黑木耳单核杂交菌株远源性的研究
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摘要
近几年来,随着人民生活水平的逐渐提高和科学技术水平的不断发展,黑木耳的食用和药用价值得到了人们的广泛认可。随着黑木耳的消费量逐年递增,带动着黑木耳栽培及下游产业的迅速发展。菌种是黑木耳产业的根本,没有好的菌种,将严重制约着黑木耳产业的发展。与其他食用菌育种方式相比,黑木耳的品种选育工作比较落后,很少有杂交品种,目前使用情况较好的菌种都是通过野生黑木耳菌种人工驯化得来的。选育黑木耳新品种已成为发展该产业的重中之重。
杂交育种是目前育种方法中最有成效的一个,黑木耳杂交育种都是用从亲本菌株分离得到的孢子单核菌株进行杂交,进而筛选并获得优良菌株。本实验首先选取HW5号和139号两株远源性大的优良菌种作为亲本菌株,然后制备HW5号菌株的原生质体菌株和139号菌株的孢子菌株,利用荧光显微镜和简单重复序列(ISSR)分子标记技术以及酯酶同工酶法来确定菌株单、双核及交配型,分别获得20个孢子单核菌株和2种交配型的原生质体单核菌株,用这两种单核菌株进行配对杂交,共获得6个杂交菌株。
进一步通过酯酶同工酶方法证实6个杂交菌株中均表现有不同程度的遗传互补性,共获得37条酶带,1条新增酶带,并用ISSR分子标记技术对杂交菌株进行分析,采用NTSYS2.10e软件计算菌株间的遗传相似系数,然后进行聚类分析。结果表明,6个杂交菌株共分成5类,Z2、Z4在遗传距离上距亲本菌株较远。Z1、Z2、Z4与亲本相比在同工酶和ISSR扩增图谱上有明显优势。通过出耳试验,获得杂交菌株的产量,耳型,抗杂菌能力,商品性能等指标,再进行综合评价,初步得到了优良的具有远源性的杂交Z1。
本论文筛选出具有双亲优秀基因的远源性菌株,为下一步能够从杂交菌株中筛选出抗逆性强、品质好、产量高、具有综合优良性状的新菌株打下基础,减少初筛栽培量、提高育种效率,不断满足国内外市场的需求,提高我国菌种的质量,为我国黑木耳产业的长足发展做出贡献。
In recent years, with the gradual increase in people's living standards and the continuous development of scientific and technological level, Auricularia auricular’s nutrition and medicinal value have been recognized widely. Following the increasing consumption of Auricularia auricular by years, its cultivation and downstream industries have been developed rapidly. Since strains are the foundation of the industry, without good strains, the industry will be restricted seriously. Now its breed methods fell behind other edible fungi. There are few cross breeding strains and the good strains normally came from artificial domestication of wild Auricularia auricular. Breeding new Auricularia species gains a top priority in the development of this industry.
Cross breeding is the most effective breed method. Now all the cross-breed Auricularia auriculars are breed by spore monocaryon strains from parent strains and then screened to get the good ones. In this paper, first two good strains HW5 and 139 with far origins were selected as the parent, then protoplast monocaryon of HW5 and spore monocaryon of 139 were prepared. The monocaryon, dual-core and mating type strains of Auricularia auricula were identified using the technique of fluorescent microscopy, and ISSR molecular marker and esterase isozyme. 20 spore monocaryon strains and 2 protoplast monocaryon mating type strains were obtained. With these cross breeding strains, 6 hybrid strains were obtained.
Furthermore, by esterase isozyme analysis it is confirmed that all the 6 hybrid strains present different levels of genetic complement, and 37 enzyme belts including a new one were obtained. At the same time, ISSR molecular marker analysis was used to screen hybrid strains. Meantime NTSYS2.10e software was applied to calculate the genetic similarity between these strains, and cluster analysis was preceded. The results showed that the six hybrid strains were divided into 5 categories, Z2, Z4 were farther away from the parent strains in genetic distance. And Z1, Z2 and Z4 have obvious advantages in isozyme and ISSR amplification patterns over parent strains. Yield, shape, anti-bacteria capacity, commodity performance and other parameters of hybrid strains were preliminary known through ear cultivation tests. After comprehensive evaluation, new good hybrid strains with far origins named Z1.
In this paper, the hybrid strains with far origins and good genes derived from parent strains were selected, which made a foundation for further screening of new strains with strong resistance, good quality, high yield and good characters, meanwhile reduce the quantity of first cultivation, improve the breeding efficiency, continuously meet the needs of domestic and foreign markets, improve the quality of Auricularia auricular strains, and contribute to the rapid development of Auricularia auricular industry in China.
杂交育种是目前育种方法中最有成效的一个,黑木耳杂交育种都是用从亲本菌株分离得到的孢子单核菌株进行杂交,进而筛选并获得优良菌株。本实验首先选取HW5号和139号两株远源性大的优良菌种作为亲本菌株,然后制备HW5号菌株的原生质体菌株和139号菌株的孢子菌株,利用荧光显微镜和简单重复序列(ISSR)分子标记技术以及酯酶同工酶法来确定菌株单、双核及交配型,分别获得20个孢子单核菌株和2种交配型的原生质体单核菌株,用这两种单核菌株进行配对杂交,共获得6个杂交菌株。
进一步通过酯酶同工酶方法证实6个杂交菌株中均表现有不同程度的遗传互补性,共获得37条酶带,1条新增酶带,并用ISSR分子标记技术对杂交菌株进行分析,采用NTSYS2.10e软件计算菌株间的遗传相似系数,然后进行聚类分析。结果表明,6个杂交菌株共分成5类,Z2、Z4在遗传距离上距亲本菌株较远。Z1、Z2、Z4与亲本相比在同工酶和ISSR扩增图谱上有明显优势。通过出耳试验,获得杂交菌株的产量,耳型,抗杂菌能力,商品性能等指标,再进行综合评价,初步得到了优良的具有远源性的杂交Z1。
本论文筛选出具有双亲优秀基因的远源性菌株,为下一步能够从杂交菌株中筛选出抗逆性强、品质好、产量高、具有综合优良性状的新菌株打下基础,减少初筛栽培量、提高育种效率,不断满足国内外市场的需求,提高我国菌种的质量,为我国黑木耳产业的长足发展做出贡献。
In recent years, with the gradual increase in people's living standards and the continuous development of scientific and technological level, Auricularia auricular’s nutrition and medicinal value have been recognized widely. Following the increasing consumption of Auricularia auricular by years, its cultivation and downstream industries have been developed rapidly. Since strains are the foundation of the industry, without good strains, the industry will be restricted seriously. Now its breed methods fell behind other edible fungi. There are few cross breeding strains and the good strains normally came from artificial domestication of wild Auricularia auricular. Breeding new Auricularia species gains a top priority in the development of this industry.
Cross breeding is the most effective breed method. Now all the cross-breed Auricularia auriculars are breed by spore monocaryon strains from parent strains and then screened to get the good ones. In this paper, first two good strains HW5 and 139 with far origins were selected as the parent, then protoplast monocaryon of HW5 and spore monocaryon of 139 were prepared. The monocaryon, dual-core and mating type strains of Auricularia auricula were identified using the technique of fluorescent microscopy, and ISSR molecular marker and esterase isozyme. 20 spore monocaryon strains and 2 protoplast monocaryon mating type strains were obtained. With these cross breeding strains, 6 hybrid strains were obtained.
Furthermore, by esterase isozyme analysis it is confirmed that all the 6 hybrid strains present different levels of genetic complement, and 37 enzyme belts including a new one were obtained. At the same time, ISSR molecular marker analysis was used to screen hybrid strains. Meantime NTSYS2.10e software was applied to calculate the genetic similarity between these strains, and cluster analysis was preceded. The results showed that the six hybrid strains were divided into 5 categories, Z2, Z4 were farther away from the parent strains in genetic distance. And Z1, Z2 and Z4 have obvious advantages in isozyme and ISSR amplification patterns over parent strains. Yield, shape, anti-bacteria capacity, commodity performance and other parameters of hybrid strains were preliminary known through ear cultivation tests. After comprehensive evaluation, new good hybrid strains with far origins named Z1.
In this paper, the hybrid strains with far origins and good genes derived from parent strains were selected, which made a foundation for further screening of new strains with strong resistance, good quality, high yield and good characters, meanwhile reduce the quantity of first cultivation, improve the breeding efficiency, continuously meet the needs of domestic and foreign markets, improve the quality of Auricularia auricular strains, and contribute to the rapid development of Auricularia auricular industry in China.
引文
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[4]武晶.应用形态学和生物化学方法对黑木耳菌属进行分类鉴定的研究[D].吉林:吉林农业大学, 2008.
[5]刘茵华.我国的食用木耳[J].中国食用菌, 1995, 3(14): 6-7.
[6]文华.黑木耳—最佳补铁食品[J].保健食品与健康, 2005, 11:39.
[7]刘雅静,刘秀河.黑木耳营养保健研究进展[J].中国食物与营养, 2010, 10:67.
[8]李时珍.本草纲目[M].北方文艺出版社. 2007. 9.
[9]张才擎.黑木耳药用研究的进展[J].中国中医药科技, 2001, 8(5): 339.
[10]付湘晋.我国天然黑色素资源研究概况[J].粮食与油脂, 2005, 12: 42-44.
[11]黄滨南,张秀娟.黑木耳多糖抗肿瘤作用的研究[J]. 2004, 20(6): 13.
[12]谢意珍,周静文.木耳降血脂作用的研究.吉林中医药[J]. 2006,26 ( 10 ): 59-60.
[13]韩春然,徐丽萍.黑木耳多糖的提取、纯化及降血脂作用的研究[J].中国食品学报, 2007, 7 ( 1 ): 54-57.
[14]陈志强,舒融,骆传环.木耳多糖的制备及辐射防护作用实验研究[J].中华放射医学与防护杂志, 2001, 21 ( 1 ): 46-47.
[15]郭素芬,成永霞,孙平.黑木耳多糖对动脉粥样硬化血管平滑肌细胞表型转化及bFGF和PDGF表达变化的研究[J].热带医学杂志, 2009, 9 ( 8 ): 868-870.
[16]王贺祥.食用菌学[M].中国农业出版社. 2004. 8.
[17]王香,苏文.黑木耳高产栽培技术[J].河南农业, 2009, 12 (上): 36-37.
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