CN114149927B - 一种秀珍菇菌株黔秀3号及其应用 - Google Patents
一种秀珍菇菌株黔秀3号及其应用 Download PDFInfo
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Abstract
本发明属于食用菌领域,具体公开了一种秀珍菇(Pleurotus pulmonarius)菌株黔秀3号,其保藏编号为CCTCC NO:M20211210。本发明还公开了该菌株在食品、保健品或饮料等方面的用途。本发明菌株黔秀3号的菌盖淡褐色,朵形饱满,商品价值高;其次,栽培周期短,黔秀3号比现有秀珍菇主栽品种生产周期至少缩短了15~20天;此外,黔秀3号产量高,其每袋平均产量为352.3g,经济效益高。本发明还公开了对该菌株具有特异性的分子标记,用该分子标记鉴定黔秀3号,结果准确、可靠;检测方法简单、速度快;适于在黔秀3号的真实性或侵权鉴定中进行现场或实时检测。
Description
技术领域
本发明属于食用菌领域,具体涉及一种秀珍菇菌株,还涉及该秀珍菇菌株的用途。
背景技术
秀珍菇(学名:肺形侧耳。别名:凤尾菇、凤尾侧耳、印度平菇或喜马拉雅平菇等;其拉丁名为:Pleurotus pulmonarius)属于侧耳科侧耳属肺形侧耳种。秀珍菇外形小巧秀美,不仅质地细嫩,味道鲜美,而且营养丰富,含有丰富的蛋白质、糖类、不饱和脂肪酸、维生素、叶酸和多种微量元素等;秀珍菇的蛋白质含量比香菇、草菇都高,其子实体中含有18种氨基酸,其中天冬氨酸和谷氨酸含量较高。另外,秀珍菇的蛋白含量高、低脂肪酸的特性,可作为减肥保健食品;其含有的多糖成分具有抗肿瘤功能,还能提高人体免疫力。故秀珍菇被视为是一种营养价值极高的珍稀食用菌,有“菇中极品”的美誉。
目前,秀珍菇生产中存在的问题:一是秀珍菇品种较少,国家认定的秀珍菇品种仅有2个,二是栽培周期长。难以满足人们高水平生活的消费需求。
我国从南到北的原始森林中具有丰富的野生食用菌资源,其中包括秀珍菇资源。对野生的秀珍菇资源进行开发,不仅可以丰富人们的餐桌,而且还可以利用食用菌药食兼用的特性,开发出附加值更高的食品、饮料和保健品等,从而大大提高菇农的收入和出口创汇能力。
发明内容
为了解决目前生产中秀珍菇品种少,生产周期长等问题,本发明目的在于提供一种秀珍菇菌株。
本发明另一目的在于提供上述秀珍菇菌株的用途。
本发明的目的通过如下技术方案实现:
本发明一种秀珍菇(Pleurotus pulmonarius)菌株黔秀3号,已保藏于中国典型培养物保藏中心,地址中国武汉武汉大学,其保藏编号为CCTCC NO:M20211210,保藏日期为2021年9月24日。
本发明还提供上述秀珍菇菌株黔秀3号在食品或保健品中的应用。
本发明还提供一种食品或保健品,所述的食品或保健品中含有上述菌株黔秀3号或黔秀3号的提取物。
本发明还提供上述菌株黔秀3号在饮料中的应用。
本发明还提供一种饮料,所述的饮料中含有上述菌株黔秀3号或黔秀3号的提取物。
上述菌株黔秀3号在制备提高人体免疫力药物上的应用。
本发明还提供一种秀珍菇的基因工程菌株,所述的秀珍菇基因工程菌株的出发菌株为上述菌株黔秀3号。
本发明还提供一种用于鉴定上述菌株黔秀3号的分子标记,所述的分子标记是长度为568bp的DNA分子片段;所述的DNA分子片段的核苷酸序列如SEQ ID No:1所示。
上述分子标记,其所述的分子标记的PCR扩增引物为通用引物ITS1和ITS4;所述的引物序列如下:
ITS1:5'-TCCGTAGGTGAACCTGCGG-3';
ITS4:5'-TCCTCCGCTTATTGATATGC-3'。
上述分子标记在菌株黔秀3号真实性或侵权鉴定中的应用。
本发明还提供了上述菌株黔秀3号的栽培方法,包括如下步骤:
(1)母种培养:将黔秀3号菌种接种于母种培养基上,在25℃、空气相对湿度60~75%、避光条件下培养8天,得黔秀3号母种;
(2)原种培养:按照1:30的质量比将步骤(1)中所得的黔秀3号母种接种于原种培养基上,在25℃、空气相对湿度60~75%,避光条件下培养25~30天,得黔秀3号原种;
(3)栽培种培养:按照1:30的质量比将步骤(2)中所得的黔秀3号原种接种到栽培培养基上,在25℃、空气相对湿度60~75%,避光条件下培养25~30天,得黔秀3号栽培种;
(4)栽培培养料接种:在无菌环境中,取出菌袋中心的塑料棒,按照1:30的质量比将步骤(3)中所得的黔秀3号栽培种接种到栽培培养料中心的棒孔中,再用套环和棉塞封口;在25℃、空气相对湿度60~75%,避光条件下培养30~35天,菌丝长满培养料;
(5)搔菌:将菌袋袋口打开,再用灭菌后的小铲刀将表面的老菌皮挖去,露出新鲜菌丝;
(6)低温刺激:将菌袋置于4℃环境中低温刺激12小时,低温刺激促进出菇整齐;
(7)出菇管理和采收:出现原基后,在温度23℃、空气相对湿度80~90%、散射光500~600lx、通风良好条件下培养6~8天;当子实体菌盖平展,孢子未弹射时,采收子实体。
上述栽培方法步骤(1)中所述的母种培养基的组成成分及其重量比为:去皮马铃薯200g取汁,葡萄糖20g,琼脂18g,磷酸二氢钾2g,硫酸镁1g,混匀后用纯净水定容至1000mL。
所述的母种培养基的制备方法:将马铃薯200g去皮切成1.5cm左右方块,放入800mLRO水中煮沸20min,3层纱布过滤取浸提液,再加入葡萄糖20g,琼脂18g,磷酸二氢钾2g,硫酸镁1g,混匀后用纯净水定容至1000mL,121℃灭菌30min;倒入直径9cm平板,备用。
上述栽培方法步骤(2)中所述原种培养基或步骤(3)中所述栽培种培养基的组成成分及其重量百分比为:玉米芯40%,棉籽壳40%,麦麸15%,豆粨2%,石灰2%,轻质碳酸钙1%,培养基含水量为60~65%。
所述步骤(2)中原种培养基或步骤(3)中栽培种培养基的制备方法:按步骤比例取玉米芯40%,棉籽壳40%,麦麸15%,豆粨2%,石灰2%,轻质碳酸钙1%,混合均匀,然后按照比例加水,搅拌均匀,装入聚丙烯塑料袋(规格15cm×33cm×0.005cm),121℃灭菌2小时,备用。
上述方法步骤(4)中所述的栽培培养料的组成成分及其重量百分比为:玉米芯40%,棉籽壳40%,麦麸15%,豆粨2%,石灰2%,轻质碳酸钙1%,培养料中含水量60~65%。
所述步骤(4)中栽培培养料的制备方法:按照重量百分比比例将玉米芯40%,棉籽壳40%,麦麸15%,豆粨2%,石灰2%,轻质碳酸钙1%,混合均匀,然后按照比例加水,搅拌均匀。栽培袋采用聚丙烯塑料袋(规格15cm×33cm×0.005cm),将混匀的栽培料装入袋中,袋口中心插入13cm空心塑料棒,3.5cm套环配套棉塞封口,121℃灭菌2小时,再冷却至常温;备用。
本发明具有的优点和有益技术效果:(1)本发明菌株黔秀3号的菌盖淡褐色,朵形饱满,商品价值高,为消费者提供了一种新的秀珍菇类型。(2)本发明黔秀3号的栽培周期短。其从栽培培养料接种到出菇采收的周期仅为55~60天,而现有的秀珍菇品种:凤尾菇、京秀1号、秀03、夏秀004、中农秀珍(国品认菌2008027)、秀珍菇5号(国品认菌2008026)”的栽培周期分别为60~80天、60~70天、70~90天、70~80、70~90天、70~90天,黔秀3号比现有秀珍菇主栽品种生产周期至少缩短15~20天。(3)本发明黔秀3号的产量高,其每袋平均产量为352.3g,经济效益高。(4)本发明分子标记对黔秀3号特异性强,检测结果准确、可靠;检测方法简单、速度快;适于在黔秀3号的真实性或侵权鉴定中进行现场或实时检测。
生物保藏:本发明秀珍菇(Pleurotus pulmonarius)菌株黔秀3号是本发明人在贵州省贵阳市花溪区阿哈湖水库地区采集,并经筛选、培育而成;该菌株已于保藏于中国典型培养物保藏中心;保藏地址为:地址中国武汉武汉大学;其保藏编号为CCTCC NO:M20211210。
附图说明:
图1.本发明菌株黔秀3号的子实体照片.
图2.本发明菌株黔秀3号的系统发育树图。
图3.采用RAPD引物S43扩增的不同秀珍菇菌种的PAPD电泳图谱;其中M为DNA分子量标准;1为黔秀3号;2为凤尾菇;3为京秀1号;4为秀03;5为夏秀004。
具体实施方式
以下通过实例对本发明作进一步说明,但是对本发明不构成任何限制。
实施例1本发明菌株黔秀3号的采集、分离驯化过程
2019年8月,本发明人在贵州省贵阳市花溪区阿哈湖水库地区,发现并采集了一株野生的类似秀珍菇的子实体。将该子实体进行组织分离,得到菌丝(菌种);将组织分离后所得菌种进行栽培出菇试验;再对朵型良好的子实体进行组织分离,接种于PDA培养基上进行培养,筛选菌丝生长速度快、长势均匀的菌株继续栽培驯化,经过数代培养,筛选出一个遗传性状稳定、出菇周期短的秀珍菇菌株,命名为:黔秀3号。
实施例2本发明菌株黔秀3号的分类鉴定
(1)黔秀3号菌株的形态学鉴定:
子实体丛生,菌盖淡褐色呈扇形,菌盖截面平整,中部稍凹,菌盖直径4.3~10.2cm,平均7.5cm;菌盖厚度适中,0.7~1.1cm,平均0.8cm;菌柄长0.9~2.1cm;菌褶无网纹,菌褶延生至菌柄顶端;子实体质地紧密。根据《中国大型菌物资源图鉴》(李玉主编,中原农民出版社,2015年出版)第990页有关秀珍菇的照片及相应特征描述,黔秀3号与秀珍菇(肺形侧耳)的形态特征一致,初步确定黔秀3号在分类上属于侧耳科侧耳属肺型侧耳种(Pleurotus pulmonarius)。
(2)黔秀3号菌株的分子生物学分类鉴定
采用新型植物基因组DNA提取试剂盒CW0531(北京康为世纪公司)提取黔秀3号菌株的基因组DNA,以ITS1和ITS4为引物进行PCR扩增。所述的ITS1和ITS4引物序列如下:
ITS1:5'-TCCGTAGGTGAACCTGCGG-3';
ITS4:5'-TCCTCCGCTTATTGATATGC-3'。
ITS-PCR反应体系为:总体积20μL:20-50ng/μL的DNA模板1μL,10×Buffer(withMg2+)2μL,2.5mM dNTP 0.5μL,5U/μl DNA聚合酶0.2μL,0.2μM引物各0.5μL,加双蒸水至20μL。ITS-PCR反应条件为:预变性94℃5min;94℃1min,60℃1min,72℃75s,30个循环;72℃延伸10min。将所得扩增产物进行凝胶电泳,结果获得长度为568bp的DNA分子片段。将所得DNA分子片段送交上海生工进行测序,该DNA分子片段的核苷酸序列如SEQ ID No:1所示。将该核苷酸序列在GeneBank上进行BLAST比对,并构建系统发育树图。结果本发明菌株黔秀3号菌株与秀珍菇(Pleurotus pulmonarius)的相似度最高,可达99.62%,说明本发明菌株黔秀3号在分类上属于侧耳属肺形侧耳种(Pleurotus pulmonarius)。从系统发育树图(见图2)也可得出相同结论。此外,从BLAST对比可知,本发明菌株黔秀3号与任何其他的秀珍菇菌株都不相同,是一种新的秀珍菇菌株。
实施例3本发明菌株黔秀3号与其他秀珍菇菌种的PAPD对比分析试验按照如下方法进行:
采用植物基因组DNA提取试剂盒CW0531(北京康为世纪公司)分别提取黔秀3号、凤尾菇(北京吉蕈园选育出的淡灰色秀珍菇)、京秀1号(北京吉蕈园选育出的淡褐色秀珍菇)、秀03(黔东南创健台江的灰色秀珍菇),夏秀004(江苏天龙菌业选育出的褐色秀珍菇)菌株的基因组DNA,选取20条RAPD引物(见表1)进行PCR扩增。
表1 RAPD随机引物序列
RAPD-PCR扩增反应体系为:总体积20μL:20-50ng/μL的DNA模板1μL,10×Buffer(with Mg2+)2μL,2.5mM dNTP 0.5μL,5U/μl EasyTaq酶0.2μL,0.2μM引物1μL,加双蒸水至20μL。RAPD-PCR反应条件为:预变性95℃5min;94℃45s,36℃1min,72℃2min,35个循环;72℃延伸10min。
从以S43为引物扩增所得的RAPD电泳图谱(见图3)可以看出,黔秀3号相对于凤尾菇、京秀1号、秀03、夏秀004均存在具有明显差异的特异性条带,说明本发明菌株黔秀3号与现有的凤尾菇、京秀1号、秀03、夏秀004等秀珍菇菌株都不同,是一种新的秀珍菇(Pleurotus pulmonarius)菌株。
实施例4本发明菌株黔秀3号的栽培试验
按照如下步骤进行:
(1)母种培养:将黔秀3号菌种(本菌种已保藏于中国典型培养物保藏中心,地址中国武汉武汉大学,保藏编号为CCTCC NO:M20211210)接种于母种培养基上,在25℃、空气相对湿度70%、避光条件下培养8天,得黔秀3号母种;母种培养基的制备方法:将200g去皮马铃薯切成1.5cm左右方块,放入800mL水中煮沸20min,3层纱布过滤取浸提液,加入葡萄糖20g,琼脂18g,磷酸二氢钾2g,硫酸镁1g,混匀后定容至1000mL,121℃灭菌30min;倒入直径90mm平板备用。
(2)原种培养:按照1:30的质量比将步骤(1)中黔秀3号母种接种于原种培养基上,在25℃、空气相对湿度70%,避光条件下培养30天,得黔秀3号原种。所述原种培养基的组成成分及其重量百分比为:玉米芯40%,棉籽壳40%,麦麸15%,豆粨2%,石灰2%,轻质碳酸钙1%,培养基含水量65%。所述原种培养基的按照如下方法制备:按照重量百分比将各组分混合均匀,然后按比例加入水,混合均匀,即为原种培养基。将其装入聚丙烯塑料袋(15cm×33cm×0.005cm),121℃灭菌2小时。
(3)栽培种培养:按照1:30的质量比将步骤(2)中所得黔秀3号原种接种到栽培培养料上。在25℃、空气相对湿度70%,避光条件下培养30天,得黔秀3号栽培种。其中栽培培养料的配制,装袋和灭菌方式与原种培养基相同,但是在在装袋时在袋口中心插入13cm空心塑料棒,3.5cm套环配套棉塞封口,121℃灭菌2小时备用。
(4)栽培培养料接种:将按照步骤(3)制备的栽培培养料,灭菌后充分冷却至常温。在无菌环境中,取出菌袋中心的塑料棒,按照1:30的质量比将步骤(3)中所得黔秀3号栽培种接种到栽培培养料中心的棒孔内,再用套环和棉塞封口,在25℃、空气相对湿度70%,避光条件下培养35天。
(5)搔菌:将步骤(4)的菌袋袋口打开,用灭菌后的小铲刀将表面的老菌皮挖去,露出新鲜菌丝。
(6)低温刺激:将菌袋置于4℃环境中低温刺激12小时,低温刺激促进菇蕾整齐。
(7)出菇管理和采收:出现原基后,在温度23℃、空气相对湿度85%、散射光500lx,通风良好条件下培养8天;当子实体菌盖平展,菌盖边缘平伸,为最适宜采收期,可采收子实体。采收时左手按住栽培袋,右手握住菌柄基部,轻轻旋钮即可完成采摘,采摘后用保鲜盒封装。
将采收后的秀珍菇黔秀3号进行肉眼观察、称重,并计算其栽培周期。
结果本发明黔秀3号的子实体(见图1)质地紧密,口感清脆,菌盖淡褐色,朵形饱满,商品价值高;其次,黔秀3号从栽培培养料接种到出菇采收的周期为55~60天,而现有主栽秀珍菇品种凤尾菇、京秀1号、秀03、夏秀004、中农秀珍(国品认菌2008027)、秀珍菇5号(国品认菌2008026)”等秀珍菇的相应栽培周期分别为60~80天、60~70天、70~90天、70~80、70~90天、70~90天,黔秀3号比现有秀珍菇主栽品种栽培周期至少缩短10~20天。此外,本发明黔秀3号农业式栽培每袋平均产量为352.3g,产量高,经济效益高。
序列表
<110> 贵州省土壤肥料研究所(贵州省生态农业工程技术研究中心)(贵州省农业资源与环境研究所)
<120> 一种秀珍菇菌株黔秀3号及其应用
<141> 2021-11-11
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 568
<212> DNA
<213> Pleurotus pulmonarius
<400> 1
gttgctggcc tctaggggca tgtgcacgct tcactagtct ttcaaccacc tgtgaacttt 60
tgatagacct gtgaagtcgt ccttcaagtc gtcagacttg gtttgctggg atttaaacgt 120
ctcggtgtga caacgcagtc tatttactta acacacccca aatgtatgtc tacgaatgtc 180
atttaatggg ccttgtgcct ataaaccata atacaacttt caacaacgga tctcttggct 240
ctcgcatcga tgaagaacgc agcgaaatgc gataagtaat gtgaattgca gaattcagtg 300
aatcatcgaa tctttgaacg caccttgcgc cccttggtat tccgaggggc atgcctgttt 360
gagtgtcatt aaattctcaa actcacattt atttgtgatg tttggattgt tgggggttgc 420
tggctgtaac aagtcggctc ctcttaaatg cattagcagg acttttcatt gcctctgcgc 480
atgatgtgat aattatcact catcaatagc acgcatgaat agagtccagc tctctaatcg 540
tccgcaagga caatttgaca attgacct 568
Claims (6)
1.一种秀珍菇(Pleurotus pulmonarius)菌株黔秀3号,已保藏于中国典型培养物保藏中心,其保藏编号为CCTCC NO:M20211210。
2.权利要求1所述的菌株黔秀3号在食品或保健品中的应用。
3.一种食品或保健品,所述的食品或保健品中含有权利要求1所述的菌株黔秀3号或黔秀3号的提取物。
4.权利要求1所述的菌株黔秀3号在饮料中的应用。
5.一种饮料,所述的饮料中含有权利要求1所述的菌株黔秀3号或黔秀3号的提取物。
6.权利要求1所述的菌株黔秀3号在制备提高人体免疫力药物上的应用。
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