球孢白僵菌Bb_bm01菌株PacC基因突变体的构建及其毒力和孢子萌发率的测定

通过敲除球孢白僵菌(Beauveria bassiana)菌株bm01的PacC基因,研究了PacC基因敲除对球孢白僵菌毒力和分生孢子萌发速率的影响。利用农杆菌(Agrobacterium tumefaciens)介导的转化方法转化Bb_bm01,通过同源重组敲除Bb_bm01的PacC基因,筛选ΔPacC基因敲除菌株,获得了遗传稳定的ΔPacC基因敲除菌株。通过毒力测定,对比了野生菌株和ΔPacC基因敲除菌株对家蚕和大蜡螟毒力的差异,发现ΔPacC基因敲除菌株对家蚕和大蜡螟毒力都减弱,差异具有统计学意义(p0.01),表明在Bb_bm01菌株中的PacC基因与它的致病性相关;另外,比较ΔPacC基因敲除菌株与野生型菌株Bb_bm01在SDB液体培养基中的孢子萌发率,发现ΔPacC基因敲除菌株孢子萌发明显减慢,表明PacC基因参与调控孢子萌发。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

黄曲霉Ste50衔接子蛋白对生长发育及致病性的影响

[目的] Ste50是真菌中重要的衔接子蛋白,在多个MAPK级联通路中起重要的信号衔接与传递作用。本研究鉴定出了黄曲霉AflSte50蛋白,并发现了其对黄曲霉的生长、产孢、致病能力和响应渗透压胁迫等方面的影响。[方法] 首先通过生物信息学方法在黄曲霉NRRL 3357中鉴定出ste50基因,并通过同源重组的方法构建了ste50基因的敲除和互补突变体菌株。而后,对基因敲除在黄曲霉生长发育、次级代谢产物合成和胁迫响应等方面的作用进行了研究。[结果] 与野生型相比,△Aflste50菌株生长速度和AFB₁合成量降低且不能产生菌核,同时对花生、玉米种子的致病能力下降。该基因在渗透胁迫条件下正调控MAP激酶的磷酸化水平,但对细胞壁胁迫无响应。[结论] Ste50（AFLA_002340）是黄曲霉衔接子蛋白,影响黄曲霉的生长、发育和AFB₁的合成,能够响应渗透压胁迫,在HOG通路中发挥作用。     

BbRho5对球孢白僵菌生长速率的作用研究

为阐明BbRho5对球孢白僵菌生防潜能的作用,构建了Bbrho5单基因敲除菌株ΔBbrho5,以野生型菌株WT作为对照,在不同培养基上测定菌落生长速率,并测定了菌株对多菌灵胁迫耐受性及对大蜡螟幼虫体壁侵染能力。进一步获取和分析了ΔBbrho5和WT细胞内基因转录组数据。结果表明,BbRho5蛋白功能缺陷显著抑制球孢白僵菌菌丝生长速率,同时微弱影响其多菌灵胁迫抗逆性及生防能力。相较于WT,ΔBbrho5中具有770个差异表达基因(DEGs),其中上调基因395个,下调基因375个。GO分析显示,ΔBbrho5 VS WT中DEGs主要富集于氧化还原酶活力(oxidoreductase activity)和单加氧酶活力(monooxygenase activity)功能。KEGG通路富集结果显示,DEGs主要富集于氮代谢及多种氨基酸代谢通路。在氮代谢通路中富集到7个功能基因,其中有5个上调,2个下调,说明敲除菌株可能采用增强氮源利用及谷氨酸合成以应对Bbrho5缺陷引起的生长迟缓。以上研究结果揭示了球孢白僵菌中小GTP酶BbRho5对球孢白僵菌生长速率具有重要影响,且氮代谢和氨基酸代谢可能为其重要的响应代谢通路。     

基因CfATG6和CfATG14参与调控果生刺盘孢细胞自噬和致病力

【目的】炭疽病是油茶的主要病害,由刺盘孢属的多种真菌引起,其中果生刺盘孢分布范围最广、分离率最高,是油茶炭疽病的主要致病菌。研究自噬相关蛋白CfAtg6和CfAtg14的生物学功能,为进一步揭示果生刺盘孢通过细胞自噬调控致病的分子机制,并为油茶炭疽病的防治提供理论基础。【方法】根据同源重组原理,通过聚乙二醇(polyethylene glycol, PEG)介导的方法,在果生刺盘孢中敲除基因CfATG6和CfATG14,并进一步获得回补菌株ΔCfatg6-C和ΔCfatg14-C。【结果】酵母双杂交试验结果显示,果生刺盘孢蛋白CfAtg6和CfAtg14可能存在互作关系。生物学表型测定结果表明,相较于野生型和回补菌株,突变体ΔCfatg6和ΔCfatg14均表现出营养生长速率显著减慢,附着胞形成率分别只有野生型的5%和18%;突变体ΔCfatg6和ΔCfatg14致病力均极显著减弱,造成的油茶叶片病斑面积少于野生型和回补菌株的1/3;CfATG6和CfATG14基因缺失突变体均丧失转运和降解CfAtg8蛋白的能力,并对细胞壁胁迫更敏感。突变体ΔCfatg6的分生孢子产量显著降低,仅为野生型的20%左右;氧化胁迫试验结果表明,相较于野生型和回补菌株,过氧化氢对突变体的生长抑制率升高10%左右。内质网压力胁迫试验表明,ΔCfatg14对二硫苏糖醇抑制率升高5%以上。【结论】自噬相关基因CfATG6和CfATG14参与调控了果生刺盘孢生长发育、细胞自噬和致病力。     

油茶果生刺盘孢组蛋白乙酰化转移酶Rtt109生物学功能

炭疽病是油茶的主要病害,造成巨大经济损失。果生刺盘孢是油茶炭疽病优势致病菌。本研究旨在探讨果生刺盘孢中组蛋白乙酰化转移酶Rtt109的生物学功能,为油茶炭疽病防治提供理论依据。通过构建基因敲除载体,根据同源重组原理,敲除目标基因CfRTT109,进一步筛选获得突变体ΔCfrtt109;构建CfRTT109基因回补质粒,转化至突变体ΔCfrtt109,通过荧光筛选回补菌株。生物学表型测定结果显示：相比于野生型,突变体ΔCfrtt109生长速率下降了51.23%,分生孢子的形成率下降了71.89%;在含有5.0mmol/L内质网胁迫剂二硫苏糖醇的PDA培养基中,突变体ΔCfrtt109的生长抑制率为50.75%,显著高于野生型和回补菌株;荧光定量PCR结果表明,与野生型和回补菌株相比,内质网相关的基因HAC1、SCJ1与PDI1基因在突变体中显著上调表达;在含有DNA损伤试剂甲基磺酸甲酯的PDA培养基中,突变体ΔCfrtt109停止生长;进一步研究发现,在甲基磺酸甲酯胁迫下,突变体ΔCfrtt109中的DNA损伤修复基因RHM52、RHM54与REV1表达量上调幅度显著低于野生型;致病力测...     

嗜水气单胞菌中转录因子AHA_1581对细菌生理功能调控机制的研究

[目的] LuxR家族转录因子能够抑制或刺激不同功能类型基因的表达,来维持细胞功能的稳定性。嗜水气单胞菌是水产养殖中重要的致病菌之一,目前对该菌中的LuxR家族转录因子功能的研究还较少。[方法] 本研究利用含有sacB标记的自杀载体pRE112和同源重组技术敲除LuxR家族转录因子AHA_1581基因。[结果] 生理表型测定结果发现,ΔAHA_1581的运动与胞外蛋白的酶活增强、生物被膜形成能力降低,且耐受低温、卡那霉素、庆大霉素胁迫,但是对K₂Cr₂O₇更加敏感。进一步对野生型Ah和ΔAHA_1581的定量蛋白质组学分析,共鉴定到2654个蛋白,其中59个蛋白下调表达,142个蛋白上调表达。生物信息学分析表明AHA_1581参与调控双组分调节系统、丙酮酸代谢、碳代谢、TCA循环等细菌重要生理过程,以及细菌耐药基因和毒力因子的差异表达。[结论] 了解AHA_1581基因在调控细菌毒力以及生物过程中所起的重要作用,对预防和控制嗜水气单胞菌引起疾病的发生和传播可能具有重要的科学意义。     

过表达基因elo1和ole1增强光滑球拟酵母Δmed15B菌株的低pH耐受能力

[目的] 研究中介体亚基Med15B（ORF CAGL0H06215g）介导的脂肪酸代谢影响光滑球拟酵母（Candida glabrata）耐受低pH胁迫的生理机制。[方法] 在菌株Δmed15B中过量表达脂肪酸延伸酶基因elo1（ORF CAGL0L08184g）和Δ9去饱和酶基因ole1（ORF CAGL0I00418g）,构建过表达菌株Δmed15B/elo1-ole1,然后与菌株Δmed15B和亲本菌株ATCC55对比分析基因elo1和ole1的表达水平、脂肪酸组分比例、细胞膜完整性和耐受能力在pH 2.0和pH 6.0条件下的差异。[结果] 发现,在pH 2.0条件下过表达菌株Δmed15B/elo1-ole1：（1）基因elo1和ole1表达水平比菌株Δmed15B分别上调了4.1倍和3.3倍,与亲本菌株ATCC55相比分别上调了2.5倍和2.2倍;（2）脂肪酸平均链长延长至17.4,高于菌株Δmed15B的16.8和亲本菌株ATCC55的17.1;不饱和脂肪酸与饱和脂肪酸的比值比菌株Δmed15B和亲本菌株ATCC55分别提高了53.1%和41.5%;（3）碘化丙啶（PI）染色细胞数占总检测细胞数的比例,与菌株Δmed15B和亲本菌株ATCC55比较分别下降了60.8%和37.7%;（4）细胞半抑制pH（胁迫）（IC₅₀）值达到pH 2.3,比菌株Δmed15B的pH 3.7和亲本菌株ATCC55的pH 3.1具有更强的耐受能力。[结论] 在菌株Δmed15B中过表达基因elo1和ole1能通过提高长链脂肪酸和不饱和脂肪酸含量,增强细胞对低pH胁迫的耐受能力。     

定量蛋白质组分析蚯蚓血红蛋白样蛋白MSMEG_3312介导的分枝杆菌耐红霉素机制

[目的]细菌耐药机制是个复杂的机制,系统生物学是系统性揭示耐药机制的有力研究手段。我们课题组前期研究结果显示,蚯蚓血红蛋白样蛋白msmeg_3312基因敲除后能够增加耻垢分枝杆菌对红霉素的耐药性,本文系统研究MSMEG_3312参与红霉素耐药性形成的机制。[方法]首先纯化MSMEG_3312蛋白,利用光谱及圆二色谱描述MSMEG-3312蛋白。利用定量蛋白质组学的方法比较分析敲除菌株Δmsmeg_3312与野生型菌株mc² 155蛋白表达的差异,并通过qRT-PCR进行验证。利用红霉素ELASA试剂盒测定Δmsmeg_3312与mc² 155的胞内药物浓度。[结果]光谱及圆二色谱分析确定MSMEG_3312是蚯蚓血红蛋白样蛋白。定量蛋白质组学分析发现,红霉素未处理的条件下,相比于野生型菌株mc² 155,敲除菌株Δmsmeg_3312有包括3种转运蛋白在内的8种蛋白表达水平上调,14种蛋白表达下调;而红霉素处理后,Δmsmeg_3312中有448种蛋白差异表达,其中有11种转运蛋白表达上调,26种蛋白与氨基酸合成通路相关。胞内药物浓度检测显示敲除菌株Δmsmeg_3312的胞内红霉素浓度显著低于野生型菌株。[结论]蚯蚓血红蛋白样蛋白MSMEG_3312调控改变了细菌对红霉素药物处理的反应网络,其介导的红霉素耐药是一种集合抗生素耐受机制。     

腺苷酸核糖基化因子BbarfA介导白僵菌孢子萌发和毒力

【目的】探究腺苷酸糖基化因子ARF在球孢白僵菌(Beauveria bassiana)中存在种类及生物学功能。【方法】利用BLASTp搜索球孢白僵菌非冗余蛋白数据库,鉴定ARF并进行聚类分析,结合表达分析、反义抑制、超量表达野生型基因和GTP解离位点与结合位点突变的基因,解析其中1个ARF与白僵菌发育分化、逆境胁迫反应和毒力的关系。【结果】球孢白僵菌中存在至少6个ARF或类似蛋白,分别聚类于酵母、人类ARF及其类似蛋白的不同类群。其中BBA_01574与人类的ARF3、ARF4和ARF5聚为一类,命名为BbarfA。BbarfA在成熟的分生孢子和球形膨大时期表达明显高于芽管伸长期。反义抑制BbarfA加速了孢子萌发,提高了菌株毒力,而超量表达BbarfA和点突变GTP解离区域的BbarfA则延迟了孢子萌发速度,降低了菌株毒力。尽管BbarfA转录受高盐、髙渗、氧化和高温胁迫的诱导,但遗传修饰的转化子与野生菌株对上述胁迫反应的敏感性无明显差异。【结论】BbarfA介导分生孢子萌发和毒力。     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Comparative study of Bifidobacteriumanimalis, Escherichiacoli, Lactobacilluscasei and Saccharomycesboulardii probiotic properties

The present work investigates some probiotic properties of four different microorganisms (Bifidobacterium animalis var. lactis BB-12, Escherichia coli EMO, Lactobacillus casei and Saccharomyces boulardii). In vitro and in vivo tests were carried out to compare cell wall hydrophobicity, production of antagonistic substances, survival capacity in the gastrointestinal tract of germ-free mice without pathological consequence, and immune modulation by stimulation of Küpffer cells, intestinal sIgA and IL-10 levels. In vitro antagonism against pathogenic bacteria and yeast was only observed for the probiotic bacteria B. animalis and L. casei. The hydrophobic property of the cell wall was higher for B. animalis and E. coli EMO, and this property could be responsible for a better ability to colonize the gastrointestinal tract of germ-free mice. Higher levels of sIgA were observed mainly for S. boulardii, followed by E. coli EMO and B. animalis, and only S. boulardii induced a significant higher level of IL-10. In conclusion, for a probiotic use, S. boulardii presented better characteristics in terms of immunomodulation, and B. animalis and L. casei for antagonistic substance production. The knowledge of the different probiotic properties could be used to choice the better microorganism depending on the therapeutic or prophylactic application.     

Probing the Drosophila retinal determination gene network in Tribolium (II): The Pax6 genes eyeless and twin of eyeless

The Pax6 genes eyeless (ey) and twin of eyeless (toy) are upstream regulators in the retinal determination gene network (RDGN), which instructs the formation of the adult eye primordium in Drosophila. Most animals possess a singleton Pax6 ortholog, but the dependence of eye development on Pax6 is widely conserved. A rare exception is given by the larval eyes of Drosophila, which develop independently of ey and toy. To obtain insight into the origin of differential larval and adult eye regulation, we studied the function of toy and ey in the red flour beetle Tribolium castaneum. We find that single and combinatorial knockdown of toy and ey affect larval eye development strongly but adult eye development only mildly in this primitive hemimetabolous species. Compound eye-loss, however, was provoked when ey and toy were RNAi-silenced in combination with the early retinal gene dachshund (dac). We propose that these data reflect a role of Pax6 during regional specification in the developing head and that the subsequent maintenance and growth of the adult eye primordium is regulated partly by redundant and partly by specific functions of toy, ey and dac in Tribolium. The results from embryonic knockdown and comparative protein sequence analysis lead us further to conclude that Tribolium represents an ancestral state of redundant control by ey and toy.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.