一种高效无选择标记的黑曲霉基因组编辑方法

黑曲霉(Aspergillus niger)是一种重要的工业生产菌株,被广泛地应用于生产酶制剂和有机酸,但仍需要进行基因组改造提高它的应用潜力。CRISPR/Cas9技术是一种被广泛采用的黑曲霉基因组编辑技术,但由于需要在基因组中整合选择标记或基因编辑效率还有待提高,影响了其在工业菌株改造中的应用。本研究建立了一种基于CRISPR/Cas9技术的高效无选择标记的基因编辑方法。首先,利用5S rRNA启动子启动sgRNA的表达,构建了一个含有AMA1(autonomously maintained in Aspergillus)复制起始片段的sgRNA和Cas9共表达质粒;同时通过敲除kusA基因构建非同源末端连接(non-homologous end joining pathway,NHEJ)修复缺陷的高效同源重组菌株;最后利用含有AMA1片段质粒的不稳定性,通过无抗平板传代丢失含有sgRNA和Cas9共表达质粒。利用该方法,在采用同源臂长度仅为20bp的无选择标记供体DNA进行基因编辑时,基因编辑效率可达到100%。该方法为黑曲霉基因功能的研究和细胞工厂的构建奠定了基础。     

阿卡波糖生物合成调控蛋白的钓取与功能解析

[目的] 发现游动放线菌Actinoplanes sp.SE50/110中阿卡波糖生物合成的调控因子,并提高其产量。[方法] 首先,利用DNA亲和层析技术,钓取与阿卡波糖生物合成基因簇2个双向启动子区域结合的调控蛋白。然后,在阿卡波糖产生菌QQ-2中强化表达或敲除这些调控蛋白编码基因,进行体内功能验证。同时,利用大肠杆菌BL21（DE3）异源表达获得可溶性蛋白,通过凝胶阻滞实验验证蛋白与启动子区域的结合能力。[结果] 经DNA亲和层析及蛋白质质谱分析,钓取出9个与双向启动子P_WV和P_AB结合的调控蛋白。在QQ-2中分别强化表达和缺失这9个调控基因后发现,基因ACPL_1889的强化表达使阿卡波糖产量提高25%,而该基因的缺失使产量降低22%;基因ACPL_5445、ACPL_3989的强化表达使阿卡波糖产量分别降低12%和39%,而这两个基因的缺失使产量分别提高15%和8%。对阿卡波糖生物合成基因转录水平的检测发现,强化表达基因ACPL_1889使acbA、acbB、acbW、acbV的转录水平升高,而缺失该基因使这4个基因的转录水平降低;敲除基因ACPL_5445使这4个基因转录水平均有提高;强化表达基因ACPL_3989使这4个基因的转录水平均下降,而其敲除使acbW和acbA的转录水平分别提高了约100倍和40倍。在凝胶阻滞实验中,ACPL_1889与ACPL_3989均能与acb基因簇的启动子区域结合。最后将正调控基因的强化表达和负调控基因的敲除进行组合,使阿卡波糖产量提升32%。[结论] 本研究发现了9个与阿卡波糖生物合成基因簇的启动子区域结合的调控蛋白,通过体内、体外实验证明ACPL_1889为阿卡波糖生物合成的正调控因子、ACPL_5445和ACPL_3989为负调控因子,不但为揭示阿卡波糖生物合成的转录调控机制奠定了基础,而且这些调控基因的改造显著提升了阿卡波糖的产量。     

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。     

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。     

Bacillus sp.ZJS3基因组测序及砷胁迫下相关基因表达分析

【背景】环境中高毒性As³⁺的微生物氧化在砷的生物地球化学循环中起重要作用,具有潜在的应用价值。【目的】Bacillus sp.ZJS3菌株是本实验室前期分离鉴定的一株As³⁺耐受菌株,而且对多种重金属具有耐受性,期望进一步明确该菌株在As³⁺胁迫下菌体形态变化及应对砷胁迫的遗传基础,为As³⁺耐受细菌的研究提供基础数据。【方法】使用单分子实时测序（single-molecule real-time sequencing,SMRT）及Illumina测序技术对Bacillus sp.ZJS3菌株进行全基因组测序,对其基因进行功能注释和生物信息学分析,并结合绝对定量PCR技术对砷抗性及砷代谢相关基因进行分析。【结果】Bacillus sp.ZJS3菌株基因组大小为5.82 Mb,GC含量为35.9%,包含染色体1个、质粒3个、CDS数量为5 981个、tRNA 104个、sRNA 136个、rRNA 42个、串联重复序列173个、基因岛13个、转运蛋白1 023个、跨膜蛋白1 717个和双组分调控基因160个。NR、Swiss-Prot、Pfam、COG、GO和KEGG数据库分别可注释Bacillus sp.ZJS3菌株基因组中97.66%、69.30%、78.52%、65.49%、67.65%和43.87%的基因。绝对定量PCR结果表明,arsC基因在砷处理条件下显著高于对照组,而arsB基因在砷处理条件下显著低于对照组。【结论】Bacillus sp.ZJS3菌株在As³⁺胁迫下可能导致细胞分裂无法正常进行,进而影响细胞形态。基因组中aqpZ、arsA、arsB、arsC等基因的存在表明该菌株具有As³⁺外排和还原As⁵⁺的能力,phoUpstBACS的存在表明菌株可以吸收As⁵⁺,但菌株受到外界环境As³⁺胁迫时arsB表达水平降低。     

克雷伯氏菌属(Klebsiella)细菌比较基因组学分析揭示多复制子抗性质粒介导广泛的抗性基因传播

[目的] 研究克雷伯氏菌与多复制子抗性质粒间的关系,分析细菌携带多复制子质粒对抗生素环境的响应机制。[方法] 以2018-2020年分离的56株不同来源克雷伯氏菌（Klebsiella sp.）分离株为研究对象,利用微量肉汤稀释法评估其多重耐药表型,对分离菌株进行全基因组测序（WGS）,通过细菌全基因组关联分析（BGWAS）技术和比较基因组学方法深入解析多复制子抗性质粒形成的机制。[结果] 耐药表型分析发现野生动物来源的菌株具有更广的耐药谱系,总体Klebsiella sp.对氨苄西林表现出很高的耐药率（80.36%）,尤其是马来穿山甲来源菌株对头孢类抗生素高度耐受,同时对氯霉素、左氧氟沙星和复方新诺明等药物耐受,基因组分析发现这些菌株携带了抗性质粒和更多的抗生素抗性基因。进一步对69个质粒序列分析,发现有28个质粒为多复制子质粒,主要携带bla_CTX-M-15、bla_CTX-M-14、bla_CTX-M-55、bla_OXA-1和bla_TEM-1等β-内酰胺酶基因。细菌携带质粒类型分析认为Klebsiella pneumoniae可能是多复制子质粒的重要宿主,质粒骨架与结构分析发现多复制子质粒多由2个或2个以上单个质粒融合而成,携带此类质粒的菌株不仅获得了更广的耐药表型,而且在全球传播扩散分布逐年增加,因此产生对抗生素环境更强的适应性。[结论] 多重耐药性细菌呈现的表型与携带的多复制子质粒有关,相比较下多复制子质粒比非多复制子质粒有更强的抗性基因携带能力,或许是细菌在强大的抗生素压力下产生的重要响应机制。本研究对于未来探索细菌抗性基因的传播扩散机制具有重要意义。     

精氨酸代谢调控蛋白ArgR调控嗜热链球菌胞外多糖合成

[目的] 研究精氨酸代谢调控蛋白ArgR对嗜热链球菌胞外多糖（EPS）合成的调控作用。[方法] 利用大肠杆菌异源表达嗜热链球菌ArgR蛋白,通过尿素变性-复性和Ni²⁺亲和层析纯化。采用凝胶电泳迁移（EMSA）和生物膜层干涉（BLI）分析ArgR和eps基因簇中P_epsA启动子的相互作用和动力学信息。构建过表达和弱化argR基因菌株,利用苯酚-硫酸法测定其合成EPS差异。[结果] 大肠杆菌异源表达的ArgR为包涵体,使用尿素变性-复性纯化可获得2.95 mg/mL可溶性蛋白;EMSA和BLI结果显示ArgR和启动子P_epsA有特异性结合,且结合因解离水平低而稳定;过表达argR基因可显著降低嗜热链球菌EPS合成,而弱化argR基因则提高EPS合成。[结论] 本研究表明ArgR能特异性结合嗜热链球菌eps基因簇启动子,并负调控EPS生物合成。     

敲除LTA4H基因的PK-15细胞系构建及其对口蹄疫病毒复制的影响

【目的】利用CRISPR/Cas9基因编辑技术构建白三烯A4水解酶(leukotriene A4 hydrolase, LTA4H)基因缺失的猪肾细胞系(porcine kidney-15, PK-15),探究LTA4H对口蹄疫病毒(foot-and-mouth disease virus, FMDV)复制的影响,为开展LTA4H功能研究及调控病毒复制机制研究提供理论依据。【方法】设计2条针对猪LTA4H基因的引导RNA (small guide RNA, sgRNA),分别构建至载体pX459-puro-MCS中;将CRISPR重组质粒转染PK-15细胞,用嘌呤霉素(puromycin)抗生素筛选,并通过有限稀释法筛选单克隆细胞,之后通过Western blotting和测序检测LTA4H基因的敲除,获得LTA4H基因功能缺失细胞系。使用Western blotting、RT-qPCR及病毒滴度测定等方法检测敲除LTA4H基因后对FMDV复制及相关蛋白的表达情况。【结果】获得的单克隆敲除细胞系与野生型细胞相比,能够显著抑制FMDV复制。【结论】本研究成功构建了LTA4H基因敲除的PK-15细胞系,证明LTA4H对FMDV的复制具有促进作用,研究结果为后续LTA4H功能研究提供理论依据。     

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。     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒（BmNPV）bacmid在大肠杆菌BW25113中快速地敲除BmNPV orf60基因。从大肠杆菌BmDH10Bac中提取BmNPV bacmid,将其电转化到含有质粒pKD46（能表达Red重组酶）的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物（5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因（cat）的首尾序列）,以pKD3质粒（含cat）为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPV bacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPV orf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

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.     

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.     

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.     

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.     

中国黑粉菌属和利罗黑粉菌属

黑粉菌属是Roussel 1806年建立的,全世界记载有三百余种,主要寄生于禾本科,是经济作物及牧草的重要致病菌·长期以来,对黑粉菌的邢子使用过各种名称,如厚垣孢子,冬孢子及黑粉孢子等.本文采用黑粉孢子以区别锈菌的冬孢子. 芳’(1979)在《中国真菌总汇》中列出黑粉菌属五十种及一个变型.作者经过显微结构和超显微结构的研究,承认其中二十九种为正确名称,八种及一变型为异名,顶黑粉菌(Ustilago acrearus Berk.)由于错拼而被废弃.埃地黑粉菌(Ustilago emodensis Berk.)被转移至利罗粉菌属(Liroa).另有十一种黑粉菌因缺少标本留待今后订正.自1979年以后,杨信东(1983)增加黑粉菌属二种我国新纪录,K.范基和郭林(1986)描述一新种,四种新纪录.在本文中,作者描述一新种:鸢尾蒜黑粉(Ustilago ixiolirii Guo L) ,孢子堆生在蒴果内,不开裂,黑色,粉末状.黑粉孢子球形,近球形,稀椭圆形, 12.5-21×10-21μm,黑褐色,壁厚1-1.Sμm,纹饰脑状.是迄今生在石蒜科植物上唯一黑粉菌的种,其它几种黑粉菌均属条黑粉菌属.本文增加七种我国新纪录.共计四十九种,寄生于六科四十四属植物,主要是禾本科和蓼科.这仅是黑粉菌属研究的初步报告,在全国范围内大量采集黑粉菌标本后,作者相信会有更多新种和我国新纪录被发现.利罗黑粉菌属(Liroa)是从黑粉菌属(Ustaligo)分出的,此属为单种属.     

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.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.