微生物学报  2017, Vol. 57 Issue (7): 1069-1082
http://dx.doi.org/10.13343/j.cnki.wsxb.20160443
中国科学院微生物研究所,中国微生物学会,中国菌物学会
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文章信息

方迪, 楼轶, 吴明德, 张静, 李国庆, 杨龙. 2017
Di Fang, Yi Lou, Mingde Wu, Jing Zhang, Guoqing Li, Long Yang. 2017
重寄生真菌盾壳霉pH信号通路中Pal相关基因的功能鉴定
Identification of pH-signaling pathway-related genes in mycopasasitic fungus Coniothyrium minitans
微生物学报, 57(7): 1069-1082
Acta Microbiologica Sinica, 57(7): 1069-1082

文章历史

收稿日期:2016-10-30
修回日期:2017-01-23
网络出版日期:2017-03-02
重寄生真菌盾壳霉pH信号通路中Pal相关基因的功能鉴定
方迪, 楼轶, 吴明德, 张静, 李国庆, 杨龙     
华中农业大学植物科学技术学院, 湖北省作物病害监测与安全控制重点实验室, 湖北 武汉 430070
摘要[目的] 研究pH信号通路(Pal)在重寄生真菌盾壳霉与寄主核盘菌互作过程中的作用。 [方法] 从盾壳霉全基因组信息中分析获得了6个Pal相关基因CmpalA、CmpalB、CmpalC、CmpalF、CmpalHCmpalI的全编码序列和氨基酸序列,通过PEG介导的原生质转化技术获得了CmpalA、CmpalB、CmpalC、CmpalFCmpalH等5个基因的敲除突变体,分析这些敲除突变体与野生型在菌落培养性状、重寄生能力、降解草酸能力、产生抗真菌物质能力等方面的差异。 [结果] 与野生型相比,在pH 6-8的条件下,5个Pal相关基因敲除突变体的菌丝生长受到显著抑制,这说明缺失Pal相关基因使盾壳霉对高pH值环境更加敏感。菌核重寄生试验发现5个Pal相关基因敲除突变体的重寄生能力均显著低于野生型。qRT-PCR试验结果表明,敲除Pal相关基因之后导致重寄生相关酶基因Cmch1Cmg1Cmsp1的表达量显著降低,而且pH信号通路下游的CmpacC基因的表达量也显著降低。Pal相关基因敲除突变体在pH 6条件下对草酸盐的降解能力显著高于野生型,同时这5个突变体在pH 8条件下产生抗真菌物质能力也显著高于野生型。 [结论] pH信号通路相关基因的缺失影响盾壳霉对环境pH的响应。pH信号通路在盾壳霉与核盘菌互作中发挥重要作用,不仅影响盾壳霉的重寄生作用,而且还影响盾壳霉的草酸降解作用和抗真菌作用。
关键词: 盾壳霉     核盘菌     pH信号通路     重寄生作用     草酸降解作用     抗真菌活性    
Identification of pH-signaling pathway-related genes in mycopasasitic fungus Coniothyrium minitans
Di Fang, Yi Lou, Mingde Wu, Jing Zhang, Guoqing Li, Long Yang     
Hubei Province Key Laboratory of Plant Pathology, College of Plant Science and Technology, Huazhong Agricultural University, Wuhan 430070, Hubei Province, China
Received 30 October 2016; Revised 23 January 2017; Published online 2 March 2017
*Corresponding author: Long Yang, Tel: +86-27-87280117; Fax: +86-27-87280670; E-mail: yanglong@mail.hzau.edu.cn
Supported by the National Natural Science Foundation of China (31272084, 31471813, 31672073)
Abstract: [Objectives] To identify pH-signalling pathway (Pal)-related genes in mycoparasitic fungus Coniothyrium minitans and to understand the role of these genes in interaction between C. minitans and its host Sclerotinia sclerotiorum. [Methods] Six Pal-related homologues were obtained from the whole genome of C. minitans and designated as CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI. PEG-mediated protoplast transformation was used to create the deletion mutants of Pal-related genes. Five Pal-related genes were knocked out individually and the mutants designated as ΔCmpalA-33, ΔCmpalB-13, ΔCmpalC-5, ΔCmpalF-50 and ΔCmpalH-26. The biological characteristics, including colony morphology, mycoparasitism, oxalate degradation and antifungal activity, were compared between knock-out mutants and the wild-type strain. [Results] Compared to the wild type strain, five Pal-related genes-deletion mutants showed significantly reduced mycelia growth between pH 6 and 8. These results indicated that the disruption of these Pal-related genes increases sensitivity to neutral or alkaline pH. The sclerotia-infection assay showed that the parasitic activities of the five Pal-related genes-deletion mutants were dramatically reduced. qRT-PCR results showed that these Pal-related genes-deletion mutants suppressed expression levels of three mycoparasitism-associated genes Cmch1, Cmg1 and Cmsp1. Meanwhile, expression of CmpacC, the pH signaling pathway downstream gene, was also reduced in the Pal-related genes-deletion mutants. The oxalate degradation of the five Pal-related genes-deletion mutants at pH 6 were increased under pH 8, and the antifungal activity of those mutants were also increased at pH 8 comparison with the wild type. [Conclusion] Disruption of the Pal-related genes resulted in impaired C. minitans responses to ambient pH. The pH-signalling pathway (Pal) plays an important role in interaction between C. minitans and S. sclerotiorum, including mycoparasitism, oxalate degradation and antifungal activity in C. minitans against S. sclerotiorum.
Key words: Coniothyrium minitans     Sclerotinia sclerotiorum     pH-signalling pathway     mycoparasitism     oxalate degradation     antifungal activity    

盾壳霉(Coniothyrium minitans)是核盘菌(Sclerotinia sclerotiorum)的一种生防真菌。这种真菌的生防机制包括重寄生作用[1-2]和抗真菌作用[3-4],即产生抗真菌物质(antifungal substances,AFS)。此外,我们小组发现盾壳霉还能降解核盘菌产生的草酸毒素[5]。草酸(oxalic acid)是一种酸性很强的有机酸,它的存在营造酸性pH环境,有利于核盘菌侵染植物。在盾壳霉与核盘菌的互作过程中,草酸可以诱导盾壳霉产生AFS[4]。与此同时,草酸诱导盾壳霉草酸脱羧酶(oxalate decarboxylase,OXDC)基因表达,并最终降解草酸[6]。草酸的降解导致环境pH值升高,促进盾壳霉重寄生相关基因(几丁质酶基因Cmch1、葡聚糖酶基因Cmg1和胞外蛋白酶基因Cmsp1)的表达[5-6],从而有利于盾壳霉寄生溃解核盘菌。可见,pH信号调控在盾壳霉-核盘菌互作系统中扮演重要角色。

前人对丝状真菌(如构巢曲霉)和酵母感应环境pH信号的分子机制进行了深入研究。研究结果表明:真菌感应环境pH信号主要由palApalBpalCpalFpalIpalHpacC这7个基因介导[7]。其中palApalBpalCpalFpalIpalH组成了信号通路,转录调控因子pacC则是这一pH信号通路(Pal)的核心,它能直接促进碱性环境下表达的基因,抑制酸性环境下表达的基因[7-9]

本课题组前期对盾壳霉pacC基因(即CmpacC)的研究结果表明:该基因对盾壳霉降解草酸毒素和抗生AFS具有负调控作用,而对盾壳霉寄生核盘菌具有正调控作用[2]。但是目前对于盾壳霉的pH信号通路,以及该信号通路对盾壳霉重寄生作用、降解草酸作用和抗真菌作用的影响等问题尚不明确。本研究从盾壳霉全基因组信息中分析获得了6个Pal基因,分别命名为CmpalACmpalBCmpalCCmpalFCmpalHCmpalI。对其进行了敲除,比较了各敲除突变体和野生型菌株Chy-1在培养特性、重寄生能力、草酸降解能力以及产生AFS等方面的差异。研究目的在于阐明盾壳霉pH信号通路在盾壳霉与核盘菌互作过程中的作用。

1 材料和方法 1.1 供试菌株、培养基以及系统进化树分析

供试盾壳霉野生型菌株Chy-1采自湖北省长阳县,本实验室保存[10]。供试核盘菌菌株为Ep-1PNA5 (简称A5),均分离自茄子[11]。从盾壳霉的全基因组信息(姜道宏老师实验室提供,尚未公开)中分析获得了6个Pal-pH上游相关基因(CmpalACmpalBCmpalCCmpalFCmpalHCmpalI)的全编码序列和氨基酸序列;将相对应的同源基因的氨基酸序列输入NCBI数据库(http://blast.ncbi.nlm.nih.gov/Blast.cgi),查找并提取其他真菌中同源基因的氨基酸序列,用MEGA软件来分析盾壳霉中6个Pal-like同源基因的系统进化树。

供试培养基包括常规马铃薯葡萄糖琼脂培养基(potato dextrose agar,PDA)、二倍PDA (2×PDA)和改良的查氏培养液(modified Czapek Dox,MCD)[2]

胡萝卜培养基:将胡萝卜洗净后切成片状置于三角瓶中,灭菌。

dPDB培养基:去皮马铃薯400 g、葡萄糖40 g、加蒸馏水定容至1000 mL。

不同pH缓冲液配方如下:dpH 3缓冲液(500 mL):柠檬酸17.23 g,柠檬酸三钠5.29 g;dpH 4缓冲液(500 mL):柠檬酸12.4 g,柠檬酸三钠12.05 g;dpH 5缓冲液(500 mL):柠檬酸7.35 g,柠檬酸三钠19.11 g;dpH 6缓冲液(500 mL):柠檬酸2.42 g,柠檬酸三钠26.02 g;dpH 7缓冲液(500 mL):Tris-base 6.06 g,用浓盐酸调节pH至7;dpH 8缓冲液(500 mL):Tris-base 6.06 g,用浓盐酸调节pH至8。

以上培养基均经过121 ℃高压灭菌20 min后使用。

1.2 原生质PEG介导Pal相关基因的敲除

根据同源重组原理,采用PEG介导的原生质体转化技术[12],用潮霉素基因替换pH信号通路各基因部分序列,获得了5个基因的敲除突变体。分别命名为∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26。但是没有获得CmpalI敲除突变体。

1.3 不同pH下菌丝生长的测定

活化培养盾壳霉野生型菌株Chy-1和各突变体,菌落边缘打孔菌丝块(直径0.5 cm)分别接种于pH为3、4、5、6、7、8的PDA固体平板培养基上培养,接种后第6天采用十字交叉法测量菌落直径。每个处理设立2个平行试验,并重复5次。

1.4 不同突变体产孢量的测定

活化培养盾壳霉野生型菌株Chy-1和各突变体,菌落边缘打孔菌丝块(直径0.5 cm)分别接种到PDA平板中央,在20 ℃下光照培养14 d后用0.5 cm的打孔器在靠近中心菌丝块的周围取4个菌丝块,置于1 mL水中,剧烈振荡1 min后用血球计数板测量孢子产量,每个处理设立3个平行试验,并重复3次。

1.5 寄生菌核试验

将野生型菌株Chy-1和各突变体分别接种至PDA培养基上,20 ℃培养30 d,然后用无菌水洗下菌落表面的分生孢子,制备成分生孢子悬浮液。用血球计数板测定孢子浓度,并用无菌水将其稀释成1×107个孢子/mL。将核盘菌菌株A5接种在胡萝卜培养基上,在20 ℃下培养30 d,收集菌核。菌核风干后,选取大小均匀的菌核(4.5 mm×3.5 mm,长×宽),用75%的酒精消毒30 min,然后用无菌水洗涤3次,每次1 min。最后将50粒菌核分别浸泡各菌株分生孢子悬浮液或无菌水(对照)中30 min。将各处理菌核置于灭过菌的潮湿砂表面。在20 ℃下培养30 d,然后取出菌核,逐一检查菌核腐烂情况。参考姜道宏等[13]对菌核腐烂程度进行分级,并计算菌核腐烂指数。每个处理设立2个平行试验,并重复3次。

1.6 基因表达试验

在PDA平板上铺一层灭过菌的玻璃纸,取上述盾壳霉野生型和突变体菌株分生孢子悬浮液(1×107个孢子/mL),涂在玻璃纸上。将平板置于20 ℃下培养3 d。然后将融化的2×PDA与pH 3、pH 5或pH 7的缓冲液以等体积混匀,制备不同缓冲pH值的PDA平板。掀起含盾壳霉菌丝的玻璃纸,转移至含缓冲液的PDA平板上,在20 ℃下培养3 d。然后将融化的2×PDA与pH 3、pH 5或pH 7的缓冲液以等体积混匀,制备不同缓冲pH值的PDA平板。掀起含盾壳霉菌丝的玻璃纸,转移至含缓冲液的PDA平板上,在20 ℃下黑暗培养12 h,刮取菌丝,提取总RNA,采用定量反转录PCR (qRT-PCR)方法检测重寄生相关基因Cmg1 (编码葡聚糖酶)、Cmch1 (编码几丁质酶)和Cmsp1 (编码蛋白酶),以及CmpacC的表达。检测Cmg1表达的PCR引物是:Glu-F:5′-TCGGCA AGCAGACAGGTGAT-3′,Glu-R:5′-TTCTCGGCGTTGATGTTCCA-3′。检测Cmch1基因表达的PCR引物是:Chitin-F:5′-CGGCTCCTGGGATACTACTT C-3′,Chitin-R:5′-TGCGGCAACACCATTAGAG-3′。检测Cmsp1表达的PCR引物是:Sp1-F:5′-CCT TCTCCAACTACGGCTCCTC-3′,Sp1-R:5′-ACTGGATGCGGGTGGTAAGA-3′。以肌动蛋白基因Cmactin为内参基因,检测其表达的qRT-PCR引物为Act7-F:5′-TCGTGACTTGACCGACTACCTC-3′,Act8-R:5′-TTGCCAATGGTGATGACCTGA-3′。检测CmpacC表达的PCR引物是:pacA-F:5′-TCT GAGTATGGACACGGAGGCA-3′,pacA-R:5′-CC AAGAACTGGTCGATGTTGAGG-3′。以野生型菌株Chy-1在pH 3的条件下各基因的表达量为1,其他突变体以之为标准进行计算,得出各基因相对表达量(relative expression)值。

1.7 降解草酸盐能力测定

将25 mL的dPDB (双倍浓度的PDB)培养液与25 mL的草酸钠(16 mmol/L)均匀混合于250 mL的三角瓶中(命名为PDB-SO),同时另外再做一个处理:在上述培养基中添加20 mmol/L的盐酸,然后分别接种100 μL盾壳霉野生型菌株或者各突变体菌株的分生孢子悬浮液(1×107孢子/mL),并于20 ℃摇床振荡培养12 d (150 r/min),以不接种盾壳霉分数孢子液的培养基为初始对照。摇培12 d后,用已称重(Wh1) 的滤纸分离每个三角瓶中的盾壳霉菌丝和培养液,然后将有菌丝的滤纸置于80 ℃烘箱中烘干后再称重(Wh2),扣除滤纸干重后就能得到各个处理的菌丝干重(Wh2-Wh1)。用pH计测定每种培养滤液的pH值,收集这些滤液并用细菌过滤器(0.45 μm)过滤,然后采用高效液相色谱(HPLC)法测定滤液中草酸根离子的含量,并计算盾壳霉野生型菌株和各突变体对草酸盐的降解率[6, 14]。每个处理设立2个平行试验,并重复3次。

1.8 抑制核盘菌菌丝生长测定

取1 mL盾壳霉菌株Chy-1和各突变体孢子悬浮液(1×106个孢子/mL)。接种至100 mL缓冲的MCD培养液中(pH 4或8)。将培养物在20 ℃下振荡培养(150 r/min) 12 d。用滤纸将各培养物过滤,收集滤液。将滤液按10%体积比添加至PDA培养基中,在对照PDA中添加等量MCD培养液,制成平板,并将活化的核盘菌菌丝接种至各平板中央。最后将培养物置于20 ℃下培养60 h,测定各平板中核盘菌菌落直径。通过与对照平板菌落直径对比,计算盾壳霉培养物滤液对核盘菌菌丝生长的抑制作用。每个处理设立2个平行试验,并重复3次。

1.9 数据统计分析

采用SAS 8.0软件中的方差分析程序(ANOVA)分析上述试验中不同处理之间的差异显著性。不同处理之间的差异采用最小显著差异法(LSD)比较(α =0.05),分析前将盾壳霉孢子产量数据通过对数(Log10) 进行转换,核盘菌菌丝生长抑制百分率通过反正弦转化后再进行差异显著性分析。

2 结果和分析 2.1 系统进化树分析

从盾壳霉的全基因组信息(姜道宏老师实验室提供,尚未公开)中分析获得了6个pal-like同源基因CmpalACmpalBCmpalCCmpalFCmpalHCmpalI 的全编码序列和氨基酸序列,并且将其提交NCBI数据库,分别获得相对应的序列登录号。这6个基因分别命名为CmpalA (GenBank登录号:KP747602)、CmpalB (GenBank登录号:KP747603)、CmpalC (GenBank登录号:KP747604)、CmpalF (GenBank登录号:KP747605)、CmpalH (GenBank登录号:KP747606)、Cmpal1 (GenBank登录号:KP747607)。分别对6个Pal-like同源基因做了序列分析,获得了相对应同源基因的内含子以及外显子的序列,对其相应的氨基酸组分属性也进行了分析(表 1)。系统进化分析结果显示:这6个基因CmpalACmpalBCmpalCCmpalFCmpalHCmpalI均与油菜黑胫病(Leptosphaeria maculans)以及小麦黄斑叶枯病菌(Pyrenophora tritici-repentis)的同源基因编码的氨基酸亲缘关系最近(图 1)。在真菌的Pal-like蛋白中,PalA蛋白和PalC蛋白的亲缘关系较近(图 1)。

表 1. CmpalACmpalBCmpalCCmpalFCmpalHCmpalI的ORF序列和氨基酸序列分析 Table 1. The ORF sequences and the amino acid sequence analysis of CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI
GenesFeature of nucleotide sequenceFeature of amino acid sequence
ORF length/bpNumber of intronNumber of exonDeduced protein lengthMolecular weight/kDaIsoelectric point
CmpalA25652382192.07.04
CmpalB3604451111123.57.95
CmpalC15811247451.76.44
CmpalF25541283389.26.22
CmpalH18362357062.68.86
CmpalI18372354861.011.06

图 1 CmpalACmpalBCmpalCCmpalFCmpalHCmpalI的系统进化树分析 Figure 1 Phylogenetic tree analysis of CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI. The phylogenetic tree was constructed by using MEGA 5.0 with homologous sequences of CmpalA, CmpalB, CmpalC, CmpalF, CmpalH and CmpalI from various strains, with their sequence IDs in the parentheses; the numbers on the horizontal lines represent evolutionary distances.

2.2 Pal相关基因的敲除

我们通过PEG介导原生质体转化的方法以及Southern blot (罗氏地高辛DNA标记和检测试剂盒)验证[6]获得了5个Pal同源基因的敲除突变体,分别是∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26 (图 2)。

图 2 盾壳霉Pal相关基因敲除突变体的Southern blot验证 Figure 2 Southern blotting confirmation of the mutants of Pal-related genes. The symbols * and ★ indicate the start codon (ATG) and the stop codon (TAA) respectively. A: southern blotting confirmation of the mutant of CmpalA gene; B: southern blotting confirmation of the mutant of CmpalB gene; C: southern blotting confirmation of the mutant of CmpalC gene; D: southern blotting confirmation of the mutant of CmpalF gene; E: southern blotting confirmation of the mutant of CmpalH gene.

2.3 Pal相关基因敲除对盾壳霉菌丝生长的影响

敲除突变体∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26在pH 3-8缓冲的PDA培养基上培养6 d后的菌落直径均小于野生型菌株(图 3图 4)。在pH 4和pH 5的条件下,各敲除突变体的菌落直径能够达到4.87-5.65 cm,而野生型菌株在pH 4和pH 5条件下的菌落直径分别达到了5.99 cm和6.06 cm。与野生型菌株Chy-1相比较,各敲除突变体的菌落直径下降了5.68%-19.64%。在pH 7和pH 8的条件下,野生型菌株的菌落直径为2.35-3.81 cm,而各敲除突变体的菌落直径仅为0.89-1.93 cm,菌落直径下降了30.64%-76.64% (图 4)。这5个Pal相关基因敲除突变体中,∆ CmpalF -50在pH 3条件下,菌落直径(2.77 cm)显著小于其他4个突变体(3.42-3.89 cm),而在pH 6-8条件下,∆ CmpalF -50的菌落直径(3.27、1.93、1.63) 显著大于其他4个突变体(1.29-1.97、0.89-1.50、0.98-1.16 cm)。

图 3 盾壳霉Pal相关基因敲除突变体在不同pH条件下的菌落生长形态(20 ℃, 6 d) Figure 3 Colonies of the Pal-related genes mutants of C. minitans on PDA with different pH (20 ℃, 6 days).

图 4 盾壳霉Pal相关基因敲除突变体在不同pH下培养6 d的菌落直径(20 ℃) Figure 4 Colony diameters of 6-day-old cultures of the Pal-related genes mutants of C. minitans on PDA buffered under different pH (20 ℃). Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

通过不同pH条件下菌丝生长测定,结果表明CmpalACmpalBCmpalCCmpalFCmpalH的敲除突变体在较低pH值(3-5) 生长受抑制程度显著低于在较高pH值(6-8) 生长受抑制程度,说明Pal相关基因敲除突变体对较高pH值(6-8) 更敏感。但是在这5个Pal相关基因敲除突变体中,CmpalF敲除突变体与其他4个(CmpalACmpalBCmpalCCmpalH)敲除突变体在不同pH条件下生长受抑制作用存在明显差异。在pH 3条件下,∆ CmpalF -50生长受抑制作用比其他4个敲除突变体更显著,而在pH 6-8条件下,∆ CmpalF -50生长受抑制作用显著低于其他4个敲除突变体。

2.4 Pal相关基因敲除对盾壳霉产孢的影响

敲除突变体∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26在PDA平板上培养14 d后的产孢量分别为3.37×107、3.07×107、2.72×107、0.97×107和0.37×107个孢子/cm2,与野生型菌株Chy-1 (3.41×107个孢子/cm2) 相比较,敲除突变体∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26的产孢量均有显著下降,而突变体∆ CmpalA -33和∆ CmpalB -13的产孢量与野生型菌株没有显著差异(图 5)。

图 5 盾壳霉Pal相关基因敲除突变体和野生型菌株产孢量比较(20 ℃, 14 d) Figure 5 Comparison of conidial production among the Pal-related genes mutants and the wild type strain Chy-1 of C. minitans (20 ℃, 14 days). Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

2.5 Pal相关基因敲除对盾壳霉寄生核盘菌菌核的影响

寄生核盘菌菌核试验结果表明,敲除突变体∆ Cm palA-33、∆ CmpalB-13、∆ CmpalC-5、∆ CmpalF-50和∆ CmpalH-26对核盘菌菌核的寄生能力均显著低于野生型菌株Chy-1。野生型菌株Chy-1寄生核盘菌菌核的致腐指数达到了81.33,而敲除突变体∆ CmpalA-33、∆ CmpalB-13、∆ CmpalC-5、∆ CmpalF-50和∆ CmpalH-26寄生核盘菌菌核的致腐指数分别为51.24、55.3、60.89、52.97和49.74 (图 6)。与野生型菌株Chy-1相比,敲除突变体∆ CmpalA-33、∆ CmpalB-13、∆ CmpalC-5、∆ CmpalF-50和∆ CmpalH-26的腐烂指数分别下降了36.9%、32.01%、25.13%、34.87%和38.84%。由此可以说明以CmpalA、CmpalB、CmpalC、CmpalF和CmpalH为主要组分的pH信号通路对盾壳霉寄生核盘菌菌核至关重要。

图 6 盾壳霉Pal相关基因敲除突变体对核盘菌菌核的寄生 Figure 6 Mycoparasitism of the Pal-related genes mutants and the wild-type of C. minitans on sclerotia of S. sclerotiorum. Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

2.6 Pal相关基因敲除对Cmch1Cmg1Cmsp1CmpacC表达的影响

在pH值为3、5和7的条件下,野生型菌株Chy-1的Cmch1相对表达量分别为1.00、1.23和1.44,而∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF-50和∆ CmpalH -26的Cmch1相对表达量分别降低至0.09-0.13、0.11-0.19和0.11-0.35 (图 7-A)。在野生型菌株Chy-1中,Cmg1在pH 3、5和7下的相对表达量分别为1.00、1.52和1.48;而∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26的Cmg1相对表达量分别降低至0.24-0.76、0.43-0.63和0.18-0.71 (图 7-B)。在野生型菌株Chy-1中,Cmsp1在pH 3、5和7下的相对表达量分别为1.00、1.38和4.45,随着pH上升,Cmsp1的相对表达量显著增加。而∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF-50和∆ CmpalH -26的Cmsp1在pH 3、5和7下的相对表达量却显著降低至0.22-0.64、0.18-0.59和0.11-0.35 (图 7-C)。可见,敲除Pal基因之后导致重寄生相关酶基因Cmch1Cmg1Cmsp1的表达量显著降低。这说明盾壳霉重寄生相关酶基因(Cmch1Cmg1Cmsp1)的表达可能受pH信号通路的正调控。

图 7 盾壳霉Pal相关基因敲除突变体和野生型重寄生相关酶基因在不同pH条件下表达分析 Figure 7 Relative transcript levels of three mycoparasitism-associated genes (Cmch1, Cmg1 and Cmsp1) in the Pal-related genes mutants and the wild-type of C. minitans under different pH. A: Relative transcript levels of Cmch1 in the Pal-related genes mutants and the wild-type of C. minitans under different pH; B: Relative transcript levels of Cmg1 in the Pal-related genes mutants and the wild-type of C. minitans under different pH; C: Relative transcript levels of Cmsp1 in the Pal-related genes mutants and the wild-type of C. minitans under different pH. Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

对pH信号通路下游的CmpacC而言,当pH为3、5和7时,CmpacC的相对表达量分别为1.00、2.45和4.66。而∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26中CmpacC的相对表达量分别降低至0.17-0.29、0.15-0.35和0.20-1.03 (图 8)。

图 8 盾壳霉Pal相关基因敲除突变体和野生型CmpacC基因在不同pH条件下的表达分析 Figure 8 Relative transcript levels of CmpacC in the Pal-related genes mutants and the wild-type of C. minitans under different pH. Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

可见,Pal相关基因的敲除显著降低了CmpacC的表达。

2.7 Pal相关基因敲除对盾壳霉降解草酸盐能力的影响

在含HCl和不含HCl的PDB-SO培养基中摇培12 d后,盾壳霉野生型菌株Chy-1和敲除突变体∆ CmpalA -33、∆ CmpalB -13、∆ CmpalF-50和∆ CmpalH -26的菌丝生物量没有显著性差异(5.1-6.5 mg/mL),仅与∆ CmpalC -5的菌丝生物量有显著差异(图 9-B)。但是与初始培养基的pH相比(含HCl和不含HCl的PDB-SO培养基的初始pH值分别为3.3和6.2),在添加HCl条件下的培养滤液pH值明显上升(pH 6.4-6.9) (图 9-A),而且对草酸根离子的降解率也显著升高(图 9-C)。在添加HCl的PDB-SO培养基下,野生型菌株Chy-1和各敲除突变体对草酸根离子的降解率达到76.0%-84.4%,显著高于不添加HCl的PDB-SO的培养基(草酸根离子的降解率为12%-48%)(图 9-C)。而在不添加HCl的PDB-SO培养基下,各敲除突变体和野生型菌株Chy-1对草酸根离子的降解能力也存在明显的差异,野生型菌株Chy-1的降解率只有12%,而各敲除突变体的降解率却能达到33%-48%,比野生型菌株Chy-1提高了21%-36% (图 9-C)。这些结果表明Pal相关基因的敲除显著增强了盾壳霉在pH 6条件下(不添加HCl)降解草酸盐的能力。这说明Pal相关基因对盾壳霉降解草酸盐具有负调控作用。

图 9 盾壳霉Pal相关基因敲除突变体和野生型在添加草酸钠的PDB (HCl存在或者不存在)中对草酸盐的降解 Figure 9 Oxalate degradation by the Pal-related genes mutants and the wild-type of C. minitans in PDB amended with sodium oxalate in absence or presence of 20 mmol/L HCl. A: pH values of the C. minitans cultures in different media; B: Mycelial dry biomass in different C. minitans cultures; C: Percentages of oxalate degradation in different C. minitans cultures. Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

2.8 Pal相关基因敲除对盾壳霉抑制核盘菌菌丝生长的影响

当pH为4时,盾壳霉野生型菌株Chy-1和敲除突变体∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF-50和∆ CmpalH -26在MCD培养基中的生物量为12-16 mg/mL (图 10-B)。培养物滤液对核盘菌菌丝生长的抑菌率为86%-91% (图 10-C)。但在pH 8的条件下,野生型菌株Chy-1和各敲除突变体对核盘菌菌丝生长的抑制作用存在差异显著。菌株Chy-1培养滤液对核盘菌菌丝生长的抑菌率为15%,而敲除突变体培养物滤液对核盘菌菌丝生长的抑菌率为73%-77% (图 10-C)。可见敲除Pal相关基因之后,盾壳霉在高pH (如pH 8) 条件下对核盘菌菌丝生长的抑制作用显著增强。这说明Pal相关基因对盾壳霉抑制核盘菌菌丝生长具有负调控作用。

图 10 盾壳霉Pal相关基因敲除突变体和野生型在pH 4和pH 8的MCD培养滤液对核盘菌的抗真菌活性 Figure 10 Antifungal activity of the filtrates from the Pal-like genes mutants and the wild-type of C. minitans cultures in the MCD medium buffered under pH 4 and pH 8 againts S. sclerotiorum. A: pH values of the C. minitans cultures in different media; B: mycelial dry biomass in different C. minitans cultures; C: percentages of inhibition of S. sclerotiorum growth in PDA amended with different C. minitans cultures. Bars in each figure headed with the same letters are not significantly different (P > 0.05) according to least significant difference test.

3 讨论

目前对真菌pH信号通路的研究报道中,构巢曲霉的pH信号通路研究得最为系统。在构巢曲霉中,pH信号通路主要包括7个Pal基因,分别是pacCpalApalBpalCpalFpalHpalI。在盾壳霉菌株中,我们仍然没有获得palI同源基因CmpalI的敲除突变体,但是系统进化树分析结果可以为我们提供一个较为清晰的证据,在盾壳霉菌株中确实存在着以CmpacC为核心的pH信号传导途径。通过对这5个敲除突变体的菌丝生长试验的分析,结果表明CmpalACmpalBCmpalCCmpalFCmpalH的敲除突变体在较低pH值(3-5) 生长受抑制作用不明显,而在较高pH值(6-8) 生长受抑制明显。这跟已报道的研究中发现palFpalC 的敲除突变体对碱性pH非常敏感的结果是一致的[15-17]。同时,构巢曲霉pH信号通路的上游相关基因敲除突变体在碱性pH下的敏感表型也基本类似[7],说明以CmpalACmpalBCmpalCCmpalFCmpalH为主要组分的盾壳霉pH信号通路可能是盾壳霉适应较高pH值(6-8) 环境的重要机制。

在Pal相关基因敲除突变体对核盘菌的重寄生能力、降解草酸和抑制核盘菌菌丝生长的试验中发现各敲除突变体对核盘菌菌核的寄生能力均具有不同程度的下降,在pH 6下降解草酸的能力和pH 8条件下对核盘菌菌丝生长的抑制作用均有显著性的增强。表达量试验为我们提供了进一步的证据表明敲除突变体∆ CmpalA -33、∆ CmpalB -13、∆ CmpalC -5、∆ CmpalF -50和∆ CmpalH -26对核盘菌寄生能力的降低可能是由于pH信号通路上游相关基因CmpalACmpalBCmpalCCmpalFCmpalH的敲除显著降低了核心转录因子CmpacC的转录表达,从而进一步下调与寄生核盘菌相关胞外酶基因(Cmch1Cmg1Cmsp1)的表达水平。前人研究报道发现构巢曲霉的同源palF基因PRR1调控着白假丝酵母中依赖pH的基因表达、丝状形成以及形态发育[18-19]。研究也发现PacC蛋白也含有核定位信号,在酸性条件下,核定位信号关闭导致PacC全蛋白不能进入细胞核中,从而不能调控靶标基因的表达。随着pH值升高,胞外的pH信号通过上游PalH和PalI膜受体传递给PalF和PalC,从而诱导PalA和PalB与Vps32蛋白形成蛋白复合体参与PacC全蛋白的剪切,形成核定位信号开放的PacC蛋白,进入细胞核参与下游基因的调控[9]

很多研究已经报道了pH信号通路调控病原真菌致病性、毒素合成以及相关的次生代谢途径。在很多病原真菌中都存在着类似的同源PacC基因以及上游相关的基因,说明在很多病原真菌中也存在着类似的pH信号通路。比如在植物病原真菌核盘菌中,草酸的分泌受到PacC的调控,从而影响核盘菌对寄主的致病能力[20]。环境pH值也调控着白色念珠菌的致病能力,并且在白色念珠菌中也克隆到了同源的PacC基因RIM101,RIM101介导的pH信号通路对白色念珠菌与寄主的互作是必要的[21]。马铃薯炭疽菌(Colletotrichum coccodes)能够分泌氨类物质调节寄主的pH值来增强炭疽菌对寄主的毒性[22]。在构巢曲霉中,葡糖糖化酶的产生、糖基化以及细胞内化学平衡均受到环境pH值的影响[23]。在pH调控次生代谢产物的合成方面,也有很多类似的报道,比如在镰刀菌中也发现类似的pH信号通路抑制镰刀菌在中性pH下合成伏马菌素[24];环境pH调控曲霉属病原菌合成柄曲霉素和黄曲霉毒素[25]。因此,对盾壳霉pH信号通路的研究对于我们了解环境pH如何调控盾壳霉重寄生、草酸毒素降解以及抗真菌物质的合成非常必要。将来,我们需要深入研究pH信号通路对次生代谢途径的调控,为建立盾壳霉pH调控网络奠定基础。

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