CN114736806B - 药植太空灵芝新菌株及其应用 - Google Patents
药植太空灵芝新菌株及其应用 Download PDFInfo
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Abstract
本发明涉及食药用菌技术领域，具体涉及药植太空灵芝新菌株及其应用。本发明提供的灵芝新菌株即灵芝(Ganoderma lucidum)药植太空灵芝1号，该菌株为由宇宙飞船搭载而经太空诱变、选育获得的优异灵芝菌株。其保藏编号为：CGMCC No.16782。本发明筛选得到一株灵芝新菌株，适用于栽培，其中灵芝多糖等有效成分含量高，可以获得优质灵芝原药材。
Description
技术领域
本发明涉及食药用菌技术领域，更确切的说，涉及一种经太空诱变获得的灵芝新菌株。
背景技术
《神农本草经》将灵芝列为上品。灵芝有“仙草”的美誉，是祥瑞、如意、富贵和长寿的象征。灵芝的人工栽培始于二十世纪六十年代，近年来产业不断发展。由于过分采集和生态环境变化,野生灵芝越来越少。而人工栽培灵芝菌种退化,产量不稳,质量下降,很多灵芝药材达不到药典的要求。中药材种子是中医药产业的源头，是中药材质量和中医疗效的根本保障。为保证灵芝质量，亟待进行灵芝优异菌株的选育。
太空育种是集航天技术、生物技术和农业育种技术于一体的农业育种新途径，是将农作物种子种苗送到太空，利用太空特殊环境(高真空，宇宙高能离子辐射，宇宙磁场、高洁净)的诱变作用，使种质产生变异，再返回地面选育新品种。
发明内容
本发明筛选得到一株灵芝新菌株，适用于栽培，其中灵芝多糖等有效成分含量高，可以获得优质灵芝原药材。
本发明提供的灵芝新菌株即灵芝(Ganoderma lucidum)药植太空灵芝1号，该菌株为由宇宙飞船搭载而经太空诱变、选育获得的优异灵芝菌株。
本发明提供的灵芝(Ganoderma lucidum)药植太空灵芝1号已于2018年10月 31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编100101)，分类命名为灵芝Ganodermalucidum，其保藏编号为：CGMCC No.16782。
本发明提供的灵芝(Ganoderma lucidum)药植太空灵芝1号(CGMCC No.16782)是由韩芝经二次太空诱变而来：首先通过神舟三号搭载，经生物学鉴定、栽培试验，筛选出一株发生正向变异、农艺性状改善的菌株，再将此菌株通过神舟七号宇宙飞船进行二次搭载，继续进行栽培试验和选育，筛选出的生长速度快、抗逆性强、产量增加、多糖含量高的灵芝菌株。灵芝多糖主要以杂糖形式存在，包括酸性/碱性多糖以及水溶性多糖等，是灵芝的次生代谢产物，灵芝最主要的有效成分，具有提高机体的免疫力和耐缺氧能力、抑制肿瘤、消除自由基等药理活性。
菌株灵芝(Ganoderma lucidum)药植太空灵芝1号(CGMCC No.16782)的形态特征如下：
子实体特征：菌盖近圆形，木栓质，宽12-23cm，厚1-1.2cm，红褐色并有油漆光泽，菌盖上具有环状棱纹和辐射状皱纹，边缘薄。菌肉淡褐色，管孔初期白色，后期变浅褐色，平均每毫米3-5个，柄侧生，与菌盖几乎垂直，长3-12cm，粗1.2-3cm，紫褐色，有光泽。孢子卵形，一端平截，10.5-11.5um×5.5-6.5um，具双层壁。外壁透明，平滑，内壁近褐色，有小刺，中间有油滴。菌落呈白色、无味、辐射状生长、浓密。菌丝有隔、无色透明，壁厚，具有锁状联合。
生物学特性：营养成分来源，药植太空灵芝1号可用改良PDA培养基(马铃薯200g，麦麸50g，葡萄糖20g，磷酸二氢钾3g，硫酸镁1.5g，维生素B1 0.1g，琼脂粉15g，蒸馏水1000mL，pH自然)保存，适宜在4℃冰箱低温冷藏保存，每3个月转接1次。
药植太空灵芝1号的菌种扩繁生产：母种生产适宜用改良PDA培养基培养；原种和栽培种生产适宜用以阔叶树木屑为主料，麦麸为辅料组成的培养基。
培养及栽培条件：菌丝生长温度10℃～38℃，最适23℃～28℃。
本发明还提供上述新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号 (CGMCCNo.16782)在栽培中的应用。可采用本领域常规方法栽培药植太空灵芝1号。
实验证明，采用新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号(CGMCCNo.16782)进行栽培，可以提高灵芝质量。
本发明还提供采用新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号 (CGMCCNo.16782)培养所得的灵芝菌丝体。
本发明还提供采用新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号 (CGMCCNo.16782)栽培所得的灵芝(子实体)。
本发明还提供上述新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号(CGMCCNo.16782)或其培养所得的灵芝菌丝体或其栽培所得的灵芝(子实体) 在制备食品、保健食品或药物上的应用。
采用新菌株灵芝(Ganoderma lucidum)药植太空灵芝1号(CGMCC No.16782)进行培养，实验证明该菌的漆酶活力、纤维素酶活力、木聚糖活力显著高于其它菌株，生长速度较快，用于菌种制作时缩短生产周期。另外该菌多糖含量较高，显示出较高的药用价值。
附图说明
图1为本发明实施例中菌株C的孢子的显微镜图。
图2表示本发明实施例中三株灵芝菌株的拮抗实验结果。
图3为本发明实施例中灵芝菌株的系统发育树。
图4为本发明实施例中三株灵芝菌株的不同培养天数的酶活性。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
实施例1
1.菌株获得
选用栽培生产上相对优良的灵芝(韩芝)菌株作为出发菌株，分别接入经灭菌处理的培养基，25℃恒温培养12天，然后将带菌丝的培养基在无菌条件下装入灭菌处理的聚丙烯塑料袋，封口，搭载组装入微生物样品盒内。对照组置于冰箱冷藏。宇宙飞船的生物搭载采用生命培养箱，由铝镁合金制成，外面有一物理保温层。经过宇宙飞船的太空飞行返回地面的灵芝菌株，转接于培养基进行培养，弃去已被太空诱变致死的菌株，将存活的菌株与对照进行对比试验、包括生物学性质比较、栽培、产量和质量测定，筛选出优质变异菌株。本发明所涉及的新菌株药植太空灵芝1号，是由栽培的韩芝菌株经神舟三号宇宙飞船(发射时间：2002 年3月25日，返回时间：2002年4月1日)搭载、经栽培、选育获得性状改善的韩芝M1菌株，再将韩芝M1经神舟七号宇宙飞船(发射时间：2008年09月 25日，返回时间：2008年09月28日)搭载，历经多年栽培试验和选育，筛选出的生长速度快、抗逆性强、产量增加、灵芝多糖含量高的优异灵芝菌株。
将该菌株编号为灵芝(Ganoderma lucidum)药植太空灵芝1号，于2018年10 月31日保藏于中国微生物菌种保藏管理委员会普通微生物中心(简称CGMCC，地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编 100101)，分类命名为灵芝Ganoderma lucidum，其保藏编号为：CGMCC No.16782。
2.菌种鉴定
为便于描述，本文将出发菌株韩芝、韩芝M1和药植太空灵芝1号菌株分别编号为菌株A、菌株B、菌株C。
2.1菌株C形态鉴定
菌盖近圆形，木栓质，宽12-23cm，厚1-1.2cm，红褐色并有油漆光泽，菌盖上具有环状棱纹和辐射状皱纹，边缘薄。菌肉淡褐色，管孔初期白色，后期变浅褐色，平均每毫米3-5个，柄侧生，与菌盖几乎垂直，长3-12cm，粗1.2-3cm，紫褐色，有光泽。孢子卵形，一端平截，10.5-11.5um×5.5-6.5um，具双层壁。外壁透明，平滑，内壁近褐色，有小刺，中间有油滴。菌落呈白色、无味、辐射状生长、浓密。菌丝有隔、无色透明，壁厚，具有锁状联合。
菌株C的孢子的显微镜图见图1。
经形态鉴定，药植太空灵芝1号属于灵芝(Ganoderma lucidum(Leyss.Ex Fr.)Karst。
2.2拮抗试验
担子菌体细胞的非亲和性(Samatic incompatibility)即高等担子菌在双核菌丝阶段发生的认识自我和排斥异己的过程。遗传背景明显不同的个体在交界区会形成明显的分界线，即拮抗反应。将不同菌株活化后，在同一平板上按“品”字形接种3个菌株，25℃恒温下培养。结果见图2。由图2可见菌株间的拮抗反应。显示药植太空灵芝1号与另2个菌株存在差异。
2.3分子鉴定：
ITS序列所用的序列片段引物为通用引物：
ITS1(5′-GGAAGTAAAAGTCGTAACAAGG-3′)和
ITS4(5′-TCCTCCGCTTATGATATGC-3′)对灵芝ITS区域进行扩增。 PCR反应采用30μL反应体系：2×EasyPCR Super Mix 15μL，10μmol/L上下游引物各1μL，DNA模板1μL，ddH2O 12μL。PCR反应扩增程序为：94℃预变性3min，94℃变性40s，54℃退火45s，72℃延伸1min，完成35个循环，最后72℃延伸10min，然后4℃保存。
供试菌株的ITS序列依次与GenBank中已登录的15条ITS序列(其中 14条为灵芝属灵芝、紫芝、树舌、四川灵芝、南方灵芝、树灵芝、韦伯灵芝等菌株的ITS序列，1条为猴头菇ITS序列)进行相似性比对，采用MEGA5.0软件对比对结果以Neighbor-Joining构建系统发育进化树。
灵芝菌株的系统发育树见图3。图3中，1.耐高温红芝；2.美国灵芝；3.韩芝； 4.信州灵芝；5.韩芝M1；6.大红芝；7.中华灵芝；9.盆景灵芝；10.霍山赤芝；11. 大片芝；12.药植太空灵芝1号；16.鹿角灵芝；17.大别山灵芝；18.黑花灵芝；19. 赤灵芝；20.紫芝；21.红花灵芝；22.黑芝；23.泰山灵芝；24.粉芝；25.元宝灵芝； 26.沪农灵芝1号；27.荷兰紫芝；28.日本紫芝；29.灵芝8号。
药植太空灵芝1号与出发菌株韩芝、韩芝M1序列与Ganoderma lucidum的相似度达到99％以上，判定为同一个种。从其系统发育树来看，韩芝与韩芝M1 更接近，药植太空灵芝1号与韩芝、韩芝M1有差异，显现出渐进式突变的特点。
使用DNAMAN分析其同源性和遗传距离发现，B与C的同源性达到99.6％，遗传距离较近为0.006；菌株A与菌株B和菌株C分别为99.1％和99.3％，遗传距离稍远,分别为0.019、0.013。三株灵芝菌株的相似性和遗传距离表见下表。
三株灵芝菌株的相似性和遗传距离表
注：表上半部分为遗传距离，下半部分为相似性，**为分隔线
实施例2生物学性质比较
1.生长速度和菌落形态
将培养基加热后定量倒入培养皿中，定量接入3株灵芝菌株，每两天在培养皿底部划线标记，划线“十字交叉法”测量菌落直径。将菌丝生长速度进行单因素方差分析后得出：韩芝、韩芝M1两菌株生长速度无显著差异，药植太空灵芝1 号菌株生长较快，与韩芝、韩芝M1两菌株存在极显著差异(P<0.01)。菌株生长速度及菌落形态见下表。
2.酶活力
灵芝是典型的木腐真菌，主要依靠分解木材获取能源和营养。灵芝的漆酶、纤维素酶和木聚糖酶，分别代表了灵芝分解木质素、纤维素和半纤维素的能力。采用配方木屑60％、麦麸18％、石灰1％、石膏1％，混匀后加水至含水量达55％，再混匀，定量分装于螺口试管中，灭菌后分别定量接入三株菌株菌丝体，置于 25℃黑暗条件下培养。观察生长状态，标记接种点至菌丝尖端的生长距离，计算生长速度。自接种日起第四天每隔3天测定菌株胞外酶活性。菌丝生长前端切取菌质，混匀后取5g加入预先配制好的缓冲液20mL，在4℃冰箱中提取12 h后于4℃10000rpm离心15min，收集上清液作为粗酶液。采用ABTS法测定漆酶活力，一个酶活单位定义为反应体系中每分钟催化1μmol ABTS氧化所需要的酶量；纤维素酶、木聚糖酶的活力测定采用3,5-二硝基水杨酸(DNS)比色法，其酶活力单位定义为每分钟催化生成1μmol·L-1产物所需要的酶量。
不同培养天数三株灵芝菌株的酶活性见下表及图4。图4中，不同小写字母代表有显著性差异(P＜0.05)。
漆酶U/g
纤维素酶U/g
木聚糖酶U/g
韩芝和韩芝M1之间显示出一些差异，说明韩芝M1也已经发生了一些正向变异。而药植太空灵芝1号的的漆酶、纤维素酶和木聚糖酶酶活力在所测定的时期内，都显著高于韩芝和韩芝M1，说明了其分解木质素、纤维素和半纤维素的能力都有所增强，生长速度快。
实施例3栽培试验：
1农艺学性状
将菌株韩芝，韩芝M1和药植太空灵芝1号按相同的方法栽培。统计这三个灵芝菌株在吉林、安徽、福建等地的段木栽培试验结果，见下表。
| 菌株 | 段木污染率 | 满段时间 | 亩产量 | 一级品率 |
| A | 6.15％ | 42.3d | 503kg | 65.2％ |
| B | 4.96％ | 38.6d | 537kg | 69.5％ |
| C | 3.55％ | 35.2d | 659kg | 78.3％ |
结果发现，菌株C的杂菌污染率低，菌丝生长最快，长势好，满段时间为 35.2天，分别比菌株A、B缩短了7.1d，3.4d。菌株C生长快，可以缩短生产周期，在子实体产量方面，菌株C的产量分别比A、B分别高31.0％，22.7％。菌株C的灵芝子实体形态好，芝体整齐，一级品率高，因此在农艺性状上超过了菌株A和B。
2有效成分含量测定
以上三菌株的子实体成分含量测定的结果见下表。结果显示,菌株C多糖含量3.57％，三萜含量0.92％。根据《中国药典》2020年版灵芝项下规定，多糖含量不得少于0.90％；三萜及甾醇以齐墩果酸，不得少于0.50％。菌株C的灵芝多糖和三萜类物质含量较高，灵芝多糖分别比菌株A、B高183％、142％。菌株C的灵芝三萜类和A、B菌株差异不大。菌株C的灵芝多糖是药典规定要求的3.97倍，显示出较高的药用价值。
| 菌株 | 灵芝多糖含量 | 灵芝三萜含量 |
| A | 1.95％ | 0.905％ |
| B | 2.51％ | 0.925％ |
| C | 3.57％ | 0.911％ |
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 中国医学科学院药用植物研究所
<120> 药植太空灵芝1号新菌株及其应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
ggaagtaaaa gtcgtaacaa gg 22
<210> 2
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
tcctccgctt atgatatgc 19
<210> 3
<211> 608
<212> DNA
<213> 灵芝(Ganoderma lucidum)
<400> 3
tgcggaagga tcattatcga gttttgaccg ggttgtagct ggccttccga ggcatgtgca 60
cgccctgttc atccactcta cacctgtgca cttactgtgg gcttcagatt gtgaggcacg 120
ctctttaccg tgcttgcgga gcatatctgt gcctgcgttt atcacaaact ctataaagta 180
acagaatgtg tattgcgatg taacacatct atatacaact ttcagcaacg gatctcttgg 240
ctctcgcatc gatgaagaac gcagcgaaat gcgataagta atgtgaattg cagaattcag 300
tgaatcatcg aatctttgaa cgcaccttgc gctccttggt atcccgagga gcatgcctgt 360
ttgagtgtca tgaattcttc aacctacaag cttttgtggt ttgtaggctt ggacttggag 420
gcttgtcggc cgttatcggt cggctcctct taaatgcatt agcttggttc gttgcggatc 480
ggctctcggt gtgataatgt ctacgccgcg accgtgaagc gtttggcgag cttctaaccg 540
tcttataaga cagctttatg acctctcacc tcaaatcagg taggactacc cgctgaactt 600
aagcatat 608
Claims (7)
1.灵芝（Ganoderma lucidum）药植太空灵芝1号，其保藏编号为：CGMCC No.16782。
2.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号在栽培中的应用。
3.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号栽培所得的灵芝子实体。
4.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号在制备食品或药物上的应用。
5.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号在制备保健食品上的应用。
6.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号栽培所得的灵芝子实体在制备食品或药物上的应用。
7.权利要求1所述的灵芝（Ganoderma lucidum）药植太空灵芝1号栽培所得的灵芝子实体在制备保健食品上的应用。
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