禾谷鐮刀菌Rab7效應(yīng)因子VPS26與VPS29的研究
發(fā)布時(shí)間：2019-03-21 19:16
【摘要】：禾谷鐮刀菌是引起小麥赤霉病的主要病原真菌,它污染谷物的同時(shí)可產(chǎn)生DON毒素,進(jìn)而嚴(yán)重?fù)p害人和牲畜健康。DON毒素的合成不僅受理化因素的影響,更主要的受細(xì)胞內(nèi)生物大分子的調(diào)控作用。Rab7可以調(diào)控DON的生物合成,Rab7功能的發(fā)揮需要結(jié)合相應(yīng)的效應(yīng)因子。本課題對(duì)禾谷鐮刀菌的Rab7假定效應(yīng)因子VPS26和VPS29進(jìn)行研究,分析了Rab7與效應(yīng)因子對(duì)禾谷鐮刀菌產(chǎn)DON毒素的調(diào)控作用,探討了VPS26和VPS29與Rab7的相互作用,為作物赤霉病的防治提供新的藥物靶標(biāo)位點(diǎn)和理論依據(jù)。利用ELISA法測(cè)定野生型和FgRab7敲除突變體在三種不同培養(yǎng)基質(zhì)中胞內(nèi)外產(chǎn)DON情況,結(jié)果發(fā)現(xiàn),無(wú)論在何種培養(yǎng)基質(zhì)中,在整個(gè)培養(yǎng)階段Rab7突變體胞外DON毒素積累量均明顯低于野生型,整個(gè)過(guò)程胞內(nèi)幾乎檢測(cè)不到DON的存在,說(shuō)明FgRab7對(duì)禾谷鐮刀菌DON毒素的生物合成有影響。為明確FgRab7調(diào)控DON毒素的分子機(jī)制,本研究利用酵母雙雜交技術(shù)分析了VPS26、VPS29與Rab7的相互作用,利用熒光定量PCR分析了VPS26、VPS29在野生型和Rab7突變體中的表達(dá)。結(jié)果顯示,禾谷鐮刀菌中,VPS26可以與激活態(tài)下的Rab7進(jìn)行相互作用,而VPS29與Rab7不具有直接相互作用的關(guān)系,表明VPS26是FgRab7的一個(gè)效應(yīng)因子,VPS29仍需進(jìn)一步研究;不同時(shí)期,野生型中VPS26與VPS29表達(dá)量逐步增高,同一時(shí)期,FgRab7缺失突變體VPS26表達(dá)量均低于野生型,其中,前14d表達(dá)量逐步增高,之后逐漸降低,35d時(shí)表達(dá)量低于野生型約8倍,VPS29表達(dá)量呈現(xiàn)先增高后降低再升高的趨勢(shì),第35d約低于野生型2倍,進(jìn)一步表明Rab7與VPS26具有直接互作關(guān)系并間接影響了VPS29的表達(dá)。進(jìn)一步對(duì)VPS26進(jìn)行功能分析,結(jié)果顯示,FgVps26敲除突變體菌絲生長(zhǎng)變慢,菌落形態(tài)變化,氣生菌絲減少,產(chǎn)孢量減少,孢子畸形,產(chǎn)DON毒素能力與野生型相比顯著下降;VPS26回補(bǔ)突變體表型缺陷可以恢復(fù),說(shuō)明VPS26在禾谷鐮刀菌的生長(zhǎng)發(fā)育及毒素合成中發(fā)揮重要的生理功能。實(shí)時(shí)熒光定量分析Rab7及VPS29在VPS26缺失突變體中的表達(dá)情況,結(jié)果表明,不同時(shí)期野生型和突變體中Rab7與VPS29表達(dá)量逐步上升,同一時(shí)期VPS26缺失使Rab7與VPS29表達(dá)量明顯下降,說(shuō)明VPS26不僅與Rab7具有相互作用關(guān)系,同樣制約著VPS29的表達(dá);FgVps26敲除突變體產(chǎn)DON毒素能力與野生型相比顯著下降,在每個(gè)檢測(cè)時(shí)期野生型DON積累量均為突變體的2~3倍,說(shuō)明Vps26的缺失影響了DON的合成。本論文證明了禾谷鐮刀菌中,VPS26是Rab7的一個(gè)效應(yīng)因子,FgRab7通過(guò)與VPS26的相互作用調(diào)控DON毒素的生物合成,FgRab7與FgVps26在DON毒素產(chǎn)生方面具有協(xié)同調(diào)控作用,研究結(jié)果在揭示Rab7調(diào)控DON毒素的分子機(jī)制方面具有一定的意義。
[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.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.121.45
本文編號(hào)：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.
【學(xué)位授予單位】：河南工業(yè)大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：S435.121.45
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