响应pH的转录因子基因PacC在灵芝菌丝生长、发育、凋亡和次级代谢过程中的功能研究

发布时间：2018-01-06 21:26

  本文关键词：响应pH的转录因子基因PacC在灵芝菌丝生长、发育、凋亡和次级代谢过程中的功能研究　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

  更多相关文章： 药用真菌 PacC/Rim101 漆酶 发育 次级代谢 离子稳态

【摘要】：灵芝(Ganoderma lucidum)是我国传统的药用大型真菌,同时,也是一种白腐真菌,是目前发现含有降解木质纤维素所需降解酶基因数目最多的担子菌。但是,由于灵芝的基础研究十分薄弱,严重阻碍了其商业价值的开发和利用。据报道,外界pH的变化会影响灵芝的生长、代谢等许多生理过程。Pal/Rim途径是真菌中特有的响应外界pH变化的信号调控途径,而PacC/Rim101是此途径中发挥关键调控作用的转录因子。本论文研究了 PacC在灵芝菌丝生长、发育、凋亡和次级代谢等生理过程中的作用。本研究发现外界pH的变化能够影响灵芝的菌丝生长和形态,并且PacC/Rim101的同源基因GlPacC的转录能够响应外界pH的变化。进一步通过基因克隆和序列分析发现,GlPacC编码807个氨基酸残基预测的蛋白分子量和等电点分别为86.15 kDa和7.76。GlPacC包含3个典型的转录因子C2H2锌指结构域、两个推测的PEST结构域、一个可能的PKA磷酸化位点和一个核定位信号基序。为了研究GlPacC如何受到外界中性或碱性pH的诱导表达,通过酵母单杂交实验证明GlPacC能够与自身基因启动子上的顺式作用元件相结合,从而调控自身基因的转录。为了进一步研究该基因在灵芝中的作用,利用基因沉默技术,筛选获得了 4个沉默效率达到55～75%的GlPaacC沉默转化子。通过平板生长实验发现GlPacC沉默菌株对外界环境pH变化更加敏感,菌丝分叉间距明显变长,表明GlPacC在灵芝菌丝生长过程中发挥重要作用。菌丝生长的改变可能会影响灵芝对各种胁迫的耐受性,通过考察对化学胁迫物质的耐受性发现,GlPacC沉默菌株对10 mM LiCl、10 mM H2O2、0.05% (w/v) SDS和1 mg/ml刚果红更加敏感,对0.4 M NaCl或KC1耐受性增强。由SDS和刚果红处理结果推测,可能是灵芝细胞壁完整性遭到破坏。进一步考察了细胞壁完整性途径的变化发现,GlPacC沉默导致Slt2和Rlml表达量下调,Slt2磷酸化水平降低,β-1, 3-葡聚糖含量降低,从而导致细胞壁变薄,最终使灵芝对细胞壁胁迫更加敏感。此外,GlPacC还能够通过调控热激转录因子和热激蛋白基因的转录,进而影响灵芝对热胁迫的耐受性。漆酶是一种重要的工业用酶,在灵芝降解木质纤维素过程中发挥关键作用。本研究发现GlPaacC沉默菌株的漆酶基因表达量和酶活力显著升高,并且GlPacC沉默菌株使发酵液pH酸化的能力增强,而培养基初始pH为3或4时,能够提高灵芝漆酶基因表达量和酶活力。在pH4或7条件下,GlPacC沉默菌株中一部分漆酶基因的表达量发生上调,一部分下调。通过酵母单杂交证实GlPacC能够与lac8和lac12基因启动子序列结合。这些结果表明GlPacC 一方面可能通过改变发酵液pH影响漆酶的合成,另一方面能够直接调控漆酶基因的转录。三萜类化合物是灵芝中一类重要的具有药理活性的次级代谢产物,其含量的多少决定了灵芝药用品质的高低。本研究发现,GlPacC沉默菌株的灵芝三萜含量、三萜合成相关基因表达量和中间代谢产物鲨烯和羊毛甾醇含量均明显提高,表明GlPacC能够负调控灵芝三萜的生物合成。通过栽培实验发现,GlPacC沉默影响了灵芝原基和子实体的形成,并且GlPacC转录水平在原基形成期达到最高,表明GlPaacC对于灵芝的有性发育过程是必需的。根据脂质过氧化检测和灵芝栽培结果推测,GlPaacC沉默菌株可能受到某种胁迫损伤。通过DNALadder、细胞原位TUNEL染色、凋亡相关基因的表达量和胞内活性氧检测共同证实GlPacC沉默导致灵芝菌丝发生了凋亡。添加ROS清除剂1 mMNAC或2 mM VC将胞内ROS清除之后,并没有缓解菌丝凋亡表型,添加8 mM H2O2诱导凋亡基因表达量变化趋势与GlPacC沉默导致的变化不一致,表明GlPaacC沉默导致灵芝菌丝凋亡并不是胞内ROS升高导致的。为了进一步探究凋亡发生的原因,对胞内pH进行检测发现,GlPacC沉默菌株胞内pH显著升高。细胞内pH的改变会导致细胞膜电势的改变,而膜电势与细胞膜上的离子通道蛋白密切相关。GlPaacC沉默菌株中负责H+外排的H+-ATPase基因Pma1表达量升高,参与K+摄入的高亲和性钾通道蛋白基因Trk1表达量下调,而参与K+或Na+外排的Na+/H+逆向转运体基因Nha1和外向整流钾通道基因Tok1表达量上调;并且胞内Na+和K+含量均明显下降,表明GlPaacC沉默导致灵芝胞内Na+和K+稳态被打破。添加0.2 M NaCl或KC1能够缓解菌丝凋亡表型,表明GlPacC通过胞内Na+和K+稳态调控灵芝菌丝凋亡过程。进一步分析Pal/Rim途径中的其它基因功能发现,灵芝只含有PalA、PalB、PalC和PalI同源基因,并且这些基因编码蛋白的结构域和在子囊菌中报道的不完全相同。外界中性和碱性pH能够诱导GlPalC和GlPalI的表达,而对GlPalA和GlPaalB的表达量无明显影响。GlPalA沉默菌株中灵芝三萜含量下降,菌丝生长和子实体发育均受到影响;而GlPalB沉默菌株中灵芝三萜含量升高,菌丝生长速率减慢,子实体发育基本不受影响。酵母双杂交实验证明GlPacC能够与GlPalA互作。PacC/Rim101是Pal/Rim途径中发挥关键调控作用的转录因子,在真菌许多生理过程中发挥重要作用,相关功能在高等担子菌中未见报道。本研究有助于进一步拓宽并完善Pal/Rim途径在整个真菌界中的作用机理,为其它担子菌Pal/Rim途径的研究提供借鉴。
[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.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S567.31

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