耀湘,林娟. 基因组重排技术选育纤维素酶高产菌株[J]. 中国食品学报, 2016, 16(11): 105-111
[24] 罗润, 林俊芳, 郭丽琼, 叶志伟, 郭天芬, 云帆. 基于CRISPR/Cas9系统的金针菇基因组编辑载体构建[J]. 食品工业科技, 2016, 37(20): 230-234
[25] 贺建超,贺聪莹,卢美欢,马英辉,王玛丽. 双孢蘑菇和姬松茸灭活原生质体融合育种研究[J]. 中国食用菌, 2014, 33(1):9-10
[26] 黎雪, 杨涛, 刘慧美, 陈琴, 龙章富. 双孢蘑菇原生质体融合子的特性与分子鉴定[J]. 四川大学学报(自然科学版), 2015, 52(4): 884-888
[27] 王波, 唐利民, 熊鹰, 姜邻. 双孢蘑菇原生质体分离与再生初探[J]. 西南农业学报, 2004, 17(2): 215-216
[28] 孙溪, 郭成金. 双孢蘑菇原生质体制备条件的优化[J]. 天津师范大学学报(自然科学版), 2006, 26(4): 32-35
[29] 王泽生, 廖剑华, 王贤樵. 双孢蘑菇原生质体融合及融合子变异研究[J]. 食用菌,1991, 1(5):10
[30] 韩业君, 曹晖, 陈明杰, 潘迎捷. 草菇原生质体制备与再生及诱变效应研究[J]. 食用菌学报, 2004, 11(2): 1-6
[31] 赖谱富, 陈君琛, 杨艺龙, 翁敏劼, 李怡彬, 沈恒胜. 超声波-内部沸腾法提取杏鲍菇多糖的工艺优化[J]. 核农学报, 2016, 30(12): 2382-2390
[32] 赖谱富, 方汝涛, 陈君琛, 杨艺龙. 超声波-酶法提取大杯蕈菇柄多糖及流变性研究[J]. 核农学报, 2015, 29(10): 1944-1953
[33] 孙姣, 陈振斌, 刘军, 刘赛武, 王小琛. 葡萄糖水溶液乳化柴油制备工艺参数优化[J]. 农业工程学报, 2015, 31(1): 228-235
[34] 马跃腾, 杜双田, 丁建, 纪晓朋, 鲍蕊, 李珍. 秦巴蛹虫草原生质体的制备及再生条件研究[J]. 西北农林科技大学学报(自然科学版), 2015, 43(9):196-202
[35] 田鸿, 张小平, 余桂荣, 李前红. 双孢蘑菇担孢子60Co-γ射线诱变效应初步研究[J]. 四川大学学报(自然科学版), 2016, 53(6): 1423-1428