CN118126845A - 一株长松萝内生真菌yafef048菌株及其分离方法和应用 - Google Patents
一株长松萝内生真菌yafef048菌株及其分离方法和应用 Download PDFInfo
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Abstract
本发明提供一株长松萝内生真菌YAFEF048菌株及其分离方法和应用,涉及微生物技术领域。所述长松萝内生真菌YAFEF048菌株的保藏编号为CCTCCM20211345,且YAFEF048菌株分离自长松萝中,YAFEF048菌株发酵液提取物可以用来制备抗耐药细菌药物。本发明克服了现有技术的不足,本发明分离得到的长松萝内生真菌YAFEF048菌株的发酵提取物能够有效对具有耐药性的细菌产生较好的抑菌活性作用,为新型抗菌药物的研究提供新方向。
Description
技术领域
本发明涉及微生物技术领域,具体涉及一株长松萝内生真菌YAFEF048菌株及其分离方法和应用。
背景技术
耐药性病原体的传播和发展,感染性疾病成为一个全球性问题,严重威胁公共卫生安全。自青霉素被发现以来,以青霉素为代表的抗生素为人类健康做出了巨大贡献,抗生素在防治耐药性细菌病原体感染方面有着不可替代的作用。如今,抗生素的大量使用导致细菌耐药性问题日趋严重,传统抗生素的研发已进入瓶颈,以控制耐药菌感染为主要目标的新结构、新靶点和新机制的新型抗菌药物的研究及开发变得极为迫切。
有研究表明,植物内生真菌的代谢产物中大多存在抗菌活性物质,次生代谢产物结构主要包含生物碱、聚酮、萜类、黄酮等类型,具有抗菌、抗病毒、抗癌、抗氧化、杀虫、抗糖尿病和免疫抑制等功能,这些种类繁多的抗菌活性物质有许多还是未开发的新物质。因此,植物内生菌因具有作为新型抗菌药物筛选源的巨大潜力,利用植物内生真菌生产活性物质可持续利用资源,减少对自然资源的破坏,缓解因自然资源匮乏等问题带来的植物源抗菌化合物开发的困难。目前已经有学者对这方面进行了研究,姚裕群等(2020)等对越南槐(Sophora tonkinensis)内生菌刺盘孢TRPH-35的进行鉴定和抗菌活性检测。华梅等(2018)对白木香(Aquilaria sinensis)茎秆和叶片中分离的10株植物内生真菌经行形态与分子鉴定,并对初提物经行致病细菌的抑菌活性检测。
长松萝(U.longissimaAch)为地衣类松萝科Usneaceae松萝属植物,松萝属地衣药用历史悠久,药用部位为全草。目前,已经从长松萝中提出许多具有抗癌、抗炎症、抗菌等生物活性的化合物,比如松萝酸(Usnic acid),拉马酸(Ramalic acid),巴尔巴地衣酸,但是对于长松萝共生菌的研究较少。
发明内容
针对现有技术不足,本发明提供一株长松萝内生真菌YAFEF048菌株及其分离方法和应用,分离得到的长松萝内生真菌YAFEF048菌株的发酵提取物能够有效对具有耐药性的细菌产生较好的抑菌活性作用,为新型抗菌药物的研究提供新方向。
为实现以上目的,本发明的技术方案通过以下技术方案予以实现:
一株长松萝内生真菌YAFEF048菌株,所述长松萝内生真菌YAFEF048菌株的保藏编号为CCTCCM20211345。
优选的,所述长松萝内生真菌YAFEF048的ITS基因序列为SEQ ID No.1所示的核苷酸序列。
长松萝内生真菌YAFEF048菌株的分离方法包括以下步骤:
(1)取长松萝样品用无菌水冲洗干净,在无菌环境下,用剪刀剪成0.5cm×0.5cm的小块,先用1L无菌水冲洗样品,再用75%乙醇消毒1min,期间不断震荡,后用无菌水冲洗3-4次,用无菌滤纸吸干样品表面多余的水分,晾干备用;
(2)将处理好的样品,放入2mL的离心管中,加入500μl的超纯水,然后在细胞破碎匀浆机中180rpm震荡2min破碎,将破碎液按十分之一、百分之一、千分之一进行稀释,取200μl子实体悬浮液涂抹在PDAKAS抗细菌培养基上,PDAKAS抗细菌培养基:马铃薯200g/L+葡萄糖20g/L+琼脂15g/L+氨苄青霉素50ug/mL+卡那霉素50ug/m,将接好的培养基放置于26℃恒温培养箱中培养;
(3)取待培养基中长出的菌丝,在显微镜下把长出的菌丝转接的到PDA培养基上培养,持续观察10d后,将所挑取的菌株进行后续分子鉴定后得到长松萝内生真菌YAFEF048菌株。
长松萝内生真菌YAFEF048菌株在制备抗耐药细菌药物中的应用,且所述应用为采用长松萝内生真菌YAFEF048的培养物的发酵液提取物、长松萝内生真菌YAFEF048细胞的超声裂解上清、长松萝内生真菌YAFEF048细胞的超声裂解沉淀中的至少一种作为抗细菌药物的活性成分。
优选的,所述长松萝内生真菌YAFEF048的培养物的发酵液提取物的制备方法包括以下步骤:
(1)加入1mL的PG液体到2mL的离心管中,并在每个离心管放3颗灭菌好的钢珠,然后将已长成型的长松萝内生真菌YAFEF048菌丝体刮取适量放入离心管,破碎90S,然后将破碎液分别接种500uL到每个装有PG液体培养基的锥形瓶中,置于28℃,转速为150r/min的摇床中培养10d进行发酵培养;
(2)发酵培养完成后,得到的真菌菌丝体及培养液,用分液漏斗将菌丝体和菌液分离,并在菌液中加入乙酸乙酯摇匀6min,菌液与乙酸乙酯的体积比为1:1,超声30min后静置12h,将分层的下层废液回收,上层萃取溶液用旋转蒸发仪冷凝回流干燥,获得长松萝内生真菌YAFEF048培养的发酵液提取物。
优选的,所述PG液体培养基由如下组分组成:马铃薯浸粉5g/L,酵母粉5g/L,葡萄糖20g/L,七水硫酸镁0.5g/L,磷酸二氢钾1g/L,维生素B10.1g/L,纯净水1L,培养基分装至100mL锥形瓶中。
本发明提供一株长松萝内生真菌YAFEF048菌株及其分离方法和应用,与现有技术相比优点在于:
本发明通过分离筛选长松萝的共生菌,得到一株具有抗菌活性的真菌YAFEF048,且该YAFEF048菌的菌液体提取物能够对具有耐药性的蜡样芽孢杆菌、缓慢芽孢杆菌、溶血性葡萄球菌、无乳链球菌、短小芽孢杆菌、大肠埃希菌等细菌产生一定的抑菌效果,有效缓解致病菌耐药性对人类健康构成的威胁,为研究开发新的抗细菌药物提供新的途径。
附图说明:
图1为本发明实施例1中YAFEF048菌丝生长情况示意图;
图2为本发明实施例1中YAFEF048菌株荧光显微镜下菌丝体示意图;
图3为本发明实施例3中YAFEF048对耐药性蜡样芽孢杆菌的抑制作用图,其中a、b、c表示对照组,d表示实验组;
图4为本发明实施例3中YAFEF048对耐药性缓慢芽孢杆菌的抑制作用图,其中a、b、c表示对照组,d表示实验组;
图5为本发明实施例3中YAFEF048对耐药性溶血性葡萄球菌的抑制作用图,a、b、c表示对照组,d表示实验组;
图6为本发明实施例3中YAFEF048对耐药性无乳链球菌的抑制作用图,a、b、c表示对照组,d表示实验组;
图7为本发明实施例3中AFEF048对耐药性短小芽孢杆菌的抑制作用图,a、b、c表示对照组,d表示实验组;
图8为本发明实施例3中YAFEF048对耐药性大肠埃希菌的抑制作用图,a、b、c表示对照组,d表示实验组。
图9为本发明实施例1中长松萝内生真菌YAFEF048的***发育树图示意图。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚,下面结合本发明实施例对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
本发明提供的菌株为长松萝内生真菌YAFEF048(Dothichiza sp.);保藏名称为Dothichiza sp.YAFEF048;保藏于中国典型培养物保藏中心,保藏地址是中国.武汉.武汉大学;保藏日期:2021年11月1日;保藏编号:CCTCCM20211345。
实施例1:
长松萝内生真菌YAFEF048菌株的分离和鉴定:
1、长松萝内生真菌的分离:
先用自来水冲洗长松萝样品24h,清除样品表面的真菌或者其他微生物,然后转入无菌工作台进行消毒处理,先用1L无菌水冲洗样品,放在培养皿中用接种刀切成小段放于50mL无菌离心管中;然后用75%的乙醇浸泡1min(期间不断震荡),倒掉乙醇后,再用无菌水冲洗3次后,用无菌滤纸吸干样品表面多余的水分,晾干备用。
用灭菌小刀取2g子实体,放入2mL的离心管中,加入500μl的超纯水,然后在细胞破碎匀浆机中180rpm震荡2min破碎,将破碎液稀释1000倍得子实体悬浮液,取200μl子实体悬浮液涂抹在PDAKAS抗细菌培养基上(PDAKAS抗细菌培养基:马铃薯200g/L+葡萄糖20g/L+琼脂15g/L+氨苄青霉素50ug/mL+卡那霉素50ug/mL),将接好的培养基放置于26℃恒温培养箱中培养。待培养基中长出的菌丝,在显微镜下把长出的菌丝转接的到PDA培养基上培养,持续观察10d后,将所挑取的菌株进行后续分子鉴定后得到长松萝内生真菌YAFEF048。
2、长松萝内生真菌的鉴定:
(1)形态的鉴定
真菌在PDA培养基上生长良好,菌落表面呈绒毛状,生长前期菌丝是白色呈放射状向周围生长,生长后期菌丝较密集,且菌层较厚,颜色微微泛红,菌落呈规则的圆形状(图1)。
(2)DNA提取
试验前先CTAB水浴65℃30min以上,提前20min打开速冻离心机;取长松萝内生菌的分离纯化培养的菌丝体于2mL离心管中,加入3颗灭菌好的钢珠,并将离心管放到装有液氮的不锈钢罐中浸泡冷却6min,用破碎机破碎1.5min,将菌丝体打碎;离心管中加入1mL的CTAB、20uLβ-巯基乙醇,摇匀(10min),放到水浴锅水浴5h,每隔10min震荡摇匀1次;水浴结束后,放入4℃、12000r/min速冻离心机离心10min;取上清液1mL转移到一个新的2mL离心管中,加入1mL(酚:氯仿:异戊醇=25:24:1),振摇10min,离心10min(重复两次);取上清液1mL转移到一个新的2mL离心管中,加入1mL(氯仿:异戊醇=24:1),震荡摇匀10min,放入4℃·12000r/min速冻离心机离心10min;取上清液500uL转移到一个新的1.5mL离心管中,加入50uLNaAc和1mL无水乙醇(-20℃),摇匀做好标记后,放入-20℃冰箱沉淀24h;沉淀完放入4℃·12000r/min速冻离心机离心10min,加入500uL 75%乙醇,轻轻摇晃5-10次,静置3min,倒掉乙醇完成洗脱(共洗脱2次);加入500uL无水乙醇洗脱1次(静置3min后倒掉无水乙醇),放入4℃·12000r/min速冻离心机离心3min,倒掉无水乙醇,放置干;加入80uL洗脱缓冲剂(TE buffer),瞬时离心1min,得到真菌YAFEF048基因组DNA。
(3)ITS分析鉴定:
用真菌通用引物LR3(5’-CCGTGTTTCAAGACGGG-3’)和ITS1(5’-CTTGGTCATTTAGAGGAAGTAA-3’)扩增真菌rDNA的间隔序列(含ITS1区、5.8S区、ITS2区)序列,所得得ITS测序序列如下所示:
TCCTAGCAGATGCGCGGACCTCAGTCCAGGCTGGTTGCATGTCGTCTCCCCTATAAGTTCTCCCCGAGAGGAGGTACATGACAGAGACCTTTATCCAACCGCCCAAACTGATGCTGGCCTGCCTATAGAAGAGTGCACCGGGTAGAAACCCGGATGAGCAACTACAGGCAAGTCTGGCTGCAAGCGCTTCCCTTTCAACAATTTCACGTGCTTTTTAACTCTCTTTCCAAAGTGCTTTTCATCTTTCGATCACTCTACTTGTGCGCTATCGGTCTCTGGCCAGTATTTAGCTTTAGAAGAAATTTACCTCCCATTTAGAGCTGCATTCCCAAACAACTCGACTCGTCGAAGGGGCTTTACACGGTAAAGGCTAGCGACCACGTACGGGATTCTCACCCTCTGTGACGTCCTGTTCCAAGGAACTTGGACCGCTGCCAATGCCAAAGCGCCCTCTGCAAATTACAACTCGGACGCCAAAGACGCCAGATTTCAAATTTGAGCTGTTGCCGCTTCACTCGCCGTTACTAGGGCAATCCCTGTTGGTTTCTTTTCCTCCGCTTATTGATATGCTTAAGTTCAGCGGGTATCCCTACCTGATCCGAGGTCAAGAGTGTAAAAATGTACTTTTGGATCGTCGTCGTTATGAGTGCAAAGCGCGAGATGTACTGCGCTCCGAAATCAATACGCCGGCTGCCAATTGTTTTGAGGCGAGTCTGCGCGCAGAGGCGAGACAAACACCCAACACCAAGCAAAGCTTGAAGGTACAAATGACGCTCGAACAGGCATGCCCCATGGAATACCAAGGGGCGCAATGTGCGTTCAAAGATTCGATGATTCACTGAATTCTGCAATTCACACTACTTATCGCATTTCGCTGCGTTCTTCATCGATGCCAGAACCAAGAGATCCGTTGTTGAAAGTTGTAACTATTAAAGTTTTTCAGACGCTGATTTCAATTACAAAGGGTTTAAGTATTGTCCAATCGGCGGGCGGACCCGCCGAGGAAACGAAGGTACTCAAAAGACATGGGTAAGAGATAGCAGGCAAAGCCTACAACTCTAGGTAATGATCCTTCCGCAGGTCACCTAC;
(4)构建发育树
基于ITS构建了长松萝内生真菌YAFEF048的***发育树,综合菌株形态学鉴定和分子生物学鉴定结果,该真菌与疡壳孢属(Dothichiza sp.)亲缘关系最近,所以将长松萝内生真菌YAFEF048鉴定为疡壳孢属(Dothichiza sp.)(如图9所示)。
实施例2:
长松萝内生真菌YAFEF048活性物质的提取:
1、设置液体发酵培养基PG:马铃薯浸粉5g/L,酵母粉5g/L,葡萄糖20g/L,七水硫酸镁0.5g/L,磷酸二氢钾1g/L,维生素B10.1g/L,纯净水1L,培养基分装至100mL三角瓶;
加入1000uL的PG液体到2mL的离心管中,并在每个离心管放3颗灭菌好的钢珠,然后将已长成型的长松萝内生真菌YAFEF048菌丝体刮取适量放入离心管,破碎90S,然后将破碎液分别接种500uL到每个装有PG液体培养基的锥形瓶中,置于28℃,转速为150r/min的摇床中培养8d。
2、上述发酵培养完成后,得到的真菌菌丝体及培养液。用分液漏斗将菌丝体和菌液分离,并在菌液中加入乙酸乙酯(1:1)摇匀6min,摇匀后静置12h,然后将分层的下层废液回收,上层萃取溶液用旋转蒸发仪冷凝回流干燥,得到长松萝内生真菌YAFEF048培养的发酵液提取物。
实施例3:
长松萝内生真菌YAFEF048培养物的发酵液提取物的对耐药性细菌的抗菌活性检测:
1、耐药细菌的选择和活化:
(1)于广东省细菌耐药性监测和质量控制中心购入具有耐药性的蜡样芽孢杆菌、缓慢芽孢杆菌、溶血性葡萄球菌、无乳链球菌、短小芽孢杆菌、大肠埃希菌并保存于实验室中;
(2)将保存的具有耐药性的蜡样芽孢杆菌、缓慢芽孢杆菌、溶血性葡萄球菌、无乳链球菌、短小芽孢杆菌、大肠埃希菌等致病细菌分别取5~6uL加入2mL离心管中,再加入700uL的LB液体培养基,然后将离心管放入37℃,转速为180r/min恒温摇床中培养12h,得到致病菌菌液,置于4℃冰箱备用,取出活化好的细菌进行稀释1/10倍(若活化好的细菌置于4℃保存超过12h则不用稀释);
2、耐药细菌的耐药性检测:
选择氨苄青霉素作为各个细菌的耐药性检测用抗生素(氨苄青霉素批号:0339,纯度:95%,购于昆明硕阳科技有限公司);
精密称取氨苄青霉素50mg,分别置于离心管中,加入1mL DMSO溶液配置成50mg/mL的抗生素DMSO溶液,备用。
3、抗菌活性的检测:
配制Luria-Bertani固体培养基,将灭菌后还未凝固的培养基倒入20mL于培养皿中冷却、凝固,吸取300uL的相应供的试菌悬液(具有耐药性的蜡样芽孢杆菌、缓慢芽孢杆菌、溶血性葡萄球菌、无乳链球菌、短小芽孢杆菌、大肠埃希菌)滴加到固体培养基上,用涂布棒均匀涂在培养基表面,制成含菌平板,然后取长松萝内生真菌YAFEF048发酵液提取物,加入DMSO(二甲亚砜)溶液,稀释到50mg/mL,待含菌LB平板晾干后,将直径5mm的滤纸圆片,经高压灭菌处理后干燥,分别滴加10uL供试液制备试验样片作为实验组(d),滴加PG纯培养基为对照组(a),滴加氨苄青霉素样片为受试菌耐药性验证的对照组(b),DMSO液作为阴性对照组(c),用无菌镊子将试验样片和3个对照样片贴放于每个培养皿表面,做好标记后盖好培养皿,将培养基正放置于置于37℃恒温培养箱中培养12h,观察是否出现抑菌圈并用十字交叉法量出抑菌圈的直径大小。
具体结果如图3-8所示,且各个受试菌中各实验组和对照组的抑菌圈大小如下表所示:
由上表可知,采用氨苄青霉素对各组细菌进行抑菌实验,没有出现抑菌圈,验证了本申请受试菌具有耐药性,而采用YAFEF048发酵液提取物对各组细菌进行抑菌实验,均有抑菌圈出现,说明YAFEF048发酵液提取物对耐药性细菌有良好的抑菌活性,其中对短小芽孢杆菌的抑菌效果最佳。
以上实施例仅用以说明本发明的技术方案,而非对其限制;尽管参照前述实施例对本发明进行了详细的说明,本领域的普通技术人员应当理解:其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或者替换,并不使相应技术方案的本质脱离本发明各实施例技术方案的精神和范围。
Claims (7)
1.一株长松萝内生真菌YAFEF048菌株,其特征在于,所述长松萝内生真菌YAFEF048菌株的保藏编号为CCTCCM20211345。
2.根据权利要求1所述的长松萝内生真菌YAFEF048菌株,其特征在于:所述长松萝内生真菌YAFEF048的ITS 基因序列为SEQ ID No.1所示的核苷酸序列。
3.如权利要求1所述的菌株的分离方法,其特征在于,所述所述分离方法包括以下步骤:
(1)取长松萝样品用无菌水冲洗干净,在无菌环境下,用剪刀剪成0.5cm×0.5cm的小块,先用1L无菌水冲洗样品,再用75%乙醇消毒1min,期间不断震荡,后用无菌水冲洗3-4次,用无菌滤纸吸干样品表面多余的水分,晾干备用;
(2)将处理好的样品,放入2mL的离心管中,加入500µl的超纯水,然后在细胞破碎匀浆机中180rpm震荡2min破碎,将破碎液按十分之一、百分之一、千分之一进行稀释,取200µl子实体悬浮液涂抹在PDAKAS抗细菌培养基上,PDAKAS抗细菌培养基:马铃薯200g/L+葡萄糖20g/L+琼脂15g/L+氨苄青霉素50ug/mL+卡那霉素50ug/m,将接好的培养基放置于26℃恒温培养箱中培养;
(3)取待培养基中长出的菌丝,在显微镜下把长出的菌丝转接的到PDA培养基上培养,持续观察10d后,将所挑取的菌株进行后续分子鉴定后得到长松萝内生真菌YAFEF048菌株。
4.如权利要求1所述的菌株在制备抗耐药细菌药物中的应用。
5.根据权利要求4所述的应用,其特征在于,所述应用为采用长松萝内生真菌YAFEF048的培养物的发酵液提取物、长松萝内生真菌YAFEF048细胞的超声裂解上清、长松萝内生真菌YAFEF048细胞的超声裂解沉淀中的至少一种作为抗细菌药物的活性成分。
6.根据权利要求5所述的应用,其特征在于,所述长松萝内生真菌YAFEF048的培养物的发酵液提取物的制备方法包括以下步骤:
(1)加入1mL的PG液体到2mL的离心管中,并在每个离心管放3颗灭菌好的钢珠,然后将已长成型的长松萝内生真菌YAFEF048菌丝体刮取适量放入离心管,破碎90S,然后将破碎液分别接种500uL到每个装有PG液体培养基的锥形瓶中,置于28℃,转速为150r/min的摇床中培养10d进行发酵培养;
(2)发酵培养完成后,得到的真菌菌丝体及培养液,用分液漏斗将菌丝体和菌液分离,并在菌液中加入乙酸乙酯摇匀6min,菌液与乙酸乙酯的体积比为1:1,超声30min后静置12h,将分层的下层废液回收,上层萃取溶液用旋转蒸发仪冷凝回流干燥,获得长松萝内生真菌YAFEF048培养的发酵液提取物。
7.根据权利要求6所述的应用,其特征在于,所述PG液体培养基由如下组分组成:马铃薯浸粉5g/L,酵母粉5g/L,葡萄糖20g/L,七水硫酸镁0.5g/L,磷酸二氢钾1g/L,维生素B10.1g/L,纯净水1L,培养基分装至100mL锥形瓶中。
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