CN103641809B - 新月旋孢酸类化合物c及其制法和用途 - Google Patents
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Abstract
本发明属于微生物工程技术领域，具体涉及从蜻蜓幼虫肠道菌--Paraphaeosphaeria？sp.QTYC11液体发酵物中提取的一种新月旋孢酸类化合物C及其制法和应用。具体而言，本发明公开了一种新月旋孢酸类化合物C，其结构式为：
Description
技术领域
本发明属于微生物工程技术领域，具体涉及从蜻蜓幼虫肠道菌--Paraphaeosphaeriasp.QTYC11液体发酵物中提取的一种新月旋孢酸类化合物C及其制法和应用。
背景技术
反枝苋是我国恶性的入侵杂草，严重危害旱田农作物。反枝苋具有很强的抗旱能力和生态适应能力，与作物争夺养分，阻碍通风，遮挡阳光，耗竭地力，从而影响作物的生长发育。若不加以防除，会降低作物产量，还会污染作物种子，影响收获。同时，它也是许多昆虫、有害螨类、病原微生物(细菌、真菌、病毒、线虫)和寄生性种子植物(列当)的寄主。因此，防治反枝苋对作物的安全生产具有重要意义。化学防治是目前治理反枝苋危害的主要措施，但化学除草剂的长期使用带来了诸如环境污染日趋严重和耐药杂草种群的上升等问题，因此，研究开发新型除草剂迫在眉睫。已有研究表明昆虫肠道菌能分泌一些消化酶帮助昆虫消化食物，也有可能合成植物毒素，这类毒素能杀死植物以利于昆虫消化吃进去的植物。因此，昆虫肠道内生菌可能是新型除草剂的重要来源，将会成为寻找新型除草剂的重要途径。
病原真菌引起的植物病害造成的损失占总产量的10％～20％，化学防治是当前防治植物病害的主要手段，然而各种有机合成杀菌剂的使用也带来很多问题，如一些耐药植物病原菌开始出现，有机合成杀菌剂引起的环境问题、农药残留问题等，都使得开发新型杀菌剂变得尤为迫切。已有研究显示微生物是新型杀菌剂的重要来源，如从担子菌中分离得到的Strobilurins杀菌剂几乎覆盖了全球主要杀菌剂市场。昆虫共生菌作为一类特境微生物，也可能是新型杀菌剂的重要来源(Y.L.Zhang,L.C.Kong,D.HJiang,etal.BioresourceTechnology.,2011,102,3575-3577.)。然而，到目前为止，尚未见从昆虫肠道菌(Phomaherbarum)分离杀菌剂或除草剂的报道。
发明内容
本发明要解决的技术问题是提供一种新月旋孢酸类化合物(lunatoicacid)C及其制备方法和用途—作为除草剂或抗菌剂。
为了解决上述技术问题，本发明提供一种新月旋孢酸类化合物C，其结构式为：
本发明还同时提供了一种Paraphaeosphaeriasp.QTYC11，其保藏号为：CCTCCNO:M2013435。该Paraphaeosphaeriasp.QTYC11能用于制备新月旋孢酸类化合物C。
本发明还同时提供了上述新月旋孢酸类化合物C的制备方法，包括如下步骤：
1)、将保藏号为CCTCCNO:M2013435的Paraphaeosphaeriasp.QTYC11接种到ME培养基上，于摇床上，在160～200rpm(较佳为180rpm)、27.5～28.5℃(较佳为28℃)条件下培养2～3天作为种子液；
2)、将种子液接种于ME培养基中，在160～200rpm(较佳为180rpm)、27.5～28.5℃(较佳为28℃)条件下发酵6.5～7.5天(较佳为7天)；
一般而言，每10mL种子液接种于350～450mL(较佳为400mL)的ME培养基中；
3)、将步骤2)所得发酵液过滤(经两层纱布过滤)，滤液用乙酸乙酯萃取(共萃取3次，每次乙酸乙酯的用量＝发酵液的体积量)，真空浓缩干燥(于0.1个负压的真空度，45℃干燥30-50分钟)，得浸膏(呈深黄色)；
4)、将步骤3)所得浸膏进行硅胶柱层析分段，采用二氯甲烷/甲醇进行梯度洗脱，所述二氯甲烷与甲醇的体积比依次为100:0、100:1、100:2、100:4、100:8、100:16、100:32；从而分别得到7个洗脱部分F1-F7；
5)、将洗脱部分F3干燥处理(于45℃干燥40-55分钟)后经凝胶柱层析分离(采用葡聚糖凝胶SephadexLH-20)，以甲醇为洗脱剂，所得的沉淀(黄色)在甲醇中重结晶；得到新月旋孢酸类化合物C。
本发明还同时提供了上述新月旋孢酸类化合物C在除草剂或抗菌剂中的应用，具体为：用于抑制反枝苋幼根的生长，所述菌为小麦赤霉病菌、黄瓜枯萎病菌、苹果腐烂病菌。
本发明具有如下有益效果：
1.本发明的新月旋孢酸类化合物C是一种新化合物，可以作为具有除草作用的新除草剂或先导化合物，还能作为抗菌剂使用。
2.本发明的新月旋孢酸类化合物C可以利用微生物进行液体发酵生产，工艺简便，周期短，成本低，来源有保证。
3.本发明利用生物法合成新月旋孢酸类化合物C对环境无污染。
本发明的新月旋孢酸类化合物C作为除草剂时的用法和用量：稀释至0.05～0.1g/L进行喷洒；每公顷的用量(有效量)约为50～90g。
具体实施方式
下面结合具体实施例对本发明做进一步的解释。
实施例1：昆虫肠道真菌Paraphaeosphaeriasp.QTYC11分离与纯化
昆虫肠道真菌草茎点霉菌的分离：
分离前让蜻蜓幼虫饥饿24h，在无菌条件下用75％(体积％)酒精表面消毒2min，无菌水漂洗3次后解剖，取出昆虫肠道，加少量无菌水在无菌研钵中研磨，用无菌水对研磨液梯度稀释成10-1、10-2、10-3，分别取各梯度稀释液0.2mL涂布于MEA培养基平板上，28℃恒温箱中培养。待菌落长出后，从组织块菌落边缘挑取少量菌丝，再次转接MEA培养基平板上，经纯化得到单菌落，经形态学与株分子生物学鉴定为Paraphaeosphaeriasp.QTYC11。
MEA培养基：麦芽提取物20g，蔗糖20g，琼脂20g，蛋白胨1g，蒸馏水定容至1L，pH7.0，1.1个大气压，121℃下灭菌20min(为常规灭菌)。
上述Paraphaeosphaeriasp.QTYC11进行了保藏，保藏单位：中国典型培养物保藏中心；保藏名称为：Paraphaeosphaeriasp.QTYC11，保藏地址：中国武汉武汉大学；保藏日期：2013.09.17；保藏号为CCTCCNO：M2013435。
上述Paraphaeosphaeriasp.QTYC11可接种至MEA斜面试管保存备用。
实施例2：昆虫肠道真菌--Paraphaeosphaeriasp.QTYC11的液体发酵
将新鲜的Paraphaeosphaeriasp.QTYC11的菌丝体块(约2～3g)接种到250mL锥形瓶中，每瓶含有150mL的ME培养基，接种5～6瓶于摇床上，在180rpm、28℃条件下培养2～3天作为种子液，然后将10mL种子液接种于装有400mL的ME培养基的1000mL锥形瓶中，在180rpm、28℃条件下发酵7天。
Paraphaeosphaeriasp.QTYC11经常规的MEA培养基活化后，即可得新鲜的Paraphaeosphaeriasp.QTYC11。
所述ME培养基的配方为：麦芽提取物20g，蔗糖20g，蛋白胨1g，蒸馏水定容至1L，自然PH，然后1.1个大气压，121℃下灭菌20min(为常规灭菌)。
实施例3、新月旋孢酸类化合物C的提取与分离
将实施例2制备而得的50L发酵液经两层纱布过滤，滤液用乙酸乙酯萃取3次(乙酸乙酯每次的用量为50L)，所得的萃取液真空浓缩干燥(于0.1个负压的真空度，45℃干燥30-50分钟)得深黄色浸膏32.6g。对该32.6g浸膏进行硅胶柱层析分段(所述硅胶柱选用200～300目的硅胶，重量为200g)，采用二氯甲烷/甲醇梯度洗脱，二氯甲烷/甲醇的体积比依次为100:0,100:1,100:2,100:4,100:8,100:16,100:32，每种洗脱液的用量分别为2700～3000、1200～1400、5600～5900、2000～2300、1200～1500、1800～2000、800～1200mL；从而分别得到7个洗脱部分F1-F7。将所得的第3种洗脱部分F3于45℃干燥40-55分钟；得8.9g固体F3。
将8.9g固体F3进行凝胶柱层析(葡聚糖凝胶SephadexLH-20)，洗脱剂为甲醇(用量为800～1200mL)，得到黄色沉淀，所得的8.9g黄色沉淀在甲醇中进行重结晶，得到针状黄色晶体即为新月旋孢酸类化合物C(约3.6g)。
其结构式为：
实施例4：新月旋孢酸类化合物C的结构鉴定
新月旋孢酸类化合物C的结构是基于它们的质谱、核磁共振谱数据分析而确定的。
光谱学数据如下：
Yellowcolorneedles.ESI-MS:m/z[M+Na]+(calcd.forC21H22O7:386.1322).1HNMR(CDCl3-d)δ:0.88(3H,t,H-6'),1.02(3H,d,H-8'),1.40(2H,m,H-5'),1.60(3H,s,H-14),1.87(3H,s,H-7'),2.44(1H,m,H-4'),5.78(1H,s,H-7),6.54(1H,d,H-12),6.55(1H,s,H-9),6.73(1H,d,H-3'),7.23(1H,d,H-11),7.94(1H,s,H-2).13CNMR(CDCl3-d)δ:193.4(C-4),192.9(C-6),169.5(C-13),167.7(C-1'),153.4(C-2),152.5(C-10),151.0(C-3'),140.8(C-8),135.2(C-11),124.9(C-2'),123.6(C-12),116.6(C-9),114.9(C-3),110.8(C-7),84.0(C-5),35.1(C-4'),29.5(C-5'),21.8(C-14),19.4(C-8'),12.4(C-7'),11.9(C-6')。
实施例5：新月旋孢酸类化合物C对反枝苋幼根的抑制作用
将新月旋孢酸类化合物C用丙酮配制成100μg/mL，10μg/mL，1μg/mL，0.1μg/mL稀释液，加5mL于铺有滤纸的培养皿中使滤纸饱和，溶剂挥发后加入5mL无菌水，对照用丙酮同样处理。选取催芽的反枝苋种子150粒(每皿10粒)放在滤纸上，每个处理重复3皿，接种后的种子放在27℃、70％相对湿度、定时光照(12h光照，12h黑暗)条件的人工气候培养箱培养。4d后测量种子根长，以平均根生长抑制率表示结果。平均根生长抑制率(％)＝[对照平均根长-处理平均根长]/对照平均根长×100％。
表1.新月旋孢酸类化合物C对反枝苋幼根抑制效果
实验结果见表1，结果表明新月旋孢酸类化合物C对反枝苋幼根有明显的抑制作用，且抑制率随着浓度的升高逐渐加强，经计算，其抑制活性的IC50为10.2μg/mL；在供试浓度为100μg/mL时，其对反枝苋幼根的抑制率可达近75％，和同等浓度(100μg/mL)的阳性对照2,4-D(2,4-二氯苯氧乙酸)的抑制效果基本相当。以上结果表明：新月旋孢酸类化合物C具有很好的除草活性，因此本发明的化合物能被用作除草剂。
实施例6：新月旋孢酸类化合物C对植物致病真菌的抑制作用
将新月旋孢酸类化合物C溶于丙酮，配成浓度为1000μg/mL，500μg/mL，250μg/mL，100μg/mL，10μg/mL，1μg/mL稀释液母液。分别吸取1mL母液与9mLMEA培养基于无菌试管中，充分振摇后倒入无菌培养皿中，使培养基中提取物的最终质量浓度为母液的十分之一，以等量丙酮作为空白对照放线菌酮为阳性对照。将活化的植物致病真菌用无菌打孔器打成直径为5mm的菌块，置于上述培养基，每处理重复3次，培养4～5天后，采用十字交叉法测量供试菌菌落直径。按如下公式计算抑制率:抑制率＝(对照菌落直径－处理菌落直径)/(对照菌落直径-5mm)×100％。
表2.新月旋孢酸类化合物C对植物致病真菌的抑制作用
实验结果见表2，从表中可看出，本发明的新月旋孢酸类化合物C对小麦赤霉病菌(IC50＝1.7μg/mL)和黄瓜枯萎病菌(IC50＝4.2μg/mL)具有很好的抑制效果，分别优于阳性对照放线菌酮对小麦赤霉病菌(IC50＝3.3μg/mL)和黄瓜枯萎病菌(IC50＝4.9μg/mL)的抑制效果，新月旋孢酸类化合物C对苹果腐烂病菌(IC50＝3.3μg/mL)也有很好的效果。
以上结果表明：新月旋孢酸类化合物C具有很好的抗菌活性，因此本发明的化合物有被用作抗菌剂的潜力。
对比例1：LunatoicacidA，其结构式如下：
将上述LunatoicacidA替代本发明的新月旋孢酸类化合物C，按照实施例5所述方法进行检测，所得结果如下表3所示。
表3.LunatoicacidA对反枝苋幼根抑制效果
将上述LunatoicacidA按照实施例6所述方法进行检测，对小麦赤霉病菌IC50＝15μg/mL。
最后，还需要注意的是，以上列举的仅是本发明的若干个具体实施例。显然，本发明不限于以上实施例，还可以有许多变形。本领域的普通技术人员能从本发明公开的内容直接导出或联想到的所有变形，均应认为是本发明的保护范围。
Claims (5)
1.新月旋孢酸类化合物C，其特征是结构式为：
2.如权利要求1所述的新月旋孢酸类化合物C的制备方法，其特征是包括如下步骤：
1)、将保藏号为CCTCCNO:M2013435的草茎点霉菌(Phomaherbarum)接种到ME培养基上，于摇床上，在160～200rpm、27.5～28.5℃条件下培养2～3天作为种子液；
2)、将种子液接种于ME培养基中，在160～200rpm、27.5～28.5℃条件下发酵6.5～7.5天；
3)、将步骤2)所得发酵液过滤，滤液用乙酸乙酯萃取，真空浓缩干燥，得浸膏；
4)、将步骤3)所得浸膏进行硅胶柱层析分段，采用二氯甲烷/甲醇进行梯度洗脱，所述二氯甲烷与甲醇的体积比依次为100:0、100:1、100:2、100:4、100:8、100:16、100:32；从而分别得到7个洗脱部分F1-F7；
5)、将洗脱部分F3干燥处理后经凝胶柱层析分离，以甲醇为洗脱剂，所得的沉淀在甲醇中重结晶；得到新月旋孢酸类化合物C。
3.如权利要求1所述的新月旋孢酸类化合物C的用途，其特征是：作为除草剂或抗菌剂。
4.根据权利要求3所述的新月旋孢酸类化合物C的用途，其特征是：用于抑制反枝苋幼根的生长。
5.根据权利要求3所述的新月旋孢酸类化合物C的用途，其特征是：所述菌为：小麦赤霉病菌、黄瓜枯萎病菌、苹果腐烂病菌。
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