寡孢节丛孢中亲和性cAMP磷酸二酯酶功能的初步研究

【作者】 马妮；

【导师】 杨金奎；

【作者基本信息】 云南大学 ， 微生物学， 2019， 硕士

【摘要】 磷酸二酯酶(Phosphodiesterases,PDEs)在细胞信号转导过程中发挥重要作用,其能够水解胞内第二信使cAMP(环磷酸腺苷),从而终止cAMP所参与的信号转导途径的功能。目前,在大多数真菌中报道的磷酸二酯酶家族包含了高亲和性磷酸二酯酶(pde2/pdeH)和低亲和性磷酸二酯酶(pdel/pdeL),它们在真菌的营养菌丝生长和致病性发育过程中发挥了重要的调控作用。捕食线虫真菌的代表菌株-寡孢节丛孢(Arthrobotrys oligospora)在饥饿条件下产生三维菌网(three-dimensional networks)作为捕食器官捕捉线虫为自身生长获取营养,但是捕食器官形成的信号调控机制还不清楚。本论文以寡孢节丛孢中与构巢曲霉等丝状真菌同源的高亲和性磷酸二酯酶(AoPdeH,AOL s00083g160)和低亲和性磷酸二酯酶(AoPdeL,AOL s00097g378)为研究对象,对磷酸二酯酶编码基因进行敲除,探究磷酸二酯酶在寡孢节丛孢营养菌丝生长和致病性发育过程中所发挥的功能。主要实验结果:1.磷酸二酯酶的功能结构域和聚类分析对寡孢节丛孢两种磷酸二酯酶的功能结构域分析表明,AoPdeH含有以下的保守结构域和位点:3’5’-环核苷酸磷酸二酯酶超家族结构域(IPR036971)、磷酸二酯酶结构域(IPR003607)、3’5’-环核苷酸磷酸二酯酶催化结构域(IPR002073)和3’5’-环核苷酸磷酸二酯酶保守位点(IPR023174);AoPdeL也含有多个保守结构域,包括是富含亮氨酸的重复结构域超家族(IPR032675)、核糖核酸酶Z/羟基酰基谷胱甘肽水解酶样(IPR036866)和cAMP磷酸二酯酶结构域(IPR000396)。聚类分析表明,AoPdeH和AoPdeL分别聚类于2个不同进化分支上,由此可以初步推断这两个蛋白在真菌中可能发挥着不同的作用。2.磷酸二酯酶敲除突变株的营养菌丝生长、抗逆性和致病性分析利用同源重组技术对两种磷酸二酯酶编码基因进行敲除,成功获得两种磷酸二酯酶基因的敲除突变株,对突变株的菌丝形态、生长速率、抗逆境胁迫能力、杀线虫能力、胞外蛋白酶活性以及三维菌网形态和数量进行了统计比较。结果发现AoPdeH基因的缺失造成其突变株生长速率显著减慢、气生菌丝出现缺陷、菌丝横膈膜增多且发生膨大变形、对环境压力敏感性增强,除此之外,ΔAoPdeH突变株失去形成成熟三维菌网的能力,杀线虫能力显著降低但并没有完全丧失杀线虫能力,对其发酵液进行蛋白酶活性分析,发现突变株胞外蛋白酶活性没有明显变化。然而,ΔAoPdeL突变株的生长速率、抗逆境胁迫能力与寡孢节丛孢野生型菌株相比没有显著差异,杀线虫能力和产生三维菌网数量有所下降,菌丝形态有膨大变形现象。以上实验结果表明AoPdeH参与了寡孢节从孢的菌丝生长、抗逆性和致病性发育相关的生物学过程的调控。3.磷酸二酯酶参与产孢过程的调控产孢量比较发现,ΔAoPdeH突变株分生孢子产量仅为野生型菌株孢子产量的2～6%,ΔAoPde 突变株的分生孢子产量与野生型菌株相比无显著差异。光学显微镜观察发现,两种磷酸二酯酶基因突变株孢子形态皆发生变形,变得狭长无横膈膜,除此之外ΔAoPdeH突变株分生孢子梗的数量明显少于野生型菌株,且孢子梗上几乎没有孢子着生,ΔAoPdeL突变株分生孢子梗数量及孢子着生方式与寡孢节丛孢野生型菌株相比无明显差异。为进一步探讨磷酸二酯酶对产孢的调控作用,选取13个产孢调控基因在产孢初期(3 days)、产孢中期(5 days)和孢子成熟期(7 days)进行荧光定量PCR分析,结果表明,ΔAoPdeH突变株13个产孢调控基因中FlbA、Acr1、FluG、VelB、NsdD、FlbC、PkaC1、Plp1、AbaA、LreA和LreB的转录水平皆下调,而在分生孢子产量与野生型菌株没有明显差异的ΔAoPdeL突变株中,13个产孢调控基因的转录水平与野生型菌株相比无明显变化。以上实验结果表明AoPdeH参与了寡孢节丛孢的产孢过程的调控。4.磷酸二酯酶参与寡孢节丛孢菌丝内cAMP水平调控通过酶联免疫法测定突变株营养菌丝生长和致病性发育过程中不同时间点的菌丝内cAMP含量,结果表明,在所选取的所有时间点ΔAoPdeH敲除株和ΔAoPdeL敲除株菌丝内cAMP含量均高于寡孢节丛孢野生型菌株。为进一步分析磷酸二酯酶对cAMP水平的调控机制,选取16个磷酸二酯酶上下游的关键基因以及参与调节cAMP含量的相关基因进行荧光定量PCR(RT-PCR)分析,其中cAMP受体样蛋白和cAMP反应原件结合蛋白、腺苷酸环化酶表达量发生了明显变化(在菌丝生长发育期间显著上调,致病性发育期间显著下调),说明磷酸二酯酶可能通过调控上述基因的表达水平从而参与寡孢节丛孢cAMP含量的调节。本文的创新点:1.首次在寡孢节丛孢中成功敲除了磷酸二酯酶家族的蛋白编码基因,发现AoPdeH参与寡孢节从孢营养菌丝生长、无性产孢、抗逆境胁迫压力、捕食器官形成和菌丝内cAMP含量调节等重要生物学过程的调控。2.通过RT-PCR比较了AoPdeH、AoPdeL、分生孢子发育调控以及cAMP合成等相关基因在ΔAoPdeH突变株、ΔAoPdeL突变株和野生型菌株中不同时间点的转录水平变化,推测磷酸二酯酶参与寡孢节丛孢中分生孢子的发育以及cAMP合成等相关基因的表达调控。更多还原

【Abstract】 Phosphodiesterases(PDEs)play the function of hydrolyzing intracellular second messenger cAMP(cyclic adenosine monophosphate),terminating the signal transduction pathways involved in these second messengers.Currently,the phosphodiesterase family reported in filamentous fungi contain high-affinity phosphodiesterase(pdeH/pde2)and low-affinity phosphodiesterase(pdeL/pdel),which play a very important regulatory role in the vegetative growth and the pathogenesis development in fungi.A representative strain of nematode-trapping fungus,Arthrobotrys oligospora,produces three-dimensional networks under starvation conditions as a predator to capture and digestive nematodes for growth,however,the signaling mechanisms of trap formation are still unclear.In this study,the genes coding for high-affinity phosphodiesterase(AoPdeH,AOL s00083g160)and low-affinity phosphodiesterase(AoPdeL,AOL s00097g378)were disrupted in Arthrobotrys oligospora,the functions of two phosphodiesterases were characterized in the vegetative growth and pathogenic development of A.oligospora.The main experimental results:1.Functional domain and cluster analysis of two phosphodiesterasesThe conserved domains of two phosphodiesterases in A.oligospora were predicted using InterProScan,AoPdeH has a 3’5’-cyclic nucleotide phosphodiesterase catalytic domain superfamily(IPR036971),a HD/PDEase domain(IPR003607),a 3’5’-cyclic nucleotide phosphodiesterase catalytic domain(IPR002073)and a 3’5’-cyclic nucleotide phosphodiesterase conserved site(IPR023174);while AoPdeL contains a leucine-rich repeat domain superfamily(IPR032675),a ribonuclease Z/hydroxyacyl glutathione hydrolase-like(IPR036866)and a cyclic-AMP phosphodiesterase classⅡ(IPR000396).Cluster analysis showed that AoPdeH and AoPdeL were divided in two different evolutionary branches,respectively,which suggested that they may play different roles in fungi.2.Analyses of vegetative growth,stress resistance and pathogenicity of phosphodiesterase mutantsThe two phosphodiesterase-encoding genes were knocked out using homologous recombination method,and two mutants were successfully obtained.The hyphal morphology,growth rate,stress tolerance ability,nematicidal ability,the extracellular protease activity,and trap formation were statistically compared between the wild-type(WT)strain and mutants.The deletion of AoPdeH caused significantly change in phenotypic traits,such as the growth rate of the mutant strain was slowe down,the hyphal septum was increased and the mutants became sensitive to the environmental pressure.In addition,the ΔAoPdeH mutants lost the ability to form mature three-dimensional networks,but the nematicidal ability only significantly reduced.Meanwhile,analysis of the proteolytic activity of the fermentation broth in theΔAoPdeH mutants showed that no significant change in proteolytic activity compared with the WT strain.However,the growth rate and stress resistance of the ΔAoPdeL mutants showed no significantly different from those of the WT strain,while the nematicidal ability and the traps number were decreased,and the hyphal morphology was swollen and deformed.Our results indicated that AoPdeH is involved in the regulation of the biological processes related to vegetative growth,stress resistance and pathogenic development in A.oligospora.3.Comparison of conidiation and sporulation-related genes between the WT and mutantsThe conidia yield of the ΔAoPdeH mutants were significantly decreased to 2-6%compared with the WT strain,and the conidia yield of the ΔAoPdeL mutants had no significantly different from that of the WT strain.The conidial morphology of the two phosphodiestera mutants was deformed and became narrow and long without septum.In addition,the conidiophores of ΔAoPdeH mutants were significantly decreased compared with the WT strain.However,the conidia yield and conidiophores of theΔAoPdeL mutants had no significantly different from the WT strain.To further investigate the regulation of phosphodiesterase on sporulation,13 sporulation-related genes were selected for real-time PCR analysis in the early period(3 days),middle period(5 days)and later period(7 days)of sporulation.The results showed that the expression levels of FlbA,Acr1,FluG,VelB,NsdD,FlbC,PkaCl,Plp1,AbaA,LreA and LreB were down-regulated in the ΔAoPdeH mutants compared with the WT strain.while the transcription levels of 13 sporulation-related genes in the ΔAoPdeL mutants showed no change significantly compared with the WT strain.The above experimental results indicated that AoPdeH is involved in the regulation of conidiation in A.oligospora.4.Phosphodiesterases involves in the regulation of intracellular cAMP levels in A oligosporaThe cAMP content in mycelia of the WT and mutants was determined by enzyme-linked immunosorbent assay(ELISA).The cAMP content in the mycelia of theΔAoPdeH and ΔAoPdeL mutants at all tested time points was higher than that of the WT strain.To further analyze the regulation mechanism of phosphodiesterase on cAMP levels,16 genes located on the upstream and downstream of phosphodiesterase or involved in the regulation of cAMP level were selected for real-time PCR analysis,in which the expression levels of cAMP receptor-like protein,cAMP reaction element binding protein and adenylate cyclase changed significantly(significantly up-regulated during mycelial growth and development,significantly downregulated during pathogenic development),indicating that phosphodiesterase participates in the regulation of cAMP level by regulating the expression of the above genes in A oligospora.Innovations in this paper:1.The coding genes of two phosphodiesterase family were successfully knocked out in the A.oligospora for the first time.It was found that AoPdeH is involved in the regulation of important biological processes,such as the vegetative growth,asexual reproduction,stress resistance,trapformation and intracellular cAMP levels.2.The transcriptional levels of several genes related to conidiation and cAMP synthesis were compared in the WT,ΔAoPdeH and ΔAoPdeL mutants,our results suggested that two phosphodiesterase may involve in the regulation of these genes in A oligospora.更多还原