禾谷镰刀菌Rab7效应因子VPS26与VPS29的研究
发布时间：2019-03-21 19:16
【摘要】：禾谷镰刀菌是引起小麦赤霉病的主要病原真菌,它污染谷物的同时可产生DON毒素,进而严重损害人和牲畜健康。DON毒素的合成不仅受理化因素的影响,更主要的受细胞内生物大分子的调控作用。Rab7可以调控DON的生物合成,Rab7功能的发挥需要结合相应的效应因子。本课题对禾谷镰刀菌的Rab7假定效应因子VPS26和VPS29进行研究,分析了Rab7与效应因子对禾谷镰刀菌产DON毒素的调控作用,探讨了VPS26和VPS29与Rab7的相互作用,为作物赤霉病的防治提供新的药物靶标位点和理论依据。利用ELISA法测定野生型和FgRab7敲除突变体在三种不同培养基质中胞内外产DON情况,结果发现,无论在何种培养基质中,在整个培养阶段Rab7突变体胞外DON毒素积累量均明显低于野生型,整个过程胞内几乎检测不到DON的存在,说明FgRab7对禾谷镰刀菌DON毒素的生物合成有影响。为明确FgRab7调控DON毒素的分子机制,本研究利用酵母双杂交技术分析了VPS26、VPS29与Rab7的相互作用,利用荧光定量PCR分析了VPS26、VPS29在野生型和Rab7突变体中的表达。结果显示,禾谷镰刀菌中,VPS26可以与激活态下的Rab7进行相互作用,而VPS29与Rab7不具有直接相互作用的关系,表明VPS26是FgRab7的一个效应因子,VPS29仍需进一步研究;不同时期,野生型中VPS26与VPS29表达量逐步增高,同一时期,FgRab7缺失突变体VPS26表达量均低于野生型,其中,前14d表达量逐步增高,之后逐渐降低,35d时表达量低于野生型约8倍,VPS29表达量呈现先增高后降低再升高的趋势,第35d约低于野生型2倍,进一步表明Rab7与VPS26具有直接互作关系并间接影响了VPS29的表达。进一步对VPS26进行功能分析,结果显示,FgVps26敲除突变体菌丝生长变慢,菌落形态变化,气生菌丝减少,产孢量减少,孢子畸形,产DON毒素能力与野生型相比显著下降;VPS26回补突变体表型缺陷可以恢复,说明VPS26在禾谷镰刀菌的生长发育及毒素合成中发挥重要的生理功能。实时荧光定量分析Rab7及VPS29在VPS26缺失突变体中的表达情况,结果表明,不同时期野生型和突变体中Rab7与VPS29表达量逐步上升,同一时期VPS26缺失使Rab7与VPS29表达量明显下降,说明VPS26不仅与Rab7具有相互作用关系,同样制约着VPS29的表达;FgVps26敲除突变体产DON毒素能力与野生型相比显著下降,在每个检测时期野生型DON积累量均为突变体的2~3倍,说明Vps26的缺失影响了DON的合成。本论文证明了禾谷镰刀菌中,VPS26是Rab7的一个效应因子,FgRab7通过与VPS26的相互作用调控DON毒素的生物合成,FgRab7与FgVps26在DON毒素产生方面具有协同调控作用,研究结果在揭示Rab7调控DON毒素的分子机制方面具有一定的意义。
[Abstract]:Fusarium graminearum (Fusarium graminearum) is the main pathogen of wheat scab. It can pollute grains and produce DON toxin, which can seriously damage the health of human and livestock. The synthesis of don toxin is not only affected by physical and chemical factors. Rab7 can regulate the biosynthesis of DON, and the function of Rab7 needs to be combined with the corresponding effect factors. In this study, the hypothetical effect factors VPS26 and VPS29 of Rab7 of Fusarium graminearum were studied. The regulatory effects of Rab7 and effect factors on the production of DON toxin by Fusarium graminearum were analyzed, and the interaction between VPS26, VPS29 and Rab7 was discussed. To provide a new drug target site and theoretical basis for the prevention and treatment of crop scab. The production of DON by wild-type and FgRab7 knockout mutants in three different culture media was determined by ELISA. The results showed that no matter in any culture medium, the growth of wild-type and FgRab7-knockout mutants produced DON in three different media. The accumulation of extracellular DON toxin in Rab7 mutant was significantly lower than that in wild type during the whole culture stage, and the presence of DON was almost absent during the whole process, indicating that FgRab7 had an effect on the biosynthesis of DON toxin from Fusarium graminearum. In order to clarify the molecular mechanism of FgRab7 regulating DON toxin, the interaction between VPS26,VPS29 and Rab7 was analyzed by yeast two hybrid technique, and the expression of VPS26,VPS29 in wild type and Rab7 mutant was analyzed by fluorescence quantitative PCR. The results showed that in Fusarium graminearum, VPS26 could interact with activated Rab7, but there was no direct interaction between VPS29 and Rab7, indicating that VPS26 was an effective factor of FgRab7, and VPS29 still needed further study. At different stages, the expression of VPS26 and VPS29 in wild type increased gradually. At the same time, the expression level of VPS26 in FgRab7 deletion mutant was lower than that in wild type. The expression level of VPS26 in the first 14 days increased gradually, then decreased gradually, and it was 8 times lower than that in wild type on the 35th day. The expression of VPS29 increased first, then decreased and then increased, which was about 2 times lower than that of wild type on the 35th day, which further indicated that Rab7 had direct interaction with VPS26 and indirectly affected the expression of VPS29. Further functional analysis of VPS26 showed that the hyphae of FgVps26 knockout mutants grew slowly, the morphology of colonies changed, aerated hyphae decreased, sporulation decreased, spores malformed, and the ability of producing DON toxin decreased significantly compared with wild type. The phenotypic defects of VPS26 complementary mutants can be recovered, indicating that VPS26 plays an important physiological role in the growth and development of Fusarium graminearum and toxin synthesis. The expression of Rab7 and VPS29 in VPS26 deletion mutants was quantitatively analyzed by real-time fluorescence analysis. The results showed that the expression of Rab7 and VPS29 in wild type and mutant increased gradually at different stages, and the expression of Rab7 and VPS29 decreased significantly after VPS26 deletion in the same period. The results indicate that VPS26 not only interacts with Rab7, but also restricts the expression of VPS29. The ability of FgVps26 knockout mutant to produce DON toxin was significantly lower than that of wild type, and the accumulation of wild-type DON in each detection period was 2-3 times that of mutant, which indicated that the deletion of Vps26 affected the synthesis of DON. This study demonstrated that VPS26 is an effective factor of Rab7 in Fusarium graminearum. FgRab7 regulates the biosynthesis of DON toxin through interaction with VPS26. FgRab7 and FgVps26 play a synergistic role in the production of DON toxin. The results have some significance in revealing the molecular mechanism of Rab7 regulating DON toxin.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.45
本文编号：2445235
[Abstract]:Fusarium graminearum (Fusarium graminearum) is the main pathogen of wheat scab. It can pollute grains and produce DON toxin, which can seriously damage the health of human and livestock. The synthesis of don toxin is not only affected by physical and chemical factors. Rab7 can regulate the biosynthesis of DON, and the function of Rab7 needs to be combined with the corresponding effect factors. In this study, the hypothetical effect factors VPS26 and VPS29 of Rab7 of Fusarium graminearum were studied. The regulatory effects of Rab7 and effect factors on the production of DON toxin by Fusarium graminearum were analyzed, and the interaction between VPS26, VPS29 and Rab7 was discussed. To provide a new drug target site and theoretical basis for the prevention and treatment of crop scab. The production of DON by wild-type and FgRab7 knockout mutants in three different culture media was determined by ELISA. The results showed that no matter in any culture medium, the growth of wild-type and FgRab7-knockout mutants produced DON in three different media. The accumulation of extracellular DON toxin in Rab7 mutant was significantly lower than that in wild type during the whole culture stage, and the presence of DON was almost absent during the whole process, indicating that FgRab7 had an effect on the biosynthesis of DON toxin from Fusarium graminearum. In order to clarify the molecular mechanism of FgRab7 regulating DON toxin, the interaction between VPS26,VPS29 and Rab7 was analyzed by yeast two hybrid technique, and the expression of VPS26,VPS29 in wild type and Rab7 mutant was analyzed by fluorescence quantitative PCR. The results showed that in Fusarium graminearum, VPS26 could interact with activated Rab7, but there was no direct interaction between VPS29 and Rab7, indicating that VPS26 was an effective factor of FgRab7, and VPS29 still needed further study. At different stages, the expression of VPS26 and VPS29 in wild type increased gradually. At the same time, the expression level of VPS26 in FgRab7 deletion mutant was lower than that in wild type. The expression level of VPS26 in the first 14 days increased gradually, then decreased gradually, and it was 8 times lower than that in wild type on the 35th day. The expression of VPS29 increased first, then decreased and then increased, which was about 2 times lower than that of wild type on the 35th day, which further indicated that Rab7 had direct interaction with VPS26 and indirectly affected the expression of VPS29. Further functional analysis of VPS26 showed that the hyphae of FgVps26 knockout mutants grew slowly, the morphology of colonies changed, aerated hyphae decreased, sporulation decreased, spores malformed, and the ability of producing DON toxin decreased significantly compared with wild type. The phenotypic defects of VPS26 complementary mutants can be recovered, indicating that VPS26 plays an important physiological role in the growth and development of Fusarium graminearum and toxin synthesis. The expression of Rab7 and VPS29 in VPS26 deletion mutants was quantitatively analyzed by real-time fluorescence analysis. The results showed that the expression of Rab7 and VPS29 in wild type and mutant increased gradually at different stages, and the expression of Rab7 and VPS29 decreased significantly after VPS26 deletion in the same period. The results indicate that VPS26 not only interacts with Rab7, but also restricts the expression of VPS29. The ability of FgVps26 knockout mutant to produce DON toxin was significantly lower than that of wild type, and the accumulation of wild-type DON in each detection period was 2-3 times that of mutant, which indicated that the deletion of Vps26 affected the synthesis of DON. This study demonstrated that VPS26 is an effective factor of Rab7 in Fusarium graminearum. FgRab7 regulates the biosynthesis of DON toxin through interaction with VPS26. FgRab7 and FgVps26 play a synergistic role in the production of DON toxin. The results have some significance in revealing the molecular mechanism of Rab7 regulating DON toxin.
【学位授予单位】：河南工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.121.45
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