红曲霉繁殖相关brlM基因鉴定及功能分析

brlA作为曲霉分生孢子形成的中心调控路径中最上游的转录因子,能够诱导下游特异基因的表达调控分生孢子的产生,brlA缺失将导致曲霉无法产生分生孢子,同时生长代谢发生改变,但对不同菌种的影响有显著差异。【目的】探究brlA同源基因brlM在红曲霉中的基因功能,探索红曲霉繁殖调控机制。【方法】从紫色红曲霉Mp-21菌株中克隆了brlA同源基因brlM。利用同源重组原理,用农杆菌介导转化法构建了brlM基因缺失突变株(△brlM),分析△brlM和野生菌株Mp-21在菌落表型、显微结构、生长速率和次生代谢产物等方面的差异,明确brlM基因在红曲霉中的主要功能。【结果】在形态水平上,brlM基因缺失菌株导致菌丝生长更加旺盛,赋予菌落更加蓬松的表型;显微观察发现△brlM失去了有性繁殖产生闭囊壳的能力,但却提升了无性繁殖产生分生孢子的能力;同时代谢产物红曲色素、莫纳可林K和桔霉素产量显著下降。【结论】红曲霉brlM基因的功能与曲霉属中的brlA并不相同,brlM基因在红曲霉有性繁殖中的作用不可替代。本研究的结果对进一步探索丝状真菌繁殖调控机制提供了新的思路。     

金黄色葡萄球菌tst-1基因敲除菌株的构建

抽提金黄色葡萄球菌834菌株的基因组DNA,PCR克隆扩增tst-1及tst-1的上、下游基因,通过将tst-1上、下游基因分别重组到载体质粒pAULA中,形成同源重组质粒pAULA Δtst-1,将pAULA-Δtst-1电转入细菌内,进行同源重组,以PCR、Western blot鉴定tst-1基因敲除菌株无tst-1基因片段,且无TSST-1蛋白表达,表明已成功构建金黄色葡萄球菌tst-1基因的敲除菌株。     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

丝状真菌Podospora anserina AA11家族裂解多糖单加氧酶基因的鉴定和功能研究

辅助活性蛋白家族(auxiliary activity family, AA family)中的裂解多糖单加氧酶(lytic polysaccharide monooxygenase, LPMO)能催化纤维素、几丁质和淀粉等多种难降解碳水化合物的氧化解聚。尽管目前对LPMO的酶学研究较多,但对LPMO基因失活的研究却鲜有报道。本研究利用同源重组方法定点敲除丝状真菌Podospora anserina中AA11家族的5个LPMO基因PaLPMO11A(Pa＿4＿4790)、PaLPMO11B(Pa＿1＿5310)、PaLPMO11C(Pa＿2＿7840)、 PaLPMO11D(Pa＿2＿8610)和PaLPMO11E(Pa＿3＿9420),分别构建了单突变体ΔPaLPMO11A (ΔA)、ΔPaLPMO11B (ΔB)、ΔPaLPMO11C (ΔC)、ΔPaLPMO11D (ΔD)和ΔPaLPMO11E (ΔE),然后通过遗传杂交构建所有多基因突变体。通过在不同碳源培养基上的表型分析、DAB和NBT染色以及纤维素酶活测定分析野生型菌株与突变型菌株在生长速率、有性生殖、氧化应激和纤维素降解...     

Candida amazonensis FLP/FRT基因敲除系统的构建及初步验证

【目的】构建一个适用于Candida amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并通过敲除C.amazonensis的丙酮酸脱羧酶基因(Pyruvate decarboxylase,PDC)对该系统进行初步验证。【方法】以gfpm(绿色荧光蛋白基因)为报告基因,通过添加相应诱导剂评估Spathaspora passalidarum来源启动子(SpXYLp、SpMAL6p、SpMAL1p、SpGAL1p)和Saccharomyces cerevisiae来源Sc GAL1p启动子在C.amazonensis中的诱导调控性能。选择严格诱导型启动子调控FLP重组酶的表达,并在FLP表达盒和潮霉素(Hygromycin B)抗性标记基因(hphm)两端添加同向重复的FRT位点,以PDC基因作为靶基因构建敲除盒PRFg HRP,转化宿主菌C.amazonensis CBS 12363,筛选得到阳性转化子后,通过添加诱导剂,表达FLP重组酶,实现FRT位点间片段切除。【结果】诱导调控实验表明启动子SpGAL1p(受半乳糖诱导)和SpMAL1p(受麦芽糖诱导)是适用于C.amazonensis的严格诱导型启动子。以SpGAL1p调控FLP基因表达,构建的敲除盒PRFg HRP成功转化宿主菌,获得阳性转化子C.amazonensis PDC01,通过添加半乳糖诱导,成功切除基因组中FLP表达盒和抗性标记盒,获得突变株C.amazonensis PDC02。【结论】首次建立了一个适用于C.amazonensis抗性标记可重复使用的FLP/FRT基因敲除系统,并利用该系统成功敲除了C.amazonensis内的PDC基因,为进一步利用代谢工程改造C.amazonensis酵母奠定了良好基础。     

sgf73基因敲除对粟酒裂殖酵母细胞有丝分裂的影响

sgf73基因编码的Sgf73蛋白对SAGA复合物的功能具有重要作用。为探究sgf73基因缺失后sgf73Δ菌株有丝分裂中纺锤体、染色体、肌动蛋白的动力学变化,以粟酒裂殖酵母（Schizosaccharomyces pombe）为材料,对sgf73Δ菌株进行生长曲线和减数分裂产孢实验;采用荧光蛋白标记和活细胞成像的方法,对sgf73Δ菌株有丝分裂进行观察。生长曲线结果分析发现,sgf73基因敲除极显著影响粟酒裂殖酵母的生长,并导致子囊孢子数目显著减少。活细胞成像结果分析发现,在有丝分裂间期,sgf73Δ菌株微管数量显著增加、微管长度趋于增长但无显著差异;sgf73Δ菌株在分裂期纺锤体出现组装缺陷,单极纺锤体极显著增加,前期和中期纺锤体伸长速率显著下降、后期纺锤体伸长速率极显著下降、分裂中期和后期时间分别显著和极显著延长,有丝分裂总时间极显著延长,细胞长度也增长。肌动蛋白分裂环在有丝分裂中维持及收缩时间出现极显著和显著延长,总速率显著降低。同时,染色体分离出现缺陷。以上结果表明,sgf73基因缺失会导致菌株微管、染色体、肌动蛋白缺陷。     

华丽龙胆GsF3′5′H和GsFNS基因的克隆及表达分析

该研究以华丽龙胆(Gentiana sino ornata)5个不同开放阶段(H₁~H₅)的蓝色花冠为试材,利用RT PCR技术克隆GsF3′5′H和GsFNS全长序列,并进行生物信息学分析,比较GsF3′5′H和GsFNS在不同组织和不同开放阶段的基因表达模式。结果显示：(1)所克隆的GsF3′5′H和GsFNS基因分别包含1 560 bp和1 590 bp开放阅读框(OFR),并分别编码520和529个氨基酸。(2)结构分析显示,GsF3′5′H和GsFNS均具有典型的F3′5′H和FNSⅡ蛋白保守结构域。(3)系统进化树分析表明,GsF3′5′H和GsFNS亲缘关系最近的物种是三花龙胆(Gentiana triflora)。(4)qRT PCR结果显示,GsF3′5′H和GsFNS基因在根、茎、叶和花冠中均表达,其中GsF3′5′H基因在花冠H₃阶段表达量最高。GsFNS在根中表达量最高,其次在花冠H₄阶段两基因的表达均较高。研究推测,GsF3′5′H基因表达产生的飞燕草素苷和GsFNS表达产生的黄酮共着色作用可能使华丽龙胆的花冠呈更稳定艳丽的蓝色,为蓝色花分子育种提供重要的基因资源。     

CRISPR/Cas9介导靶向敲除拟南芥GGB基因突变体的鉴定

GGB是抗旱负调控基因。为了获得拟南芥ggb突变体材料,构建了以拟南芥U6启动子驱动GGB sgRNA的CRISPR/Cas9基因组编辑载体。将构建好的编辑载体利用农杆菌介导的浸花法转化野生型拟南芥。对转基因后代GGB基因的测序结果分析发现,在靶位点处有缺失4个碱基和增加1个T碱基的2种突变体产生。分别对野生型拟南芥和上述2种ggb突变体进行半定量RT PCR分析结果显示,突变体材料中几乎检测不到GGB基因表达,说明获得了GGB基因敲除突变体。对野生型和ggb突变体叶片失水率、耐旱表型及单株种子量的测定结果表明,与野生型相比,拟南芥GGB基因突变后,叶片失水率显著减少,抗旱性明显增强,而单株种子量却并没有改变。研究表明,GGB是一种理想的作物分子育种的候选靶基因,获得的突变体为今后从农作物中克隆的GGB同源基因进行功能互补验证提供了有用的遗传材料。     

百合LbCAT和LbGPX基因的克隆与表达分析

以切花百合(Lilium brownii var. viridulum)‘卡瓦纳’cDNA为模板,克隆了过氧化氢酶(LbCAT)和谷胱甘肽过氧化物酶(LbGPX)基因。序列分析表明,这2个基因分别包含1 479 bp和519 bp的开放阅读框(ORF),编码492个和172个氨基酸。进化分析结果表明,LbCAT蛋白与岷江百合CAT蛋白的氨基酸序列相似性最高(99.19%),且亲缘关系最近;LbGPX蛋白与油棕GPX蛋白的氨基酸序列相似性最高(78.61%),亲缘关系最近。qRT PCR结果显示,LbCAT和LbGPX在百合根、鳞茎、叶和花中都有表达。LbCAT在叶中表达量最高,LbGPX在花中表达量最高。这2个基因在百合花蕾的生长发育过程中均有表达,且表达量逐渐增加;在PEG处理后2个基因的转录水平升高,但独角金内酯(SLs)处理却显著降低了这2个基因的转录水平;该结果为百合抗逆性机理研究以及抗逆育种奠定了基础。     

马尾松PmPGK1和PmGPIC基因的克隆和表达分析

为了解马尾松（Pinus massoniana）磷酸甘油酸激酶1（PGK1）与胞质溶胶葡萄糖磷酸异构酶（GPIC）的功能，采用RACE技术克隆了PmPGK1和PmGPIC基因，并进行了生物信息学分析与亚细胞定位，采用实时荧光定量PCR技术分析PmPGK1和PmGPIC的表达特性。结果表明，PmPGK1和PmGPIC全长为2 106和1 848 bp，分别编码507和566个氨基酸。PmPGK1和PmGPIC分别定位于叶绿体和胞质溶胶。PmPGK1表达量为新叶 > 老叶 > 新茎 > 根 > 花；而PmGPIC为老叶 > 花 > 新叶 > 新茎 > 根。低温胁迫24 h，PmPGK1和PmGPIC的表达量均随时间延长先降低后升高，且PmGPIC的表达量在处理2 h后即降至较低水平；高浓度CO₂胁迫24 h，PmPGK1的表达量随时间延长呈降低-升高-再降低的变化趋势，PmGPIC的表达下调但变化较不显著。因此，推测PmPGK1主要参与卡尔文循环及叶绿体/质体糖酵解，PmGPIC主要参与细胞质基质糖酵解；PmPGK1、PmGPIC活性在低温胁迫下均受抑制；PmPGK1活性在CO₂胁迫下受到显著抑制，而PmGPIC活性的影响不大。     

转录因子PaPho与丝状真菌Podospora anserina中磷元素的吸收和利用研究

作为生物体必需的营养元素之一,磷在物质代谢、信号传导和能量储存中起着关键作用。【目的】研究丝状真菌Podospora anserina中调控磷酸盐代谢相关转录因子的作用,可进一步阐明真核微生物中磷元素吸收的调控机制。【方法】利用同源重组的方法定点敲除P.anserina中2个磷代谢相关转录因子PaPho1和PaPho2,遗传杂交构建双重突变体ΔPaPho1ΔPaPho2;通过表型分析、无机磷含量测定和酸性磷酸酶活性测定分析各突变菌株的变化;利用实时定量聚合酶链反应(real-time quantitative polymerase chain reaction,RT-qPCR)分析磷代谢相关基因的表达情况。【结果】在无机磷作为唯一磷来源的培养基上,ΔPaPho1ΔPaPho2无法生长;在添加有机磷的培养基中,ΔPaPho1ΔPaPho2和野生型菌株生长无显著性差异。在同时添加有机磷和无机磷的培养基中,ΔPaPho1ΔPaPho2的无机磷含量和酸性磷酸酶活性比野生型菌株的分别下降了25.0%和61.9%,ΔPaPho1ΔPaPho2中无机磷酸盐转运蛋白基因的表达水平显著降低。【结论】在P...     

Isolation and characterization of a laccase gene fromPodospora anserina

The genome of the filamentous ascomycetePodospora anserina contains at least four non-adjacent regions that are homologous to the laccase gene ofNeurospora crassa. One of these regions contains a gene (lac2) encoding a protein that displays 62% identity with theN. crassa laccase. In shaken cultures,lac2 mRNA is present at low basal levels throughout the growth phase but increases at least 20-fold at the beginning of the autolytic phase and decreases again thereafter. Addition of aromatic xenobiotics (guaiacol, hydroquinone, benzoquinone) to the medium during the growth phase results in a rapid, drastic and temporary increase in the abundance oflac2 mRNA. The promoter region oflac2 contains two sequences which display complete homology with the eukaryotic Xenobiotic Responsive Element and two sequences homologous to the eukaryotic Antioxidant Responsive Element. The identity and function of the laccase encoded bylac2 are discussed.     

A novel family of linear plasmids with homology to plasmid pAL2-1 of Podospora anserina

Three recently isolated wild-type strains of the ascomycete Podospora anserina were analyzed for the presence of linear mitochondrial plasmids. In one of these strains, designated Wa6, at least 12 distinct plasmid-like elements were identified. From molecular analyses a minimum number of 78 individual linear molecules with proteins bound to their 5 ends was estimated. In addition, the different members of this family of typical linear plasmids were shown to possess a common central region and terminal sequences which differ from one plasmid to another due to the presence of different numbers of a 2.4 kb sequence module. Finally, the pWa6 plasmids share a high degree of sequence similarity with pAL2-1, a linear plasmid previously identified in mitochondria of a long-lived mutant of P.anserina. A mechanism is proposed which explains the generation of these distinct, closely related extrachromosomal genetic traits.     

Deletion of the mating-type sequences in Podospora anserina abolishes mating without affecting vegetative functions and sexual differentiation

The mating-type locus of Podospora anserina controls fusion of sexual cells as well as subsequent stages of development of the fruiting bodies. The two alleles at the locus are defined by specific DNA regions comprising 3.8 kb for mat+ and 4.7 kb for mat–, which have identical flanking sequences. Here we present the characterization of several mutants that have lost mat+-specific sequences. One mutant was obtained fortuitously and the other two were constructed by gene replacement. The mutants are deficient in mating with strains of either mat genotype but are still able to differentiate sexual reproductive structures. The loss of the mating type does not lead to any discernible phenotype during vegetative growth: in particular it does not change the life span of the strain. The mutants can recover mating ability if they are transformed with DNA containing the complete mat+ or mat– information. The transformants behave in crosses as do the reference mat+ or mat– strains, thus indicating that the transgenic mat+ and mat– are fully functional even when they have integrated at ectopic sites.     

Evidence for a life span-prolonging effect of a linear plasmid in a longevity mutant of Podospora anserina

The linear mitochondrial plasmid pAL2-1 of the long-lived mutant AL2 of Podospora anserina was demonstrated to be able to integrate into the high molecular weight mitochondrial DNA (mtDNA). Hybridization analysis and densitometric evaluation of the mitochondrial genome isolated from cultures of different ages revealed that the mtDNA is highly stable during the whole life span of the mutant. In addition, and in sharp contrast to the situation in certain senescence-prone Neurospora strains, the mutated P. anserina mtDNA molecules containing integrated plasmid copies are not suppressive to wild-type genomes. As demonstrated by hybridization and polymerase chain reaction (PCR) analysis, the proportion of mtDNA molecules affected by the integration of pAL2-1 fluctuates between 10% and 50%. Comparative sequence analysis of free and integrated plasmid copies revealed four differences within the terminal inverted repeats (TIRs). These point mutations are not caused by the integration event since they occur subsequent to integration and at various ages. Interestingly, both repeats contain identical sequences indicating that the mechanism involved in the maintenance of perfect TIRs is active on both free and integrated plasmid copies. Finally, in reciprocal crosses between AL2 and the wild-type strain A, some abnormal progeny were obtained. One group of strains did not contain detectable amounts of plasmid pAL2-1, although the mtDNA was clearly of the type found in the long-lived mutant AL2. These strains exhibited a short-lived phenotype. In contrast, one strain was selected that was found to contain wild-type A-specific mitochondrial genomes and traces of pAL2-1. This strain was characterized by an increased life span. Altogether these data suggest that the linear plasmid pAL2-1 is involved in the expression of longevity in mutant AL2.     

Identification of PaPKS1, a polyketide synthase involved in melanin formation and its use as a genetic tool in Podospora anserina

In the filamentous fungus Podospora anserina, many pigmentation mutations map to the median region of the complex locus ‘14’, called segment ‘29’. The data presented in this paper show that segment 29 corresponds to a gene encoding a polyketide synthase, designated PaPKS1, and identifies two mutations that completely or partially abolish the activity of the PaPKS1 polypeptide. We present evidence that the P. anserina green pigment is a (DHN)-melanin. Using the powerful genetic system of PaPKS1 cloning, we demonstrate that in P. anserina trans-duplicated sequences are subject to the RIP process as previously demonstrated for the cis-duplicated regions.     

蕨麻愈伤组织原生质体制备条件的优化

以青海‘蕨麻4号’诱导培养的愈伤组织为材料,采用4因素3水平L9(34)正交实验,研究酶类组合、酶解时间、甘露醇浓度及离心速度等主要因素对蕨麻原生质体分离的影响,建立高效、稳定的蕨麻原生质体分离体系,为进一步通过原生质体融合、基因工程等方法对蕨麻进行品种改良奠定基础。结果表明:各因素对蕨麻原生质体产量的影响顺序为:酶类组合酶解时间甘露醇浓度离心速度;青海‘蕨麻4号’愈伤组织原生质体的最适酶解条件为:2.0%纤维素酶+0.75%果胶酶,40r/min振荡酶解10h,甘露醇浓度为0.5mol/L,离心转速为1 000r/min时原生质体的产量达最大(8.96×10~5 cells/g),活力为92.77%。