【摘要】：核盤菌(Sclerotinia sclerotiorum(Lib.)de Bary)是油菜菌核病的病原菌,每年造成油菜大量減產(chǎn),經(jīng)濟(jì)損失巨大。重寄生真菌盾殼霉(Coniothyrium minntans)對核盤菌專性寄生,對核盤菌的生物防治有著廣闊的開發(fā)前景。實驗室前期研究發(fā)現(xiàn)盾殼霉能降解核盤菌產(chǎn)生的草酸毒素,這一過程中可以通過產(chǎn)生氨來緩解酸化的環(huán)境,也說明這其中伴隨著盾殼霉的氮源代謝調(diào)控,而參與該氮源代謝的調(diào)控基因和功能還未見報道。盾殼霉氮源代謝調(diào)控基因(cmareA)對其生長發(fā)育,盾殼霉重寄生,降解草酸和產(chǎn)生抗真菌的影響也尚不清楚。因此,該課題在實驗室前期研究的基礎(chǔ)上,對盾殼霉的氮源代謝調(diào)控基因cmareA進(jìn)行了深入研究,獲得的主要研究成果如下:從盾殼霉Chy-1中克隆獲得cmareA部分序列,比對盾殼霉基因組數(shù)據(jù)庫獲得全部cmareA基因序列,基因全長2811 bp,除去一個內(nèi)含子之后的外顯子序列大小為2754 bp,該開放閱讀框編碼917個氨基酸。系統(tǒng)進(jìn)化樹分析結(jié)果表明cmareA編碼的氨基酸與小麥穎枯病菌(Parastagonospora nodorum)的同源areA編碼的氨基酸親緣關(guān)系最近,同源性高達(dá)85%�；虻谋磉_(dá)分析證明cmareA基因只有在優(yōu)先氮源不足或氮源饑餓狀態(tài)下,cmareA的轉(zhuǎn)錄調(diào)控才會被激活,cmareA基因才會表達(dá)。通過Split-Marker技術(shù)對cmareA基因進(jìn)了敲除與互補(bǔ),獲得敲除突變體ΔcmareA-129和ΔcmareA-133,相對應(yīng)的互補(bǔ)轉(zhuǎn)化子ΔcmareA-129C和ΔcmareA-133C。對其生物學(xué)特性的研究發(fā)現(xiàn),cmareA的缺失會減慢盾殼霉在PDA培養(yǎng)條件下的菌絲生長發(fā)育,同時會減低其產(chǎn)孢量。另外,cmareA的缺失會導(dǎo)致盾殼霉在以硝酸和銨鹽為氮源的MCD培養(yǎng)條件下的菌絲生長受到抑制,生長速率顯著下降。通過平板對峙試驗和菌核沙皿寄生菌核試驗,測定各突變體寄生核盤菌的能力。結(jié)果顯示,cmareA敲除后,敲除轉(zhuǎn)化子ΔcmareA-129和ΔcmareA-133對核盤菌菌絲和菌核的寄生能力減弱。進(jìn)一步測定重寄生相關(guān)酶(胞外蛋白酶、葡聚糖酶和幾丁質(zhì)酶)活性,結(jié)果發(fā)現(xiàn)敲除突變體的重寄生能力的減弱可能是由于cmareA缺失導(dǎo)致幾丁質(zhì)酶活性降低所引起的。通過液相色譜的方法,測定各突變體降解草酸的能力,結(jié)果顯示,cmareA缺失后盾殼霉降解草酸的能力也同樣減弱。在不同氮源的培養(yǎng)條件,發(fā)現(xiàn)可能由于cmareA敲除突變體的生長速度減慢導(dǎo)致產(chǎn)生的抗真菌物質(zhì)的能力也有不同程度的降低。另外,在研究氮源與pH對盾殼霉抗菌作用的交互影響實驗中,發(fā)現(xiàn)盾殼霉以硝酸鹽為氮源時產(chǎn)生AFS,酸性pH是必須的,cmareA也是必須的。綜上所述,盾殼霉氮源代謝調(diào)控基因cmareA不僅參與了盾殼霉的生長發(fā)育、硝酸和銨類氮源的調(diào)控,cmareA基因還能正調(diào)控著盾殼霉的重寄生,草酸的降解,同時還調(diào)控抗真菌物質(zhì)的產(chǎn)生。
[Abstract]:Sclerotinia sclerotiorum (Lib.) de Bary) is the pathogen of sclerotinia sclerotiorum. (Coniothyrium minntans), a highly parasitic fungus, is a specific parasitic fungus to Sclerotinia sclerotiorum, and has a broad prospect for biological control of Sclerotinia sclerotiorum (Sclerotinia sclerotiorum). Previous laboratory studies have found that the bacteria can degrade oxalic acid toxin produced by Sclerotinia sclerotiorum, which can alleviate the acidified environment by producing ammonia, which also shows that the nitrogen source metabolism regulation is accompanied by chitosan. However, the regulatory genes and functions involved in the metabolism of this nitrogen source have not been reported. The effects of (cmareA) on its growth and development, the high parasitism, the degradation of oxalic acid and the production of antifungal were also unclear. Therefore, on the basis of the previous research in laboratory, the cmareA gene regulating nitrogen source metabolism was deeply studied. The main research results were as follows: the partial sequence of cmareA was cloned from Chy-1. The whole cmareA gene sequence was obtained by comparing the genomic database of Trichoderma auratus. The exon sequence of the gene with a total length of 2811 bp, excluding an intron was 2754 bp,. The open reading frame encodes 917 amino acids. Phylogenetic tree analysis showed that the amino acid encoded by cmareA was closely related to the amino acid encoded by homologous areA of (Parastagonospora nodorum) of wheat glume blight, and the homology was as high as 85g. Gene expression analysis showed that cmareA gene can only be activated by cmareA transcription regulation and cmareA gene expression only when the priority nitrogen source is insufficient or the nitrogen source is hungry. The complementary transformants 螖 cmareA-129 and 螖 cmareA-133, corresponding to the knockout mutants 螖 cmareA-129 and 螖 cmareA-133, were obtained by Split-Marker. The complementary transformants 螖 cmareA-129C and 螖 cmareA-133C. were obtained. The study of its biological characteristics showed that the absence of cmareA could slow down the growth and development of the hyphae and reduce the sporulation under the condition of PDA culture. In addition, the absence of cmareA resulted in the inhibition of mycelium growth and the decrease of growth rate in MCD cultured with nitric acid and ammonium salt as nitrogen source. The ability of each mutant to parasitize Sclerotinia sclerotiorum was determined by plate confrontation test and nuclear test of Sclerotinia sclerotiorum parasite. The results showed that after cmareA knockout, the parasitic ability of knockout transformants 螖 cmareA-129 and 螖 cmareA-133 on sclerotia and sclerotia decreased. The activity of hyperparasitic related enzymes (extracellular protease, glucanase and chitinase) was further determined. It was found that the decrease in the ability of heavy parasitism of knockout mutants may be due to the decrease of chitinase activity due to the absence of cmareA. The ability of the mutants to degrade oxalic acid was determined by liquid chromatography. The results showed that the ability of chitosan to degrade oxalic acid was also weakened after the absence of cmareA. Under different nitrogen sources, it was found that the ability of antifungal substances produced by cmareA knockout mutants was also decreased in varying degrees due to the slow growth rate of the mutant. In addition, in the experiment of the interaction between nitrogen source and pH, it was found that it is necessary to produce AFS, acid pH when the nitrate is used as nitrogen source, and cmareA is also necessary. In conclusion, the nitrogen source regulation gene cmareA not only participates in the growth and development of chitosan, but also in the regulation of nitric acid and ammonium nitrogen source. CmareA gene can also regulate the hyperparasitism and the degradation of oxalic acid. At the same time, it also regulates the production of antifungal substances.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S476

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