CN103992955B - 一株黑曲霉ny-1及提高其杀虫活性的改进技术和应用 - Google Patents
一株黑曲霉ny-1及提高其杀虫活性的改进技术和应用 Download PDFInfo
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Abstract
本项发明表观遗传修饰，具体讲是一株黑曲霉NY-1及提高其杀虫活性的改进技术和应用。黑曲霉NY-1(Aspergillus？niger)保藏于中国典型培养物保藏中心，保藏日期：2014年4月28日，保藏号：CCTCC？NO:M2014169。所述化学表观遗传修饰技术用于提高NY-1发酵产物的杀虫活性并应用于微生物农药。本项发明涉及菌株分离自山东安丘采集的牛蒡，通过PCR扩增ITS区段，测序结果与Genebank数据库中菌株Aspergillus？niger(Accession？number:KF358715.1)的相似度为100％。本发明采用化学表观修饰剂激活一株黑曲霉NY-1的杀虫沉默基因，提高NY-1的杀虫活性，并用于微生物农药。较基因调控和其它沉默基因激活技术，该方法具有快速、低成本、效果明显等优点。
Description
技术领域
本发明涉及化学表观遗传修饰技术，更具体地，是一株黑曲霉菌NY-1菌株及提高其杀虫活性的改进技术和应用。菌株NY-1在被化学表观遗传修饰剂作用后，其发酵液的提取物具有更高的杀虫活性。
背景技术
共生真菌表现出丰富的次生代谢能力，部分次生代谢产物也表现出杀虫活性，但是应用药用植物内生菌发酵法生产天然杀虫药物时，一方面内生菌脱离植物体后，往往次生代谢停止或延缓；另一方面内生菌在自生和共生条件下代谢途径会有所差异。BokJ.W等人在研究巢曲霉(Aspergillusnidulans)的基因组时发现，该菌基因组内有可产生1个萜类、2个吲哚生物碱、14个非核糖体多肽以及27个聚酮类化合物的基因存在，但是当在实验条件下发酵培养时，却只能表达出几个次级代谢产物，严重阻碍了真菌天然药物的研究与发展(ChemBiol,2006,13(1):31-37.)。
曲霉属(Aspergillus)是一类在陆地、海洋等各种环境中都广泛分布的真菌，其体内丰富多样的代谢途径使得该属的很多菌株被用于生产活性酶。除此之外已有的文献报道表明该属的有些菌株还具有特异的杀虫活性。黄曲霉(Aspergillusflavus)代谢产生的黄曲霉毒素就是一种昆虫化学绝育剂；曲酸不仅能影响昆虫的变态，而且可以提高烟碱的杀虫效果；杂色曲霉(Aspergillusversicolor)形成的杂烯亚胺对果蝇成虫有击倒作用。化学表观遗传修饰剂的在天然药物领域的应用为这一问题的解决提供了一种具有前景的方法。表观遗传学(Epigenetics)是指在DNA序列不发生改变的情况下，基因的表达和功能发生了可遗传的改变，而最终导致了生物表型的变化，这种改变在发育和细胞增殖过程中能够稳定地遗传下去。相对于分子生物学基因调控和其它沉默基因激活技术，该方法具有快速、简便、效果明显、成本低等优点。化学表观遗传修饰是最近几年正在逐渐发展的真菌次生代谢产物研究新技术，其主要是应用化学小分子(组蛋白脱乙酰酶抑制剂和DNA甲基化酶抑制剂)修饰真菌异染色质，激活沉默次生代谢途径基因簇转录，以获得新型的功能分子(Nat.Prod.Rep.2010,27(1),11–22.)。WilliamsR.B等利用5-AZA诱导Cladosporiumcladosporioides得到三种氧化脂类化合物(OrgBiomolChem,2008,6(11):1895-1897.)。JonC等利用SAHA处理黑曲霉(Aspergillusniger)得到一种从未在任何生物体内合成的化合物NygeroneA(Org.Biomol.Chem.,2009,7:435–438.)。RobertH.C的课题组主要对真菌的小分子表观遗传修饰剂的作用进行研究。该课题组利用9种DNMT和HDAC小分子抑制剂对12种真菌(包括：Aspergillusflavus、Aspergilluswesterdijkiae、Cladosporiumcladosporioides、Clonostachyssp.、Diatrypesp.、Penicilliumchrysogenum、Penicilliumcitrinum、Rhizopussp.、Verticilliumpsalliotae以及另外三种不明丝状真菌)进行诱导，结果表明，其中11种均对表观遗传修饰做出了反应，有的部分次级代谢产物产量提高，有的激活了新的化合物合成等(Nat.Prod.Rep.2010,27,11-22.)。
本发明采用化学表观遗传修饰技术激活一株黑曲霉NY-1的杀虫功能性沉默次生代谢途径，提高黑曲霉NY-1的杀虫活性。使用化学表观遗传修饰剂激活菌株的次级代谢产物，从而提高菌株的杀虫活性的方法具有靶点明确、效果明显的特点，可应用于微生物资源的开发利用中。
发明内容
本项发明目的在于提供一株黑曲霉NY-1菌株及提高其杀虫活性的改进技术和应用。
为充分达到以上目的，本发明采用的技术方案为：一株黑曲霉NY-1菌株，黑曲霉NY-1(Aspergillusniger)保藏于中国典型培养物保藏中心，保藏日期：2014年4月28日，保藏号：CCTCCNO:M2014169。
将所述黑曲霉于发酵培养基中静置或摇床发酵培养15-30天，发酵产物待用。所述发酵培养基比例为马铃薯200重量份，葡萄糖20重量份，酵母浸膏3重量份，蛋白胨5重量份，蒸馏水1000重量份，5-氮胞苷8.133×10-6重量份。所述黑曲霉NY-1发酵培养基中的5-氮胞苷用于激活黑曲霉NY-1的沉默代谢途径，提高黑曲霉NY-1发酵液的杀虫活性。
将所述黑曲霉NY-1于加入5-氮胞苷的发酵培养基中静置或摇床发酵培养15-30天，发酵产物待用。将激活后的发酵产物用有机溶剂提取，杀虫活性实验表明提取物具有较正常生长条件下更高的杀虫活性，为正常生长条件下的4.45倍。所述有机溶剂为：石油醚、乙酸乙酯、氯仿、丙酮、甲醇、乙醇或水中的一种或几种。所述发酵培养基中加入的5-氮胞苷的浓度为8.133×10-6重量份。经化学表观遗传修饰剂5-氮胞苷作用后，黑曲霉NY-1发酵液的杀虫活性半致死浓度LC50为0.037mg/mL，是正常生长条件下杀虫活性的4.45倍。
本发明所具有的优点：
本发明所述的黑曲霉NY-1菌株分离自山东安丘采集的牛蒡，通过PCR扩增18S-28SrDNA的ITS区段，测序结果与Genebank数据库中已知菌株相应序列进行比对，与菌株Aspergillusniger(Accessionnumber:KF358715.1)的相似度为100％。此种改进菌株NY-1发酵产物杀虫活性的方法，相对于分子生物学基因调控和其它沉默基因激活技术，其具有简便、快速、低成本、效果明显等优点。
具体实施方式
为阐明对本项发明特征的理解，下面结合一些非限定性的实施实例对本发明做进一步阐述。本项发明所述的黑曲霉NY-1(Aspergillusniger)，保藏于中国典型培养物保藏中心，保藏日期：2014年4月28日，保藏号：CCTCCNO:M2014169，保藏单位地址：武汉市武昌珞珈山。
实施例1：滨海植物来源黑曲霉(Aspergillusniger)NY-1菌株分类。
1)菌种培养
按照微生物学实验室常规培养方法，挑取少量保存于葡萄糖-琼脂培养基的黑曲霉(Aspergillusniger)NY-1，接种于PDA平板表面，25℃培养4天，初期菌落呈蔓延生长，整体为黑色，有白菌丝体，后期孢子黑色，为地毯状，放射型。
菌种初步鉴定
按照文献方法(东北农业大学学报，2007,38,(1):101-106)，通过PCR扩增18S-28SrDNA的ITS区段，测序结果与Genebank数据库中已知菌株相应序列进行比对，与菌株Aspergillusniger(Accessionnumber:KF358715.1)的相似度为100％。序列信息如下：ACTGGGATCTACTGATCGAGGTCACCTGGAAGAATGGTTGGAAAACGTCGGCAGGCGCCGGCCAATCCTACAGAGCATGTGACAAAGCCCCATACGCTCGAGGATCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCCGGAGAGGGGGACGGCGACCCAACACACAAGCCGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTAACTGATTGCATTCAATCAACTCAGACTGCACGCTTTCAGACAGTGTTCGTGTTGGGGTCTCCGGCGGGCACGGGCCCGGGGGGCAGAGGCGCCCCCCCGGCGGCCGACAAGCGGCGGGCCCGCCGAAGCAACAGGGTACAATAGACACGGATGGGAGGTTGGGCCCAAAGGACCCGCACTCGGTAATGATCCTTCCGCAGGTAC。
实施例2：黑曲霉(Aspergillusniger)NY-1菌株的发酵培养。
1)发酵培养
菌种培养：首先按照微生物学实验室常规培养方法，挑取少量保存于葡萄糖-琼脂培养基的黑曲霉NY-1(Aspergillusniger)，接种于PDA平板表面，25℃培养4天，作为规模发酵培养的菌种，待用。
其次切取种子板上4cm2大小的黑曲霉NY-1(Aspergillusniger)菌种，投入已灭菌、盛有液体培养基的2000mL锥形瓶中，室温静置培养22天，随即以乙酸乙酯灭菌，待用。
所述液体培养基为马铃薯60g/瓶，葡萄糖6g/瓶，酵母浸膏0.9g/瓶，蛋白胨1.5g/瓶，5-氮胞苷2.44μg/瓶，蒸馏水300mL/瓶。
2)发酵产物分离与纯化
首先将上述黑曲霉液体培养基的发酵产物以乙酸乙酯提取3次，合并提取液，减压蒸馏除去溶剂，得到浸膏。其次将所述发酵产物的浸提取物膏进行柱层析分离，以石油醚:乙酸乙酯体系梯度洗脱。再次收集石油醚:乙酸乙酯体积比20:1和5:1的组分A和B可用于制备微生物农药杀虫剂。
实施例3：杀虫活性试验。
卤虫(brineshrimp)又名盐水丰年虫，分类学上属节肢动物门、甲壳纲、无甲目、盐水丰年虫科、卤虫属。厦门大学的肖永堂等以卤虫为毒理实验对象，研究了采集自浙江和福建省红树林的共188株海洋真菌代谢产物的抗虫活性(厦门大学学报,2005,44(6):847-850)；刘永等(生态毒理学报,2011,6(5):557-560)测定了7种除虫菊酯对非靶性生物-卤虫的安全性和生态毒理评价。因此卤虫作为毒物急性毒性的实验方法简单、操作方便、快捷、可行性强。(PlantMedica,1991,45:31-34；农药,2011,50(4):261-262,275)。
1)卤虫的孵化
取一药匙约200mg卤虫卵置于500ml陈化海水中通气孵化，温度维持在28℃-30℃，自然光照条件。24h后及时除去卵壳和未孵化的卵，继续孵化24h，待用。整个实验过程中不饲喂卤虫。
2)待测样品制备
将上述实施例分离纯化的黑曲霉发酵产物总提取物、柱层析后组分A和B分别加入低于0.1％含量的DMSO助溶，将提取物配制成梯度浓度(mg/mL)：0.005、0.014、0.024、0.038、0.048、0.095、0.143、0.190、0.238、0.381、0.476、0.619、0.714、0.857、0.952mg/ml的浓度梯度，待用。
3)实验方法
按照文献报道的方法，取卤虫幼虫悬浮液200μL，加入到96孔板中，每孔加卤虫14-16条。空白对照组和各浓度梯度组分别设六个平行。空白组包括海水对照和助溶剂DMSO对照(Bioorg.Med.Chem.Lett.2010,20:5677-5680)。25℃恒温培养24h后，最后统计卤虫死亡个数。利用公式计算死亡率。
卤虫致死活性以校正死亡率表示，公式表示如下：
校正死亡率(％)＝(处理死亡率-对照死亡率)/(100-对照死亡率)×100，并计算出半致死浓度LC50。
实验结果显示激活后总提取物、组分A和B的半致死浓度LC50分别为0.037mg/mL，0.017mg/mL，0.024mg/ml，较之激活前总提取物半致死浓度LC50(0.1645mg/ml)，激活后表现出更好的杀虫活性。
上述实验结果证明本发明所涉及的黑曲霉的发酵产物具有较好的杀虫活性，可以用于微生物农药杀虫剂的制备。
序列信息：
ACTGGGATCTACTGATCGAGGTCACCTGGAAGAATGGTTGGAAAACGTCGGCAGGCGCCGGCCAATCCTACAGAGCATGTGACAAAGCCCCATACGCTCGAGGATCGGACGCGGTGCCGCCGCTGCCTTTCGGGCCCGTCCCCCCGGAGAGGGGGACGGCGACCCAACACACAAGCCGGGCTTGAGGGCAGCAATGACGCTCGGACAGGCATGCCCCCCGGAATACCAGGGGGCGCAATGTGCGTTCAAAGACTCGATGATTCACTGAATTCTGCAATTCACATTAGTTATCGCATTTCGCTGCGTTCTTCATCGATGCCGGAACCAAGAGATCCATTGTTGAAAGTTTTAACTGATTGCATTCAATCAACTCAGACTGCACGCTTTCAGACAGTGTTCGTGTTGGGGTCTCCGGCGGGCACGGGCCCGGGGGGCAGAGGCGCCCCCCCGGCGGCCGACAAGCGGCGGGCCCGCCGAAGCAACAGGGTACAATAGACACGGATGGGAGGTTGGGCCCAAAGGACCCGCACTCGGTAATGATCCTTCCGCAGGTAC
555rDNA黑曲霉(Aspergillusniger)。
Claims (11)
1.一株黑曲霉菌株，其特征在于：其为黑曲霉NY-1、拉丁名为Aspergillusniger，保藏于中国典型培养物保藏中心，保藏号：CCTCCNO:M2014169。
2.如权利要求1所述的黑曲霉菌株的发酵产物，其特征在于：将所述黑曲霉在发酵培养基中静置或摇床发酵培养15-30天。
3.如权利要求2所述的黑曲霉菌株的发酵产物，其特征在于培养基包括：马铃薯200重量份，葡萄糖20重量份，酵母浸膏3重量份，蛋白胨5重量份，5-氮杂胞苷8.133×10-6重量份和蒸馏水1000重量份。
4.一种提高菌株黑曲霉NY-1抗虫活性的方法，其特征在于：在发酵培养基中加入12.2×10-6重量份的5-氮杂胞苷。
5.一种权利要求1所述的黑曲霉作为微生物农药杀虫剂的应用。
6.如权利要求5所述的黑曲霉作为微生物农药杀虫剂的应用，其特征在于：所述黑曲霉置于发酵培养基中静置或摇床发酵培养15-30天，得到发酵产物。
7.如权利要求6所述的黑曲霉作为微生物农药杀虫剂的应用，其特征在于：将发酵产物用有机溶剂提取，提取物经柱色谱分离后，其产物用于制备生物农药杀虫剂。
8.如权利要求7所述的黑曲霉作为微生物农药杀虫剂的应用，其特征在于：所述溶剂为：石油醚、乙酸乙酯、氯仿、丙酮、甲醇、乙醇或水中的一种或几种。
9.如权利要求8所述的黑曲霉作为生物农药杀虫剂的应用，其特征在于：所述发酵产物进行柱层析分离，以石油醚:乙酸乙酯体系梯度洗脱，收集石油醚:乙酸乙酯体积比20:1和5:1用于制备微生物农药杀虫剂。
10.如权利要求9所述的黑曲霉作为生物农药杀虫剂的应用，其特征在于：所述层析柱为硅胶、反相、凝胶或大孔树脂中的一种或几种。
11.如权利要求9所述的黑曲霉作为生物农药杀虫剂的应用，其特征在于：所述石油醚:乙酸乙酯体系洗脱梯度为体积比20:1至1:1。
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