【作者】 张凡；

【导师】 徐晓勇；

【作者基本信息】 华东理工大学 ， 制药工程与技术， 2017， 硕士

【摘要】 植物线虫每年对农业生产造成巨大损失,但能有效控制植物线虫的杀线虫剂却远远不足。研究表明,大环内酯类的阿维菌素及环肽类Emodepside具有杀线虫活性。环六肽的白僵菌素因具有独特的作用机制和广泛的生物活性,而杀虫真菌球孢白僵菌和玫烟色拟青霉等杀虫真菌均能够生物合成白僵菌素。植物寄生线虫每年造成巨大的粮食损失,但能够用于植物线虫有效防控的药物种类十分有限。本文主要从白僵菌素的生物合成、化学结构修饰以及植物根结线虫的抑制活性等方面对白僵菌素进行了详细研究。白僵菌素生物合成的研究是以球孢白僵菌（Bb 2860）和玫烟色拟青霉（ISF 2679）为研究对象,文中分别对球孢白僵菌的C6转录因子基因BBA₀9723,玫烟色拟青霉的C6转录因子ISF₀0192和组蛋白去乙酰化酶基因ISF₀4479进行了敲除。最后将上述三个敲除子与BBA₀9723过表达的球孢白僵菌以及球孢白僵菌和玫烟色拟青霉野生型的白僵菌素生物合成能力进行了对比。研究结果表明基因ISF₀0192和基因BBA₀9723对白僵菌素的合成起到负调控的作用,基因ISF₀4479使玫烟色拟青霉菌基本丧失了白僵菌素的生物合成能力。测试真菌对大蜡螟的杀虫毒力。测试结果表明,各个转化子对大蜡螟的杀虫毒力均优于相应的野生型真菌。这说明上述研究的基因对真菌的杀虫毒力有影响。白僵菌素作为一种具有潜力的药物先导,其生物制备和分离纯化是关键。本论文探索了发酵玫烟色拟青霉提取白僵菌素的方法以及白僵菌素分离纯化的条件。利用化学合成手段对白僵菌素进行了结构修饰,将吗啉环引入白僵菌素的结构中,并测试了白僵菌素与化学修饰得到的白僵菌素衍生物对植物根结线虫的抑制活性。研究结果表明在白僵菌素化学结构中的苯环上衍生吗啉环能部分提高化合物对植物根结线虫的抑制活性,衍生物中组分3在10 mg/L浓度下对南方根结线虫活体抑制活性病害指数为1.5,比对照白僵菌素本身的1.8活性略好,可以进一步对白僵菌素进行结构修饰。更多还原

【Abstract】 Plant nematodes cause great loss to food production every year,but the kinds of nematicide which can control it are far from enough.The researh previously shows that Avermectin and Emodepside which belong to macrolide and cyclopeptide compound respectively can control the nematodes efficiently.Beauvericin has special mechanism and broad bioactivity.Cordyceps bassiana（Bb2860）and Isaria fumosorosea（ISF2679）which both are insecticidal fungi can biosynthesize Beauvericin.The study in this article is mostly about the biosynthesis regulation,chemical modification and nematocidal activity of Beauvericin.The research of biosynthesis regulation on Beauvericin is based on the Cordyceps bassiana（Bb2860）and Isaria fumosorosea（ISF2679）.The gene BBA₀9723 of Cordyceps bassiana,ISF₀0192 and ISF₀4479 of Isaria fumosorosea were knocked out respectively.BBA₀9723 and ISF₀0192 are speculated to be gene of C6 transcription factor repectively,and ISF₀4479 is the gene of histone acetylation enzyme.Beauviricin biosynthesis ability of the three deletion mutant,the BBA₀9723 overexpression mutant and the wild type of Cordyceps bassiana and Isaria fumosorosea were compared.The toxicity of these fungi to G mellonella were tested too.The result show that ISF₀0192 and BBA₀9723 negatively control the Beauvericin biosynthesis,and the deletion of ISF₀4479 causes it obsolutely losed.The mutations’toxicity against G.mellonella are better than the relating wild type respectively,and this shows that the genes all above can effect the toxicity of fungi.Beauvericin is a promising drug lead,and its biosynthesis and extraction is the key process.In the part,we studied the fermentation of Isaria fumosorosea,extraction and purification of Beauvericin.Beauvericin was chemical modificated by chemical synthesis method,and morpholinyl was introducted into the structure.At last,bioassay was done to compared the inhibitory bioactivity against with nematode in vitro and in vivo.The bioassay results showed that the introdution of morpholinyl can increase the inhibition activity of the compound.The inhibition index of the separation 3 of the derivative is 1.5 which is better than that of the Beauvericin.And this indicated that the chemical modefication need further exploration.更多还原