CN110172408B - 一株桃儿七的内生真菌及其应用 - Google Patents
一株桃儿七的内生真菌及其应用 Download PDFInfo
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Abstract
本发明提供了一株桃儿七内生真菌，粉红粘帚霉fhnzm(Clonostachys rosea)，保藏号CGMCC No.17070，保藏于中国普通微生物菌种保藏管理中心。该菌株能够提高桃儿七根中鬼臼毒素的含量，抑制桃儿七根的生长，抑制植物种子的萌发和胚根的伸长，能够作为核心菌株来生产用于促进桃儿七鬼臼毒素积累的菌剂，对于绿色防治桃儿七病虫害和提高人工栽培桃儿七的品质，有效提高桃儿七鬼臼毒素的积累具有重大意义。
Description
技术领域
本发明涉及农业和工业生物技术领域，具体的是一株桃儿七的内生真菌及其应用。
背景技术
桃儿七(Sinopodophyllum hexandrum Royle)是小檗科桃儿七属草本植物，国家三级保护植物，主要分布在我国西部2800米以上的林缘及灌丛中。由于自然资源的匮乏和濒危，被收录入《中华珍稀濒危植物名录》和《中国植物红皮书》。
桃儿七在民间主要用于痛疖肿毒、风湿性骨痛、气管炎等症，现代药学研究表明，其根及根茎中主要含有木脂素类、黄酮类、皂苷、多糖等成分，其中木脂素类的鬼臼毒素含量最高。鬼臼毒素具有广泛的药学活性，如0.5％鬼臼毒素酊在治疗尖锐湿疣方面具有起效快、治愈率高、安全性好等特点，1990年世界卫生组织推荐其为一线药物，1994年我国《国家基本药物》皮肤类抗病毒唯一入选药物。鬼臼毒素还是合成抗癌药物GP7、VP-16、VM- 26、NK611等及抗艾滋病药物的前体物质。已应用于临床的VP-16和VM-26对小细胞肺癌、淋巴癌等多种肿瘤疾病均有很好的疗效。
由于鬼臼毒素类物质化学结构复杂，对其进行化学全合成的难度较大；基因工程学手段构建工程菌也由于参与形成该类物质的酶数量过多而无法进行。目前，鬼臼毒素的来源主要是通过採挖野生桃儿七，从其根和根茎中提取。近年来，随着野生资源的锐减及封山育林等生态保护政策的实施，野生资源已经很难获取。提高桃儿七种植海拔可以有效提高其品质，但产量低、农事操作困难、对生态环境破坏严重，事实上，在高海拔区域(如：2800米以上)人工种植很难实施。桃儿七人工驯化栽培已有很多报道，但栽培周期长、产量低、药材收购价格低，农民没有种植的积极性。通过桃儿七组培苗胚根及愈伤组织生产鬼臼毒素有零星报道，但难以放大培养，鬼臼毒素含量低等问题限制了推广应用。通过桃儿七内生菌生产鬼臼毒素已有报道，虽然容易放大培养，但是通过内生菌发酵生产鬼臼毒素存在含量低、菌种稳定性差、随着菌种的传代生产鬼臼毒素的能力丧失等问题。因此，提高桃儿七鬼臼毒素含量问题亟待解决。
发明内容
本发明的目的是提供一种可克服现有技术不足的技术方案，即提供一株桃儿七的内生真菌及其应用。
本发明的目的一是提供一株桃儿七内生真菌粉红粘帚霉fhnzm(Clonostachysrosea)，其保藏号为CGMCC No.17070，保藏于中国普通微生物菌种保藏管理中心。
本发明的目的二是提供fhnzm的培养方法，具体步骤如下：
无菌环境下挑取fhnzm菌丝接入马铃薯葡萄糖液体培养基中，温度27-30℃，转速120- 160rpm，发酵时间7-10天。
马铃薯葡萄糖液体培养基配方如下：马铃薯200g，放入水中煮沸30分钟，用双层纱布过滤，取其滤液加20g葡萄糖，补足水至1L，自然PH。
作为优选，所述发酵条件为温度30℃，转速160rpm，发酵时间9天。
本发明的目的三是提供fhnzm的用途，具体内容如下：
(1)抑制桃儿七生长，尤其是根的伸长；
(2)抑制其他经济作物种子的萌发和胚根的伸长，降低种子的发芽指数，可用于开发种苗的生长抑制剂，起到蹲苗、壮苗并且防治病虫害的作用；
(3)促进桃儿七鬼臼毒素的积累，提高桃儿七根中鬼臼毒素含量，包括组织培养的桃儿七器官和自然生长的桃儿七植株。
本发明的目的四是提供fhnzm菌株在促进桃儿七鬼臼毒素积累方面的应用，具体为提供以fhnzm为核心菌剂的促进桃儿七鬼臼毒素积累的方法及相关产品。
粉红粘帚霉是一种分布广泛的重寄生菌，具有生长快、产孢量大、寄主范围广、寄生能力强、生防功能多样，包括拮抗、重寄生和诱导植物抗性及毒杀线虫等，是目前已发现的拮抗微生物中最具潜力的植物病害生防因子，对多种植物病原菌都有很强的抑制作用。本发明提供的粉红粘帚霉fhnzm除具有基于上述粉红粘帚霉菌的共同特性外，还可以促进桃儿七鬼臼毒素的积累，抑制桃儿七及其他作物生根。因此，本发明提供的粉红粘帚霉fhnzm可以有效应用于桃儿七驯化栽培，对于绿色防治桃儿七病虫害和提高人工栽培桃儿七的品质，有效提高桃儿七鬼臼毒素的积累具有重大意义。
保藏说明：
菌种名称：粉红粘帚霉
拉丁名：Clonostachys rosea
菌株编号：fhnzm
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号
保藏日期：2019年1月14日
保藏中心登记入册编号：CGMCC No.17070
附图说明
图1为实施例1分离获得的fhnzm菌株的显微照片。图2为fhnzm的MEGA7构建进化树结果。图3是实施例3fhnzm发酵液处理的桃儿七幼苗生根情况。图4是实施例3中PDA处理的桃儿七幼苗生根情况。
具体实施方式
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。
下述实施例中所使用的桃儿七种子，均采自兰州市兴隆山海拔2300米以上，经鉴定为桃儿七种子，均为新鲜种子。
下述实施例中所使用的材料、试剂等，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培养基如下：
MS培养基：大量元素：NH4NO3 1650mg/L，KNO31900 mg/L，CaCl2·2H2O 440 mg/L，MgSO4·7H2O 370mg/L，KH2PO4 700mg/L；微量元素：KI 0.83mg/L，H3BO3 6.2 mg/L，MnSO4·4H2O 22.3mg/L,ZnSO4·7H2O 8.6mg/L，Na2MnO4·2H2O 0.25 mg/L，CuSO4·5H2O 0.025mg/L，CoCl2·6H2O 0.025mg/L，FeSO4·7H2O 27.8mg/L，Na2- EDTA·2H2O 37.3mg/L；有机成分：肌醇100mg/L，烟酸0.5mg/L，盐酸吡哆醇(维生素B6)0.5mg/L，盐酸硫胺素(维生素B1)0.5mg/L，甘氨酸2mg/L。
PDA培养基：马铃薯200g，放入水中煮沸30分钟，用双层纱布过滤，取其滤液加20g葡萄糖，补足水至1L，自然PH。
实施例1：fhnzm的分离与鉴定
桃儿七种子表面消毒后，种植于灭过菌的混合基质中，混合基质组成为稻壳炭：蛭石＝1:1，待幼苗子叶展开后收集幼苗，自来水冲洗干净，70％的酒精浸泡30sec,0.1％的升汞表面消毒15min，用灭过菌的蒸馏水冲洗2-3遍，将胚根切成1-2cm的小段，置于MS固体培养基，23℃，黑暗条件下培养；取冲洗水1ml于MS培养基，置于相同条件下培养，作为对照。10天后观察到内生真菌的生长，挑取生长速度快、侵染率高的优势菌株，在PDA固体培养基上划线纯化。送上海生工生物工程股份有限公司进行18srDNA测序，NCBI数据库BLAST比对，MEGA7构建进化树。MEGA7构建进化树结果见图2。
NCBI数据库BLAST比对结果如下：
上述结果证明所分离的内生真菌为粉红粘帚霉，是子囊菌门、子囊菌纲、肉座菌目、生赤壳科、粘帚霉属、粉红粘帚霉，真菌菌株命名为fhnzm。
实施例2：fhnzm菌体及发酵液中鬼臼毒素测定
将实施例1分离得到的fhnzm接入PDA培养基，30℃，160r/min振荡培养，培养9d后，培养液即为菌液。菌液用滤纸过滤，滤液3000r/min离心15min，上清液经0.22um滤膜过滤，收集所得滤液即为发酵液，-20℃保存备用。过滤出的菌体研磨后，用体积比1:1的甲醇/ 氯仿混合液超声提取1h，(功率250W，频率70Khz),过滤，挥干，甲醇定容，高效液相色谱法检测菌体及发酵液中鬼臼毒素含量(色谱条件：色谱柱：Shim-pack GIST C18柱 (4.6mm×250mm,5.0um)，流动相：甲醇-水(50:50),流速1ml/min，检测波长290nm，柱温30℃)。
结果表明fhnzm菌体及发酵液都不含有鬼臼毒素。
实施例3：fhnzm菌液抑制桃儿七幼苗的生长
桃儿七种子用1000倍液的多菌灵浸泡1小时，再用浓度为80mg/L的GA4+7在室温浸泡48小时，晾干或擦干种子表面水分，播种于灭过菌的混合基质，混合基质组成为稻壳炭：蛭石＝1:1。室温条件下培养，60天左右，种子发芽整齐，子叶完全展开后将幼苗分为两部分，各100株幼苗，一部分幼苗浇灌如实施例2所述方法制得的fhnzm菌液，每5天浇灌1次，共2次，另一部分幼苗浇灌PDA培养液，1个月后观察幼苗生根情况。
实验结果如图3、图4所示，从图中可以看出，通过粉红粘帚霉fhnzm菌液处理的桃儿七幼苗的根的长度明显短于未经处理的对照组的根的长度，证明粉红粘帚霉fhnzm菌液能够明显抑制桃儿七根的生长。
实施例4：fhnzm菌液抑制黄瓜和萝卜种子生长
在9cm培养皿中垫上两张滤纸，均匀放入10粒大小基本一致、饱满的黄瓜(或萝卜)种子，加入实施例2所述方法制得的fhnzm菌液5mL，盖上皿盖，在25℃的培养箱中避光培养48h，统计发芽率、测量根长。对照加入液体PDA培养基5mL。试验设置重复3次。
种子发芽指数(GI)计算公式：
式中：
A1—fhnzm菌液处理的种子平均发芽率，％；
A—fhnzm菌液处理的种子平均根长，mm；
B1—对照种子平均发芽率，％；
B2—对照种子的平均根长，mm。
实验结果表明通过fhnzm菌液处理后，黄瓜和萝卜种子的GI指数分别为74％和42％，一般认为发芽指数GI低于85％，对种子发芽具有抑制作用。因此可以证明，fhnzm菌液对黄瓜和萝卜种子的发芽具有明显的抑制作用。
实施例5：fhnzm菌液促进桃儿七幼苗鬼臼毒素含量的积累
桃儿七种子用1000倍液的多菌灵浸泡1小时，再用浓度为80mg/L的GA4+7在室温浸泡48小时，晾干或擦干种子表面水分，播种于灭过菌的混合基质，混合基质组成为稻壳炭：蛭石＝1:1。室温条件下培养，60天左右，种子发芽整齐，子叶完全展开后将幼苗分为两部分，各100株幼苗，一部分幼苗浇灌如实施例2所述方法制得的fhnzm菌液，每5天浇灌1次，共2次，另一部分幼苗浇灌液体PDA培养基。15天后，分别收集幼苗胚根，洗净、烘干、研磨，胚根用体积比1:1的甲醇/氯仿混合液超声提取1h,(功率250，频率70Khz),过滤，挥干，甲醇定容，高效液相色谱法检测鬼臼毒素含量(色谱条件：色谱柱：Shim-pack GIST C18柱 (4.6mm×250mm,5.0um)，流动相：甲醇-水(50:50),流速1ml/min，检测波长290nm，柱温30℃)。
经检测，通过粉红粘帚霉fhnzm菌液处理的幼苗鬼臼毒素含量为6.52mg/g，而PDA对照组仅为4.22mg/g，粉红粘帚霉fhnzm菌液的处理使得鬼臼毒素含量提高了54.5％。实验结果表明，粉红粘帚霉fhnzm菌液处理能够显著促进桃儿七幼苗鬼臼毒素的积累。
实施例6：fhnzm发酵液促进桃儿七愈伤不定根鬼臼毒素的积累
取长度5cm的组培不定根接入1/2MS基本培养液、蔗糖浓度为20g/L，并按体积比20％添加实施例2方法制得的粉红粘帚霉fhnzm发酵液，对照添加PDA培养基，在黑暗条件下，温度20℃，培养7天，分别收集不定根，洗净、烘干、研磨，用体积比1:1的甲醇/氯仿混合液超声提取1h，(功率250，频率70Khz)，过滤，挥干，甲醇定容，高效液相色谱法检测鬼臼毒素含量(色谱条件：色谱柱：Shim-pack GIST C18柱(4.6mm×250mm,5.0um)，流动相：甲醇-水(50:50),流速1ml/min，检测波长290nm，柱温30℃)。
由于本实施例采用组织培养实验方式，为满足组培实验无菌操作环境的要求，故采用滤除fhnzm菌体的发酵液进行实验。经检测，通过粉红粘帚霉fhnzm发酵液处理的桃儿七组培不定根鬼臼毒素含量为6.48mg/g，而PDA对照组的鬼臼毒素含量仅为4.29mg/g，粉红粘帚霉fhnzm发酵液处理后相对于对照，鬼臼毒素含量提高了51％。实验结果表明，粉红粘帚霉fhnzm发酵液处理能够显著促进桃儿七组培不定根鬼臼毒素的积累。
序列表
<110> 甘肃省科学院生物研究所
<120> 一株桃儿七的内生真菌及其应用
<130> 1
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1307
<212> DNA
<213> 粉红粘帚霉(Clonostachys rosea)
<400> 1
atacggcgaa actgcgaatg gctcattata taagttatcg tttatttgat agtaccttac 60
tacttggata accgtggtaa ttctagagct aatacatgct taaaatcccg acttcggaag 120
ggatgtattt attagattaa aaaccaatgc ccttcggggc tctctggtga ttcatgataa 180
cttctcgaat cgcatggcct tgcgccggcg atggttcatt caaatttctt ccctatcaac 240
tttcgatgtt tgggtattgg ccaaacatgg ttgcaacggg taacggaggg ttagggctcg 300
accccggaga aggagcctga gaaacggcta ctacatccaa ggaaggcagc aggcgcgcaa 360
attacccaat cccgactcgg ggaggtagtg acaataaata ctgatacagg gttcttttgg 420
atcttgtaat tggaatgagt acaatttaaa tcccttaacg aggaacaatt ggagggcaag 480
tctggtgcca gcagccgcgg taattccagc tccaatagcg tatattaaag ttgttgtggt 540
taaaaagctc gtagttgaac cttgggcctg gctggccggt ccgcctcacc gcgtgtactg 600
gtccggccgg gcctttccct ctgtggaacc ctatgccctt cactgggtgt agcggggaaa 660
caggactttt actttgaaaa aattagagtg ctccaggcag gcctttgctc gaatacatta 720
gcatggaata ataaaatagg acgtgtggtt ctattttgtt ggtttctagg accgccgtaa 780
tgattaatag ggacagtcgg gggcatcagt attcaattgt cagaggtgaa attcttggat 840
ttattgaaga ctaactactg cgaaagcatt tgccaaggat gttttcatta atcaggaacg 900
aaagttaggg gatcgaagac gatcagatac cgtcgtagtc ttaaccataa actatgccga 960
ctagggatcg gacgatgtta aaaattgact cgttcggcac cttacgagaa atcaaagtgc 1020
ttgggctcca gggggagtat ggtcgcaagg ctgaaactta aagaaattga cggaagggca 1080
ccaccagggg tggagcctgc ggcttaattt gactcaacac ggggaaactc accaggtcca 1140
gacacaatga ggattgacag attgagagct ctttcttgat tttgtgggtg gtggtgcatg 1200
gccgttctta gttggtggag tgatttgtct gcttaattgc gataacgaac gagaccttaa 1260
cctgctaact agcccgtatt gctttggcag tacgctggct tcttaga 1307
Claims (7)
1.一株桃儿七的内生真菌粉红粘帚霉(Clonostachys rosea)fhnzm，其在中国微生物菌种保藏管理委员会普通微生物中心的保藏编号为CGMCC No.17070。
2.根据权利要求1所述的一株桃儿七的内生真菌粉红粘帚霉fhnzm，其特征在于所述菌株能够提高桃儿七根中鬼臼毒素的含量。
3.根据权利要求1所述的一株桃儿七的内生真菌粉红粘帚霉fhnzm，其特征在于所述菌株能够抑制桃儿七根的生长。
4.根据权利要求1所述的一株桃儿七的内生真菌粉红粘帚霉fhnzm，其特征在于所述菌株能够抑制植物种子的萌发和胚根的伸长。
5.根据权利要求1所述的一株桃儿七的内生真菌粉红粘帚霉fhnzm，其特征在于所述菌株的培养方法为菌丝接入马铃薯葡萄糖液体培养基中，温度27-30℃，转速120-160rpm，发酵时间7-10天。
6.根据权利要求5所述的一株桃儿七的内生真菌粉红粘帚霉fhnzm，其特征在于所述菌株的培养温度为30℃，转速160rpm，发酵时间9天。
7.一种促进桃儿七鬼臼毒素积累的菌剂，其特征在于该菌剂中包含权利要求1所述的桃儿七的内生真菌粉红粘帚霉fhnzm。
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