本文關(guān)鍵詞：響應(yīng)pH的轉(zhuǎn)錄因子基因PacC在靈芝菌絲生長、發(fā)育、凋亡和次級代謝過程中的功能研究　出處：《南京農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 藥用真菌 PacC/Rim101 漆酶 發(fā)育 次級代謝 離子穩(wěn)態(tài)

【摘要】：靈芝(Ganoderma lucidum)是我國傳統(tǒng)的藥用大型真菌,同時,也是一種白腐真菌,是目前發(fā)現(xiàn)含有降解木質(zhì)纖維素所需降解酶基因數(shù)目最多的擔(dān)子菌。但是,由于靈芝的基礎(chǔ)研究十分薄弱,嚴重阻礙了其商業(yè)價值的開發(fā)和利用。據(jù)報道,外界pH的變化會影響靈芝的生長、代謝等許多生理過程。Pal/Rim途徑是真菌中特有的響應(yīng)外界pH變化的信號調(diào)控途徑,而PacC/Rim101是此途徑中發(fā)揮關(guān)鍵調(diào)控作用的轉(zhuǎn)錄因子。本論文研究了 PacC在靈芝菌絲生長、發(fā)育、凋亡和次級代謝等生理過程中的作用。本研究發(fā)現(xiàn)外界pH的變化能夠影響靈芝的菌絲生長和形態(tài),并且PacC/Rim101的同源基因GlPacC的轉(zhuǎn)錄能夠響應(yīng)外界pH的變化。進一步通過基因克隆和序列分析發(fā)現(xiàn),GlPacC編碼807個氨基酸殘基預(yù)測的蛋白分子量和等電點分別為86.15 kDa和7.76。GlPacC包含3個典型的轉(zhuǎn)錄因子C2H2鋅指結(jié)構(gòu)域、兩個推測的PEST結(jié)構(gòu)域、一個可能的PKA磷酸化位點和一個核定位信號基序。為了研究GlPacC如何受到外界中性或堿性pH的誘導(dǎo)表達,通過酵母單雜交實驗證明GlPacC能夠與自身基因啟動子上的順式作用元件相結(jié)合,從而調(diào)控自身基因的轉(zhuǎn)錄。為了進一步研究該基因在靈芝中的作用,利用基因沉默技術(shù),篩選獲得了 4個沉默效率達到55～75%的GlPaacC沉默轉(zhuǎn)化子。通過平板生長實驗發(fā)現(xiàn)GlPacC沉默菌株對外界環(huán)境pH變化更加敏感,菌絲分叉間距明顯變長,表明GlPacC在靈芝菌絲生長過程中發(fā)揮重要作用。菌絲生長的改變可能會影響靈芝對各種脅迫的耐受性,通過考察對化學(xué)脅迫物質(zhì)的耐受性發(fā)現(xiàn),GlPacC沉默菌株對10 mM LiCl、10 mM H2O2、0.05% (w/v) SDS和1 mg/ml剛果紅更加敏感,對0.4 M NaCl或KC1耐受性增強。由SDS和剛果紅處理結(jié)果推測,可能是靈芝細胞壁完整性遭到破壞。進一步考察了細胞壁完整性途徑的變化發(fā)現(xiàn),GlPacC沉默導(dǎo)致Slt2和Rlml表達量下調(diào),Slt2磷酸化水平降低,β-1, 3-葡聚糖含量降低,從而導(dǎo)致細胞壁變薄,最終使靈芝對細胞壁脅迫更加敏感。此外,GlPacC還能夠通過調(diào)控?zé)峒まD(zhuǎn)錄因子和熱激蛋白基因的轉(zhuǎn)錄,進而影響靈芝對熱脅迫的耐受性。漆酶是一種重要的工業(yè)用酶,在靈芝降解木質(zhì)纖維素過程中發(fā)揮關(guān)鍵作用。本研究發(fā)現(xiàn)GlPaacC沉默菌株的漆酶基因表達量和酶活力顯著升高,并且GlPacC沉默菌株使發(fā)酵液pH酸化的能力增強,而培養(yǎng)基初始pH為3或4時,能夠提高靈芝漆酶基因表達量和酶活力。在pH4或7條件下,GlPacC沉默菌株中一部分漆酶基因的表達量發(fā)生上調(diào),一部分下調(diào)。通過酵母單雜交證實GlPacC能夠與lac8和lac12基因啟動子序列結(jié)合。這些結(jié)果表明GlPacC 一方面可能通過改變發(fā)酵液pH影響漆酶的合成,另一方面能夠直接調(diào)控漆酶基因的轉(zhuǎn)錄。三萜類化合物是靈芝中一類重要的具有藥理活性的次級代謝產(chǎn)物,其含量的多少決定了靈芝藥用品質(zhì)的高低。本研究發(fā)現(xiàn),GlPacC沉默菌株的靈芝三萜含量、三萜合成相關(guān)基因表達量和中間代謝產(chǎn)物鯊烯和羊毛甾醇含量均明顯提高,表明GlPacC能夠負調(diào)控靈芝三萜的生物合成。通過栽培實驗發(fā)現(xiàn),GlPacC沉默影響了靈芝原基和子實體的形成,并且GlPacC轉(zhuǎn)錄水平在原基形成期達到最高,表明GlPaacC對于靈芝的有性發(fā)育過程是必需的。根據(jù)脂質(zhì)過氧化檢測和靈芝栽培結(jié)果推測,GlPaacC沉默菌株可能受到某種脅迫損傷。通過DNALadder、細胞原位TUNEL染色、凋亡相關(guān)基因的表達量和胞內(nèi)活性氧檢測共同證實GlPacC沉默導(dǎo)致靈芝菌絲發(fā)生了凋亡。添加ROS清除劑1 mMNAC或2 mM VC將胞內(nèi)ROS清除之后,并沒有緩解菌絲凋亡表型,添加8 mM H2O2誘導(dǎo)凋亡基因表達量變化趨勢與GlPacC沉默導(dǎo)致的變化不一致,表明GlPaacC沉默導(dǎo)致靈芝菌絲凋亡并不是胞內(nèi)ROS升高導(dǎo)致的。為了進一步探究凋亡發(fā)生的原因,對胞內(nèi)pH進行檢測發(fā)現(xiàn),GlPacC沉默菌株胞內(nèi)pH顯著升高。細胞內(nèi)pH的改變會導(dǎo)致細胞膜電勢的改變,而膜電勢與細胞膜上的離子通道蛋白密切相關(guān)。GlPaacC沉默菌株中負責(zé)H+外排的H+-ATPase基因Pma1表達量升高,參與K+攝入的高親和性鉀通道蛋白基因Trk1表達量下調(diào),而參與K+或Na+外排的Na+/H+逆向轉(zhuǎn)運體基因Nha1和外向整流鉀通道基因Tok1表達量上調(diào);并且胞內(nèi)Na+和K+含量均明顯下降,表明GlPaacC沉默導(dǎo)致靈芝胞內(nèi)Na+和K+穩(wěn)態(tài)被打破。添加0.2 M NaCl或KC1能夠緩解菌絲凋亡表型,表明GlPacC通過胞內(nèi)Na+和K+穩(wěn)態(tài)調(diào)控靈芝菌絲凋亡過程。進一步分析Pal/Rim途徑中的其它基因功能發(fā)現(xiàn),靈芝只含有PalA、PalB、PalC和PalI同源基因,并且這些基因編碼蛋白的結(jié)構(gòu)域和在子囊菌中報道的不完全相同。外界中性和堿性pH能夠誘導(dǎo)GlPalC和GlPalI的表達,而對GlPalA和GlPaalB的表達量無明顯影響。GlPalA沉默菌株中靈芝三萜含量下降,菌絲生長和子實體發(fā)育均受到影響;而GlPalB沉默菌株中靈芝三萜含量升高,菌絲生長速率減慢,子實體發(fā)育基本不受影響。酵母雙雜交實驗證明GlPacC能夠與GlPalA互作。PacC/Rim101是Pal/Rim途徑中發(fā)揮關(guān)鍵調(diào)控作用的轉(zhuǎn)錄因子,在真菌許多生理過程中發(fā)揮重要作用,相關(guān)功能在高等擔(dān)子菌中未見報道。本研究有助于進一步拓寬并完善Pal/Rim途徑在整個真菌界中的作用機理,為其它擔(dān)子菌Pal/Rim途徑的研究提供借鑒。
[Abstract]:Ganoderma lucidum (Ganoderma lucidum) is a traditional Chinese medicinal macrofungi, at the same time, is also a kind of white rot fungus, is found to contain the degradation of lignocellulose degrading enzyme gene required for the largest number of basidiomycetes. However, the basic research of Ganoderma lucidum is very weak, seriously hindered the development of its commercial value and utilization. According to reports, changes in the external pH can affect the growth of fungus, the metabolism of many physiological processes such as.Pal/Rim pathway regulation in response to external changes of pH fungi in specific ways, and PacC/Rim101 is the transcription factor play a key role in this way. This paper studies on the PacC in the mycelium growth, development, apoptosis and secondary metabolism in the role of. The study found that the changes of the external pH can affect the mycelial growth and morphology of Ganoderma lucidum, and homologous gene transcription of GlPacC PacC/Rim101 in response to external pH Change. Through further analysis of gene cloning and sequence discovery, prediction of GlPacC encoding 807 amino acid residues of the protein molecular weight and isoelectric point were 86.15 kDa and contains 3 typical 7.76.GlPacC transcription factor C2H2 zinc finger domain, PEST domain two putative PKA phosphorylation sites, a possible and a a nuclear localization signal motif. In order to study how GlPacC expression induced by external neutral or alkaline pH, by the yeast one hybrid experiment proved that GlPacC can be cis elements on the combination and its gene promoter, and transcriptional regulation of their genes. In order to further study the role of this gene in Ganoderma lucidum, using gene silencing screening technology, obtained 4 silencing efficiency reached 55 ~ 75% GlPaacC silencing transformants. GlPacC silencing strains found more sensitive to pH changes in the external environment by plate growth experiment, divided mycelium The fork spacing was longer, play an important role in the growth of GlPacC showed that the mycelia of Ganoderma lucidum mycelium growth process. The change may influence of Ganoderma lucidum on tolerance to various stresses, through the study of tolerance to stress chemical substances found in GlPacC mM LiCl silent strains of 10, 10 mM H2O2,0.05% (w/v) SDS and 1 mg/ml Congo red more sensitive to 0.4 M NaCl or enhanced KC1 tolerance. By SDS and Congo red that the Ganoderma lucidum may be cell wall integrity is destroyed. Further study found that changes in cell wall integrity pathway, GlPacC silencing expression of Slt2 induced by Slt2 and Rlml, decreased phosphorylation levels, beta -1 reduce the 3- leading to cell wall glucan content, thinning, the Ganoderma lucidum is more sensitive to cell wall stress. In addition, GlPacC can also through transcriptional regulation of heat shock transcription factor and heat shock protein gene, and the effect of Ganoderma lucidum Tolerance to heat stress. Laccase is an important industrial enzyme that plays a key role in Ganoderma lucidum during lignocellulose degradation. The study found that the expression of laccase gene silencing of GlPaacC strains and the enzyme activity was significantly increased, and enhance the ability of GlPacC silencing strain pH fermentation broth acidification, and medium initial pH 3 or 4, can improve the laccase gene expression and enzyme activity in pH4 or 7. Under the condition that the expression of GlPacC was part of a silent laccase gene were up-regulated, down regulated. A part of the yeast one hybrid showed that GlPacC can combine with lac8 and lac12 gene promoter sequences. These results show that the GlPacC hand by changing the fermentation liquid of pH effect of laccase synthesis, on the other hand can directly regulate the transcription of laccase gene. Three triterpenoids in Ganoderma lucidum is an important class of pharmacologically active secondary metabolites The product, the content of which determines the level of medical quality of Ganoderma lucidum. This study found that Ganoderma lucidum three terpene content of GlPacC silencing strains, three terpene synthesis related gene expression and the intermediate metabolite of squalene and lanosterol content were increased significantly, showing that GlPacC can negatively regulate the biosynthesis of Ganoderma three terpene. Through cultivation experiment GlPacC, silent and affecting the formation of primordium and fruiting body of Ganoderma lucidum, and the transcription level of GlPacC reached the highest in the formation of primordia, suggesting that GlPaacC is required for the development process of Ganoderma lucidum sexual. According to lipid peroxidation and detection of Ganoderma cultivation results suggested that GlPaacC silencing strains may be subject to some stress damage. Through the DNALadder, TUNEL in situ staining. ROS apoptosis related gene expression and intracellular detection confirmed that GlPacC silencing resulted in apoptosis of mycelium of Ganoderma Lucidum with the ROS scavenger mMNAC or 1. 2 mM VC intracellular ROS after removal, did not alleviate the mycelial apoptotic phenotype, adding 8 mM H2O2 induced apoptosis gene expression variation tendency with GlPacC silencing leads to inconsistent, showed that GlPaacC silencing leads to apoptosis and intracellular mycelium is not ROS increase. In order to further explore the reasons for the occurrence of apoptosis. Intracellular pH were detected, GlPacC silencing strain pH was significantly increased. The intracellular pH changes will lead to the cell membrane potential changes and H+-ATPase gene of Pma1 ion channel membrane potential and cell membrane protein is closely related to.GlPaacC silencing strains responsible for H+ efflux expression elevated high potassium channel protein gene Trk1 affinity expression involved in K+ uptake, Na+/H+ antiporter gene Nha1 and outward rectifying potassium channel gene Tok1 in K+ or Na+ efflux up-regulated; and the content of Na+ and K+ in cells were Declined obviously, showed that GlPaacC silencing leads to intracellular K+ homeostasis and Na+ of Ganoderma lucidum was broken. Adding 0.2 M NaCl or KC1 can relieve the apoptosis phenotype of hyphae by Na+ and K+ GlPacC, showed that the homeostasis of mycelium of Ganoderma lucidum apoptosis cells. Further analysis of other genes in the Pal/Rim pathway found that Ganoderma lucidum only contains PalA, PalB, and PalC PalI homologous gene, and the gene encoding protein domain and in ascomycetes reported is not the same. The external expression of neutral and alkaline pH can induce GlPalC and GlPalI, and the expression of GlPalA and the amount of GlPaalB had no significant effect on the decline of.GlPalA silencing terpene content of Ganoderma lucidum three strains, mycelium growth and development affected; and three GlPalB strains of Ganoderma lucidum terpene content of silence increased, the mycelium growth rate slowed down, fruitbody development affected. Yeast two hybrid experiment proved that GlPacC can interact with GlPalA .PacC/Rim101 is a transcription factor play a key role in the Pal/Rim pathway, play an important role in many physiological processes in fungi, related functions reported in higher basidiomycetes. This study helps to further expand and improve the Pal/Rim pathway in fungi by the mechanism, to provide reference for the study of other basidiomycete Pal/Rim pathway.

【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S567.31

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