CN113265339B - 一种可分泌蜂蜜曲菌素的菌株lx911及其应用 - Google Patents
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Abstract
本发明属于微生物学技术领域,一种可分泌蜂蜜曲菌素的菌株LX911及其应用。一种可分泌蜂蜜曲菌素的菌株LX1911,其分类学名为Arthrinium arundinis,于2021年5月8日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC No.61639。本发明的菌株LX1911在培养过程中可以分泌蜂蜜曲菌素,利用其这一特性可以用于制备蜂蜜曲菌素,为蜂蜜曲菌素的开发利用提供参考。
Description
技术领域
本发明属于微生物学技术领域,具体涉及的是一种可分泌蜂蜜曲菌素的菌株LX911及其应用。
背景技术
蜂蜜曲菌素分子量为178.063,其分为(R)-蜂蜜曲菌素和(S)-蜂蜜曲菌素,结构式分别如下:
已有相关研究报道,蜂蜜曲菌素具有杀幼虫、细胞毒性、丙肝病毒蛋白酶抑制剂、抗菌、抗肿瘤、抗白血病、抗病毒和作为除草剂的用途。
发明人在研究霉变甘蔗过程中,发现一株可以分泌蜂蜜曲菌素的菌株即菌株LX1911,在现有技术中该菌株从未在甘蔗中发现过。利用该菌株能分泌蜂蜜曲菌素的特性可以用于制备蜂蜜曲菌素,具有重要研究意义。
公开于该背景技术部分的信息仅仅旨在增加对本发明的总体背景的理解,而不应当被视为承认或以任何形式暗示该信息构成已为本领域一般技术人员所公知的现有技术。
发明内容
本发明的目的是提供一种可分泌蜂蜜曲菌素的菌株LX911及其应用。
一种可分泌蜂蜜曲菌素的菌株LX1911,其分类学名为Arthrinium arundinis,于2021年5月8日保藏于广东省微生物菌种保藏中心,保藏号为GDMCC No.61639,其基因序列如SEQ ID No.3所示。
本发明提供的可分泌蜂蜜曲菌素的菌株LX1911是霉变的甘蔗样品中分离得来,经形态学和分子生物学鉴定为Arthrinium arundinis,于2021年5月8日保藏于保藏于广东省微生物菌种保藏中心,(简称GDMCC,地址:广州市先烈中路100号大院59号楼5楼,广东省微生物研究所,邮编:510070),保藏号为GDMCC No.61639。
本发明还提供了所述的菌株LX1911在制备蜂蜜曲菌素上的应用。
与现有技术相比,本发明具有以下有益效果:
本发明对菌株进行分离纯化,通过形态学观察、菌株鉴定和分子鉴定,确定获得的菌株为节菱孢菌芦竹节菱孢Arthrinium arundinis;其次还对菌株液体培养基培养过程中的产物进行测定,发现液体培养基中蜂蜜曲菌素的相对含量达到9.31%,说明本发明的菌株LX1911在培养过程中会分泌蜂蜜曲菌素,利用其这一特性可以用于制备蜂蜜曲菌素,具有重要研究意义。
保藏信息说明
Arthrinium arundinis LX1911,保藏编号为GDMCC No.61639,保藏日期为2021年5月8日,保藏单位为广东省微生物菌种保藏中心(GDMCC),保藏地址为广州市先烈中路100号大院59号楼5楼。
附图说明
图1为菌株LX1911的菌落形态图;
图2为液体培养基中挥发性成分总离子流图。
具体实施方式
下面结合具体的实施例对本发明做进一步的详细说明,所述是对本发明的解释而不是限定。
下述实施例中所用的材料和试剂,如无特殊说明,均可从商业途径得到。下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
实施例
一、菌株筛选
从全国不同省区采集霉变甘蔗样品,用无菌操作切取甘蔗块,点种于马铃薯葡萄糖琼脂培养基上,28℃培养,待长出菌落后,挑取菌落边缘形态一致的菌丝进行多次纯化,采用PDA斜面4℃保存纯化菌株;
二、菌落形态观察
对菌落进行显微观察拍照,并且对菌落的形态结构进行描述,菌落的形态如图1所示。
从图1可以看出,PDA菌落灰白色,平展,致密棉絮状;基质菌丝微黄,菌丝由光滑、透明、分枝、有间隔的菌丝组成,分生孢子梗直立,有隔,淡棕色,光滑,退化为分生细胞;分生细胞呈淡棕色,光滑,分生孢子深褐色,光滑,表面看呈球形,直径5-7μm,从侧面看呈双凸镜状,宽2-4μm,表面有一条白色的赤道狭缝。
根据菌落形态、孢子形态和大小等特征,初步鉴定菌株为节菱孢属Arthrinium。
三、分子鉴定
1.按照Biospin真菌基因组DNA提取试剂盒的操作步骤,得到Arthrinium真菌的基因组DNA。
2.以Arthrinium真菌的基因组DNA为模板,采用引物对ITS进行PCR扩增,得到PCR扩增产物;其中,引物对ITS的序列见下表1,PCR反应体系见下表2。
表1引物对ITS引物序列
表2 PCR反应体系
PCR反应程序:94℃变性3min;94℃变性30s、56℃退火10s、72℃延伸30s,共35个循环;72℃延伸5min。
3.对PCR扩增产物进行测序,得到其核苷酸序列见SEQ ID No.3。
4.Genebank数据库比对,菌株LX1911与Arthrinium arundinis同源性在99%以上;Mega6.0分析,确定菌株LX1911为节菱孢菌芦竹节菱孢Arthrinium arundinis,菌株代号为LX1911,因此将其命名为Arthrinium arundinis LX1911。
四、菌株培养
1.选取筛选纯化的节菱孢霉菌Arthrinium arundinis LX1911菌株接种在PDA培养基平板上,28℃培养7天。用5mm打孔器打孔3个菌丝饼,接入PYS(马铃薯-酵母膏-蔗糖)液体培养基,28℃,130r/min震荡培养21天。
2.PYS液体培养基中蜂蜜曲菌素GC-MS分析
GC条件:DB-5ms毛细管色谱柱(30m×250μm×0.25μm),不分流模式,进样口温度250℃,载气氦气,流速1mL/min;
梯度升温程序:初始温度40℃,保持5min,以4℃/min速度升温至120℃,保持3min,再以10℃/min速度升温至220℃,保持10min,最后以30℃/min速度升温至280℃,保持15min;
MS条件:采用全扫描模式,离子源温度230℃,四级杆温度150℃,质量扫描范围15-600amu;
定性方法:所得质谱数据与标准信息库NIST17.L进行检索比对,对匹配度大于80的鉴定结果予以确认;
定量方法:利用工作站数据处理系统,按峰面积归一化法进行定量分析,得到各成分的相对含量,结果见下表3和图2。
表3液体培养基中挥发性成分相对含量
从表3和图2可以看出,菌株LX1911在培养过程中可以分泌出蜂蜜曲菌素,利用菌株LX1911这一特性可以用于制备蜂蜜曲菌素。
综上所述,本发明对菌株进行分离纯化,通过形态学观察、菌株鉴定和分子鉴定,确定获得的菌株为节菱孢菌芦竹节菱孢Arthrinium arundinis;在对其培养过程中的产物进行测定时,发现本发明的菌株LX1911在培养过程中会分泌蜂蜜曲菌素,利用其这一特性可以用于制备蜂蜜曲菌素,为制备蜂蜜曲菌素提供另一重要来源,具有重要研究意义。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式,并且很显然,根据上述教导,可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用,从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
序列表
<110> 广西壮族自治区农业科学院
<120> 一株可分泌蜂蜜曲菌素的节菱孢菌及其应用
<130> 2021
<160> 3
<170> SIPOSequenceListing 1.0
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<213> 人工序列(Artificial Sequence)
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tccgtaggtg aacctgcgc 19
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<213> 人工序列(Artificial Sequence)
<400> 2
tcctccgctt attgatatgc 20
<210> 3
<211> 558
<212> DNA
<213> 节菱孢霉菌(Arthrinium arundinis)
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tagcttacac tccataccat ctgttaacct acccagttat gcctcggcgt aagctcggtt 60
ggaggcacct gcagctaccc tgtagttgcg gactgccaac tccagccgcg gcccgccggc 120
ggtacactaa actctgtttt attttatatt ctgagcgtct tattttaata agttaaaact 180
ttcaacaacg gatctcttgg ttctggcatc gatgaagaac gcagcgaaat gcgataagta 240
atgtgaattg cagaattcag tgaatcatcg aatctttgaa cgcacattgc gcccatcagt 300
attctggtgg gcatgcctgt tcgagcgtca tttcaaccct taagcctagc ttagtgttgg 360
gaatctgctg tactgcagtt ccttaaagac agtggcggag cggcggtagt cctctgagcg 420
tagtaattta tttctcgctt ttgtcaggct ctgtcctccc gccataaaac ccccaatttt 480
ttagtggttg acctcggatc aggtaggaat acccgctgaa cttaagcata tctaaagccg 540
ggaggaaaaa gtaaaatg 558
Claims (2)
1.一种可分泌蜂蜜曲菌素的菌株LX1911,其特征在于,其分类学名为Arthriniumarundinis,于2021年5月8日保藏于广东省微生物菌种保藏中心,保藏号为GDMCCNo.61639,其基因序列如SEQ ID No.3所示。
2.根据权利要求1所述的菌株LX1911在制备蜂蜜曲菌素上的应用。
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