双孢蘑菇单孢子杂交及杂交菌株酯酶同工酶研究
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摘要
为了发展双孢蘑菇(Ayaricus bisporus)生产,选育优良的蘑菇菌种是个关键的问题之一。在改良蘑菇菌种和选育优良新菌种的方法中,以单孢菌株选育和单孢杂交选育为比较重要和值得应用的方法,可以使双孢蘑菇的单产较大幅度的提高。
本文采用了引自加拿大的双孢蘑菇品种Ta、Tb,以及国内主栽品种As2796,通过对双孢蘑菇菌农艺学性状的测定,对国外引进品种、国内主栽品种的生产性能作了评价。同时选用引自加拿大的Tb和国内普遍栽培的As2796双孢蘑菇为亲本菌株进行单孢分离研究,找到一种较为简单易行、高效、选择性强的单孢分离法。对这些单孢菌株的培养性状和自发营养缺陷型等进行研究,利用营养缺陷型单孢菌株进行杂交,以营养缺陷因子互补作为杂合体选择标记。并进行酯酶同工酶电泳分析。结果如下：
(1)通过引进品种与国内主要栽培种的农艺学性状比较,从加拿大引进的Tb表现出高产性状、出菇早、且出菇整齐,子实体肉质细嫩,口味较好。但在抗逆性和适应性方面,国内品种As2796优于Tb。
(2)对传统的单孢分离方法进行了改进,加入吐温使分散的孢子在无菌水中分布均匀,简单易行,提高了单孢分离的效率。抗生素的加入有效的降低了杂菌的污染。注射器的使用使得整个稀释过程完全在一个密闭无菌的环境中进行,进一步降低了染菌率,试验中污染率为5.30%。双孢蘑菇的单孢子不容易萌发。双孢蘑菇单孢子的萌发不仅取决于其自身的内在因素外,还与其外界的培养条件密切相关。高密度的孢子和同种刺激菌丝体的存在能显著的提高单孢子的萌发率,双孢蘑菇孢子的质量对单孢子萌发产生很大影响,而添加刺激物对孢子萌发影响并不大。
(3)在双孢蘑菇的单孢培养物中存在着自发营养缺陷突变,试验中从429株Tb单孢菌株中筛选到48株营养缺陷型菌株,发生率为11.3%；从127株As2796单孢菌株中筛选到13株营养缺陷型菌株,发生率为10.2%。共有61株被鉴定为营养缺陷型,平均发生率10.8%。其中蛋氨酸、腺嘌呤营养缺陷型占有很大比重,蛋氨酸占营养缺陷菌株的34.4%,腺嘌呤占营养缺陷菌株的21.3%,组氨酸占有8.2%,其他的缺陷因子型占有的比例很小。营养缺陷菌株杂交试验中,不同品系的菌株之间的杂交率很低,为18.1%。最后鉴定为营养缺陷互补的杂交菌株为13株。并观察其生长情况,淘汰生长不好的杂交菌株,保留下长势较好的杂交菌株有编号：AT035011、AT045121、AT061243、AT110355、AT035341、AT078121、AT022352、AT116345、AT056129共9株。
(4)酯酶同工酶分析结果表明,杂交菌株出现了双亲没有的新谱带(Rf=0.40),这条谱带出现的原因可能与杂交种的杂交优势有关。酯酶同工酶电泳结果说明杂交菌株既遗传了亲本的大部分遗传物质,同时也因为杂交发生了一些变异。结合前面的分析,杂交菌株AT061243、AT035011、AT116345、AT035341可能综合了双亲各自的优良性状,Tb菌株高产优质和As2796菌株适应力强的优点。但也要看到AT061243、AT035341、AT116345三菌株的差异很小。其中AT116345与AT035341具有完全相同的酶带,说明二者可能是相同的种。
In order to develop the Ayaricus bisporus producing, selecting good strain is the key factor. The technique of single spore isolation and crossbreeding are the most improtant and valuable method, and we can promot the yield of mushroom by this way.
Taking two introduced varities of Agaricus bisporus Ta and Tb, which are introduced from Canada, and two main domestic varieties As2796 as research projects, by way of mensurating the agronomic characteristics of Agaricus bisporus to appraise the production performance of the strains which introduced overseas and that of main domestic varieties. At the same time, takeing Ayaricus bisporus Tb, and Ayaricus bisporus As2796 as reseach objects, by way of monospore isolation and breeding, reseach on the factor which influence on monospore sprout, besides improved the method of monospore isolation, adopted a kind of more simple, effective and selective measure, also study on growing characteristics and spontaneous auxotroph of their monospore strains. Then use auxotrophic monospore strains crossbreeding each other and take advantage of auxotrophic complementary as selective mark of hybrids. Finally hybrids proved by esterase isozyme electrophoresis. The result indicate that:
(1) Comparing the agronomic characteristics of introduced varieties with that of main domestic varieties, we could see that strains Tb, which were introduced from Canada, showed its with high production, tender meat, good shape, very delicious. But As2796 have good adaptability and resistibility than Tb.
(2) Application of tween-80 and penicillin, effectual improve efficiency of Single spore isolation, besides greatly reduce pollution of Bacteria. Pollution rate only 5.30% in experiment. Ayaricus bisporus monospore germinate very difficult. Momospore ger-minate depend not only on their own internal factors, but also with the outside cultiva-teng conditions. Living mycelia of same strain and high-dense spores can effectual st-imulate monospore to germinate. Qulity of Ayaricus bisporus spores greatly influence on monospore to germinate, but stimulus without greatly influence.
(5) The spontaneous auxotrophic mutant have been finded in monospore strains. Author find 48 auxotrophic strains from 429 Tb monospore strains, which mutation rate is 11.3%, and find 13 auxotrophic strains from 127 As2796 monospore strains, which mutation rate is 10.2%, and mutation average rate is 10.8%.Auxotrophic strain-s crossbreeding rate is very low only 13%. Met- and Ade- account for very large prop-ortion, Met- account for 34.4% in auxotrophic strains, Ade- account for 21.3% in aux-otrophic strains, His- account for 8.2% in auxotrophic strains, other auxotrophic fator-'s stains rarely find. Then,13 hybrid strains have been proved auxotrophic compleme-ntary hybrid strains in cross-breeding experiment. Finally, author reserve 9 growth better strains:AT035011, AT045121,AT061243,AT110355,AT035341,AT078121, AT 022352, AT116345,AT056129.
(6) The esterase isozyme electrophoresis patterns showed not only most of the genetic material of the parents are descended, but also there were some genetic differences between the hybrid strains and the parent strains. Author find new esterase isozyme electrophoresis band(Rf=0.40) in hybrid strains. Also the results show hybrid strains:AT061243, AT035011, AT116345, AT035341 may integrated the merits of their parents.
本文采用了引自加拿大的双孢蘑菇品种Ta、Tb,以及国内主栽品种As2796,通过对双孢蘑菇菌农艺学性状的测定,对国外引进品种、国内主栽品种的生产性能作了评价。同时选用引自加拿大的Tb和国内普遍栽培的As2796双孢蘑菇为亲本菌株进行单孢分离研究,找到一种较为简单易行、高效、选择性强的单孢分离法。对这些单孢菌株的培养性状和自发营养缺陷型等进行研究,利用营养缺陷型单孢菌株进行杂交,以营养缺陷因子互补作为杂合体选择标记。并进行酯酶同工酶电泳分析。结果如下：
(1)通过引进品种与国内主要栽培种的农艺学性状比较,从加拿大引进的Tb表现出高产性状、出菇早、且出菇整齐,子实体肉质细嫩,口味较好。但在抗逆性和适应性方面,国内品种As2796优于Tb。
(2)对传统的单孢分离方法进行了改进,加入吐温使分散的孢子在无菌水中分布均匀,简单易行,提高了单孢分离的效率。抗生素的加入有效的降低了杂菌的污染。注射器的使用使得整个稀释过程完全在一个密闭无菌的环境中进行,进一步降低了染菌率,试验中污染率为5.30%。双孢蘑菇的单孢子不容易萌发。双孢蘑菇单孢子的萌发不仅取决于其自身的内在因素外,还与其外界的培养条件密切相关。高密度的孢子和同种刺激菌丝体的存在能显著的提高单孢子的萌发率,双孢蘑菇孢子的质量对单孢子萌发产生很大影响,而添加刺激物对孢子萌发影响并不大。
(3)在双孢蘑菇的单孢培养物中存在着自发营养缺陷突变,试验中从429株Tb单孢菌株中筛选到48株营养缺陷型菌株,发生率为11.3%；从127株As2796单孢菌株中筛选到13株营养缺陷型菌株,发生率为10.2%。共有61株被鉴定为营养缺陷型,平均发生率10.8%。其中蛋氨酸、腺嘌呤营养缺陷型占有很大比重,蛋氨酸占营养缺陷菌株的34.4%,腺嘌呤占营养缺陷菌株的21.3%,组氨酸占有8.2%,其他的缺陷因子型占有的比例很小。营养缺陷菌株杂交试验中,不同品系的菌株之间的杂交率很低,为18.1%。最后鉴定为营养缺陷互补的杂交菌株为13株。并观察其生长情况,淘汰生长不好的杂交菌株,保留下长势较好的杂交菌株有编号：AT035011、AT045121、AT061243、AT110355、AT035341、AT078121、AT022352、AT116345、AT056129共9株。
(4)酯酶同工酶分析结果表明,杂交菌株出现了双亲没有的新谱带(Rf=0.40),这条谱带出现的原因可能与杂交种的杂交优势有关。酯酶同工酶电泳结果说明杂交菌株既遗传了亲本的大部分遗传物质,同时也因为杂交发生了一些变异。结合前面的分析,杂交菌株AT061243、AT035011、AT116345、AT035341可能综合了双亲各自的优良性状,Tb菌株高产优质和As2796菌株适应力强的优点。但也要看到AT061243、AT035341、AT116345三菌株的差异很小。其中AT116345与AT035341具有完全相同的酶带,说明二者可能是相同的种。
In order to develop the Ayaricus bisporus producing, selecting good strain is the key factor. The technique of single spore isolation and crossbreeding are the most improtant and valuable method, and we can promot the yield of mushroom by this way.
Taking two introduced varities of Agaricus bisporus Ta and Tb, which are introduced from Canada, and two main domestic varieties As2796 as research projects, by way of mensurating the agronomic characteristics of Agaricus bisporus to appraise the production performance of the strains which introduced overseas and that of main domestic varieties. At the same time, takeing Ayaricus bisporus Tb, and Ayaricus bisporus As2796 as reseach objects, by way of monospore isolation and breeding, reseach on the factor which influence on monospore sprout, besides improved the method of monospore isolation, adopted a kind of more simple, effective and selective measure, also study on growing characteristics and spontaneous auxotroph of their monospore strains. Then use auxotrophic monospore strains crossbreeding each other and take advantage of auxotrophic complementary as selective mark of hybrids. Finally hybrids proved by esterase isozyme electrophoresis. The result indicate that:
(1) Comparing the agronomic characteristics of introduced varieties with that of main domestic varieties, we could see that strains Tb, which were introduced from Canada, showed its with high production, tender meat, good shape, very delicious. But As2796 have good adaptability and resistibility than Tb.
(2) Application of tween-80 and penicillin, effectual improve efficiency of Single spore isolation, besides greatly reduce pollution of Bacteria. Pollution rate only 5.30% in experiment. Ayaricus bisporus monospore germinate very difficult. Momospore ger-minate depend not only on their own internal factors, but also with the outside cultiva-teng conditions. Living mycelia of same strain and high-dense spores can effectual st-imulate monospore to germinate. Qulity of Ayaricus bisporus spores greatly influence on monospore to germinate, but stimulus without greatly influence.
(5) The spontaneous auxotrophic mutant have been finded in monospore strains. Author find 48 auxotrophic strains from 429 Tb monospore strains, which mutation rate is 11.3%, and find 13 auxotrophic strains from 127 As2796 monospore strains, which mutation rate is 10.2%, and mutation average rate is 10.8%.Auxotrophic strain-s crossbreeding rate is very low only 13%. Met- and Ade- account for very large prop-ortion, Met- account for 34.4% in auxotrophic strains, Ade- account for 21.3% in aux-otrophic strains, His- account for 8.2% in auxotrophic strains, other auxotrophic fator-'s stains rarely find. Then,13 hybrid strains have been proved auxotrophic compleme-ntary hybrid strains in cross-breeding experiment. Finally, author reserve 9 growth better strains:AT035011, AT045121,AT061243,AT110355,AT035341,AT078121, AT 022352, AT116345,AT056129.
(6) The esterase isozyme electrophoresis patterns showed not only most of the genetic material of the parents are descended, but also there were some genetic differences between the hybrid strains and the parent strains. Author find new esterase isozyme electrophoresis band(Rf=0.40) in hybrid strains. Also the results show hybrid strains:AT061243, AT035011, AT116345, AT035341 may integrated the merits of their parents.
引文
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