结核分枝杆菌同源重组基因敲除系统的构建和应用

【目的】结核分枝杆菌同源重组效率很低,突变株的构建需要半年之久。本研究的目的在于构建一种用于在结核分枝杆菌中进行基因快速敲除、且易于筛选的高效同源重组系统。【方法】野生型结核分枝杆菌转化含有SacB反向选择标记、且能诱导表达两种同源重组酶gp60和gp61的质粒pSL002。然后分别将靶基因的两个同源臂克隆入到含有hyg(潮霉素)抗性基因和gfp(绿色荧光蛋白)基因的重组质粒pSL001中,再将靶基因同源臂-loxP-hyg-gfp-loxP片段从pSL001切下,转化含有pSL002的野生型结核分枝杆菌,一步得到双交换突变株。再将含有SacB反向选择标记、且表达Cre重组酶的质粒pSL003转化入结核分枝杆菌双交换突变株中,切除两个loxP之间的hyg抗性基因和gfp基因,得到无痕缺失突变株。最后利用含有2%蔗糖的琼脂糖平板去除含有SacB反向选择标记的质粒pSL002和pSL003。【结果】在结核分枝杆菌中成功构建了高效同源重组系统,利用该系统构建了rv1364c、pstP跨膜区、pstP胞外区三个突变株,得到双交换突变株的效率为25%-62.5%,从双交换突变株得到无痕缺失突变株的效率为100%。通过gfp作为荧光标记基因,利用NightSea BlueStar蓝光手电筒和滤光眼镜,可以对平板上的基因缺失株直接进行快速判定。【结论】该同源重组系统利用gp60和gp61重组酶,在时间上将在结核分枝杆菌中无痕缺失突变株的构建从6个月缩短到3个月。这是目前为止在结核分枝杆菌中构建突变株最快且效率最高的方法,为加速分枝杆菌功能基因组的研究提供了新的遗传工具。     

谷氨酸棒状杆菌CRISPR-Cpf1和Cre/loxP基因敲除技术的比较

【背景】谷氨酸棒状杆菌的基因敲除系统较为匮乏且效率不高,难以对其进行代谢工程改造,不利于高性能工业菌株的构建及规模生产。【目的】分别采用CRISPR-Cpf1和Cre/loxP基因敲除系统对谷氨酸棒状杆菌ATCC13032(CorynebacteriumglutamicumATCC13032)基因组上的argR和argF基因进行敲除,比较两种敲除方法的优缺点,为合理选择敲除系统提供依据。【方法】特异性重组的Cre/loxP敲除系统是首先利用同源重组将基因组上的靶基因替换为两端带有重组位点loxP的kanR片段,然后由重组酶Cre识别loxP位点并发生重组反应,从而去除替换到基因组上的kanR片段,进一步利用质粒的温敏特性将其消除,从而实现靶基因的敲除。CRISPR-Cpf1敲除系统是利用Cpf1对pre-crRNA进行加工,形成的成熟crRNA引导Cpf1识别和结合到靶DNA的特定序列上并切割双链DNA分子,通过同源重组作用去除靶基因,基于质粒自身的温敏特性将其消除,从而完成基因敲除的整个过程。【结果】Cre/lox P系统可在8N+2 d内完成N轮迭代基因敲除,而CRISPR-Cpf1系统可在5N+2d内完成N轮迭代基因无痕敲除,理论上还可以一次对多个靶位点进行编辑,效率更高,但存在同源重组效率较低、假阳性率高等缺点。【结论】与Cre/loxP系统相比,CRISPR-Cpf1辅助的同源重组基因敲除方法可省时、省力地实现基因的无痕敲除,理论上还可实现多个基因的同时敲除、总体效率更高,然而编辑效率还有提高的空间。     

炭疽芽胞杆菌mntA基因缺失突变株的构建及鉴定

目的:通过同源重组的方法敲除炭疽芽胞杆菌减毒AP422株的mntA基因,使菌株进一步减毒,用于构建新的疫苗候选株。方法:利用PCR方法扩增mntA基因上下游同源臂后与温敏质粒连接,构建打靶载体,并转化炭疽芽胞杆菌减毒AP422株;利用抗生素和温度2种选择压力实现同源重组,敲除目标基因mntA,然后利用Cre-LoxP系统去除抗性筛选标记,得到无抗性标记的缺失突变株,并利用PCR和Western印迹等方法对重组菌进行系统鉴定,最后分析突变株的生物学性状。结果:敲除了AP422株的mntA基因,获得了无抗性标记的缺失突变株,突变株的生存竞争能力比原始菌株明显减弱。结论:突变株获得了进一步减毒,可用于构建新的疫苗候选株。     

Pseudomonas plecoglossicida NyZ12基因无痕敲除方法的建立

旨在建立一种适合环己胺降解菌NyZ12基因无痕敲除的可靠方法。通过overlapping PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pEX18km上,将重组质粒转化到大肠杆菌S17pir中,再通过接合转移到假单胞菌NyZ12菌株内,经pEX18km质粒上sacB基因的反向筛选得到突变株并通过PCR方法和测序鉴定。结果显示,成功构建了假单胞菌NyZ12菌株orf4637的基因突变株(NyZ12Δ4637)。通过自杀载体同源重组可以成功获得敲除的无痕突变株,且突变株基因组上没有任何抗性筛选标记残留,为环己胺降解菌NyZ12基因功能研究提供了可靠的基因敲除技术。     

利用Red重组系统敲除鲍曼不动杆菌asa A基因

目的利用Red重组系统敲除鲍曼不动杆菌ATCC 17978的asaA。方法设计上下游引物中包含asaA的同源序列,并以pKD4质粒为模板,扩增含有卡那霉素抗性基因的DNA片段。将该片段转化于表达重组酶的17978感受态细胞中,在卡那霉素筛选压力下,得到经两次同源双交换的具有卡那霉素基因标记的突变菌株。随后,在重组酶的作用下将抗性基因去除,最终得到无抗性基因标记的突变菌株ΔasaA。结果通过该重组系统,首先将卡那霉素抗性基因的DNA片段替换了基因组中asaA的DNA片段,然后将卡那霉素抗性基因的DNA片段消除,最终获得了asaA缺失的突变体。结论通过Red重组系统为鲍曼不动杆菌中其他基因的缺失突变提供方法与思路。     

副溶血性弧菌基因敲除方法的建立及应用

目的摸索出一套副溶血性弧菌基因敲除的可靠方案,副溶血性弧菌致病相关基因的敲除对深入研究其致病机制有重要意义。方法通过融合PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pDS132上,将重组质粒转化大肠杆菌S17λpir中,再接合转移到副溶血性弧菌菌株内,经pDS132质粒上sacB基因的反向筛选得到突变株。结果成功构建了副溶血性弧菌RIMD2210633菌株ΔopaR,ΔtoxR和ΔaphA三个基因突变株。结论通过自杀载体同源重组成功获得精确敲除的无痕突变株更有利于基因功能的研究,使后续副溶血性弧菌突变株与野生株的对比研究成为可能。     

肠出血性大肠杆菌O157∶H7 z1445基因缺失株的构建

目的:利用Red重组系统敲除肠出血性大肠杆菌(EHEC)O157∶H7的z1445基因,构建大肠杆菌z1445基因缺失突变株。方法:以O157∶H7为模板,PCR扩增目的基因两侧的同源臂序列,分别经酶切后连接到p UC19-kan质粒的卡那霉素抗性基因kan两侧,PCR获得中间嵌合kan基因(带有FRT位点)的同源线性片段,利用质粒p KD46敲除z1445基因,利用质粒p CP20去除抗性标记基因;PCR鉴定及测序验证目的基因缺失后,测定缺失株及野生株的生长曲线。结果:敲除了z1445基因,突变株与野生株的生长曲线接近。结论:构建了z1445基因缺陷型菌株,为进一步分析z1445基因在O157∶H7与宿主的相互作用中发挥的作用提供了材料。     

两步PCR介导的Red重组技术快速敲除鼠疫耶尔森菌sRNA及染色体大片段

【目的】利用Red同源重组系统,通过二步PCR法建立一种适合鼠疫耶尔森菌s RNA和大片段染色体基因敲除的方法。【方法】第一步PCR先扩增出目的基因的上、下游同源臂(600–1000 bp)及卡那抗性盒,再以上、下游同源臂及卡那抗性盒等摩尔混合物为模板,通过融合PCR获得含上下游同源臂及卡那抗性盒的线性突变盒,再将此突变盒的PCR产物电转到含有pKD46质粒的鼠疫201菌株,在阿拉伯糖的诱导下,p KD46质粒表达Red重组酶,促使卡那抗性盒替换目的基因,最后对获得的重组克隆进行PCR鉴定。【结果】本研究通过两步PCR法构建600–1000 bp的同源臂,提高了同源重组效率,并将鼠疫菌sRNA RyhB1(108 bp)和RyhB2(106 bp)和染色体大片段47-2(10.4 kb)、47-3(21.6 kb)、47-3a(9.2 kb)及47-3b(6.1 kb)成功敲除。【结论】基于Red重组系统构建的二步法突变技术,是一种简单、高效的精确修饰鼠疫菌s RNA及大片段染色体的方法,适合于鼠疫菌全基因组的基因敲除,为鼠疫菌基因表达与调控、致病和毒力等研究提供有力的工具。     

一种基于温敏质粒的新型基因敲除方法

基于温敏型质粒而不用线性DNA的方法用于快速敲除沙门氏菌染色体上的目的基因。以伤寒沙门氏菌S.ty2基因组为模板扩增得到的ssaV基因的上下游同源臂,与两端带有FRT位点的卡那霉素抗性基因片段连接到温敏型质粒pHY304,共同构建同源重组载体;然后转化S.ty2,通过筛选得到带有抗性标记的重组菌。通过转入重组酶表达质粒pCP20,去除抗性标记,得到ssaV基因缺失的重组菌,并在 DNA 水平进行了鉴定。建立了一种改进的基于温敏质粒的沙门氏菌的基因敲除方法,此方法也值得在其他革兰氏阴性菌的基因敲除中尝试应用。     

肠出血性大肠杆菌Ⅲ型分泌系统ATP水解酶escN基因突变株的构建

目的:利用λ噬菌体的Red重组系统敲除肠出血性大肠杆菌O157∶H7的Ⅲ型分泌系统ATP水解酶Esc N,构建大肠杆菌esc N基因缺失突变株。方法:以O157∶H7为模板,PCR扩增目的基因两侧的同源臂序列,分别酶切连接于p UC19-kan质粒上,PCR获得中间嵌合卡那霉素抗性基因(带有FRT位点)的同源线性片段,利用质粒p KD46和p CP20介导的重组技术敲除esc N基因,并去除抗性标记;PCR及测序验证目的基因缺失后,测定缺失株及野生菌株的生长曲线。结果:敲除了肠出血性大肠杆菌O157∶H7的esc N基因,突变株与野生株的生长曲线相近。结论:构建了Ⅲ型分泌系统缺陷菌株,为进一步研究Ⅲ型分泌系统因子在肠出血性大肠杆菌致病过程中的作用奠定了基础。     

铜绿假单胞菌中快速基因操作工具的开发和应用

针对基因的操作,包括缺失和插入基因、替换基因元件(如启动子)、融合荧光蛋白基因、构建原位的基因报告系统,是大多数生物技术实验室的必备技术。目前广泛使用的基于2次同源重组的基因操作方法,在构建质粒、转化和筛选方面较为繁琐。另外使用该方法进行长片段敲除的效率较低。为了简化基因操作的流程,本研究构建了一个最小化的铜绿假单胞菌(Pseudomonas aeruginosa)整合型质粒pln2,只需将目标基因内部一段序列克隆到pln2质粒并导入细菌,由于质粒不能在细菌内自我复制而只能通过单次等位基因交换整合到基因组上,从而使目的基因断裂为两部分而失去活性。在pln2的基础上开发了一整套工具质粒适用于基因组的不同操作,包括融合荧光蛋白基因、替换基因元件(如启动子)、构建原位的转录型荧光报告系统。此外,本研究借助该系统成功实现超长片段基因簇的敲除,单次可以敲除长达270 kb的片段。     

Marker rescue from the Nicotiana tabacum plastid genome using a plastid/Escherichia coli shuttle vector

We recently reported an 868-bp plastid DNA minicircle, NICE1, that formed during transformation in a transplastomic Nicotiana tabacum line. Shuttle plasmids containing NICEI sequences were maintained extrachromosomally in plastids and shown to undergo recombination with NICE1 sequences on the plastid genome. To prove the general utility of the shuttle plasmids, we tested whether plastid genes outside the NICE1 region could be rescued in Escherichia coli. The NICE1-based rescue plasmid, pNICER1, carries NICE1 sequences for maintenance in plastids, the CoIE1 ori for maintenance in E. coli and a spectinomcyin resistance gene (aadA) for selection in both systems. In addition, pNICERl carries a defective kanamycin resistance gene, kan*, to target the rescue of a functional kanamycin resistance gene, kan, from the recipient plastid genome. pNICERl was introduced into plastids where recombination could occur between the homologous kan/kan* sequences, and subsequently rescued in E. coli to recover the products of recombination. Based on the expression of kanamycin resistance in E. coli and the analysis of three restriction fragment polymorphisms, recombinant kan genes were recovered at a high frequency. Efficient rescue of kan from the plastid genome in E. coli indicates that NICE 1-based plasmids are suitable for rescuing mutations from any part of the plastid genome, expanding the repertoire of genetic tools available for plastid biology.     

新型酵母蛋白表位标记和基因敲除质粒系统的构建及可行性验证

【目的】构建一套用于酿酒酵母基因功能研究的质粒。该套质粒结合pUG系列和pFA6a系列的优点,同时采用同尾酶实现蛋白表位标签的串联插入。【方法】利用PCR技术分别克隆pUG系列质粒的lox P位点、pFA6a质粒多酶切位点和ADH1终止子模块;通过重组连接各片段,构建pCLHN-TRP和pCLHN-URA质粒。在此基础上利用同尾酶实现多种蛋白表位标签的单个或串联重复插入,获得一系列蛋白表位标记质粒。最后,以ATG1、COX4和NHX1为例验证本质粒系列的性能。【结果】在本项工作中,我们共构建2种基因敲除用质粒和17种表位标记用质粒(涵盖1-8 FLAG、1-12 V5、3-9 HA、2-8MYC、GFP和m Cherry)。在几个靶基因上的应用证实了本套质粒的实用性。尤其值得指出的是,通过组合采用不同重复度的串联表位标签,在同一张膜上同时检测表达差异极大的不同蛋白而不使高表达蛋白信号饱和成为可能。【结论】本文所构建的pCLHN质粒系列是对现有酵母质粒工具的有益补充。     

Mapping of the sor genes for l-sorbose degradation in the chromosome of Klebsiella pneumoniae

Summary A series of mutants was isolated in Klebsiella pneumoniae strain 1033, among them mutants unable to grown on l-sorbose. Different R' plasmids carrying the sor genes and other surrounding chromosomal genes were also isolated. Each plasmid contained the structural genes sorA for an Enzyme II of the phosphoenolpyruvate-dependent carbohydrate: phosphotransferase system, sorD for a d-glucitol 6-phosphate dehydrogenase, sorE for an l-sorbose 1-phosphate reductase, and the corresponding regulator gene sorR. These structural genes are coordinately expressed and inducible by l-sorbose. Cis-dominant and pleiotropic mutations rendering the expression of the sor genes constitutive or eliminating it were isolated. Complementation of a series of mutations in Escherichia coli K12 and K. pneumoniae by various R' and F' plasmids and by P1 transduction in K. pneumoniae located the sor genes within the following gene sequence: rbs rha pfkA metB ppc argH ilv btuB rpoB metA ace sor pgi malB uvrA. The rbs-ilv gene loci tightly linked in E. coli K12 at 84 min, are separated in the map of K. pneumoniae 1033 and located at 86 and 89 min, respectively.     

谷氨酸棒杆菌中基于CRISPR/Cas9的多位点碱基编辑系统的优化

作为新型的基因组编辑工具,碱基编辑技术结合了CRISPR/Cas系统的定位功能和碱基脱氨酶的编辑功能,可实现特定位点的碱基突变,具有不产生双链DNA断裂,无需外源模板且不依赖染色体DNA同源重组的优势.目前,研究者们已在重要的工业生产菌株谷氨酸棒杆菌(Corynebacterium glutamicum)中开发了多种碱...     

The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli

The CRISPR/Cas adaptive immune system provides resistance against phages and plasmids in Archaea and Bacteria. CRISPR loci integrate short DNA sequences from invading genetic elements that provide small RNA-mediated interference in subsequent exposure to matching nucleic acids. In Streptococcus thermophilus, it was previously shown that the CRISPR1/Cas system can provide adaptive immunity against phages and plasmids by integrating novel spacers following exposure to these foreign genetic elements that subsequently direct the specific cleavage of invasive homologous DNA sequences. Here, we show that the S. thermophilus CRISPR3/Cas system can be transferred into Escherichia coli and provide heterologous protection against plasmid transformation and phage infection. We show that interference is sequence-specific, and that mutations in the vicinity or within the proto-spacer adjacent motif (PAM) allow plasmids to escape CRISPR-encoded immunity. We also establish that cas9 is the sole cas gene necessary for CRISPR-encoded interference. Furthermore, mutation analysis revealed that interference relies on the Cas9 McrA/HNH- and RuvC/RNaseH-motifs. Altogether, our results show that active CRISPR/Cas systems can be transferred across distant genera and provide heterologous interference against invasive nucleic acids. This can be leveraged to develop strains more robust against phage attack, and safer organisms less likely to uptake and disseminate plasmid-encoded undesirable genetic elements.     

微嗜酸寡养单胞菌中的漆酶对黄曲霉毒素B1降解脱毒的生物活性

【目的】探究微嗜酸寡养单胞菌中的漆酶对AFB1的降解活性,并确定漆酶在菌株CW117降解代谢AFB1过程中的贡献。【方法】从微嗜酸寡养单胞菌基因组中,共筛选到两个漆酶基因lc1和lc2,并用大肠杆菌BL21外源表达蛋白rLC1和rLC2,在体外检测其对AFB1的降解活性。同时参考前人报道,研究了氧化性辅剂对漆酶AFB1降解的促进作用。在体外实验基础上,利用自杀质粒pK18mobsacB,以同源重组方法构建了两株漆酶缺失株CW117Δlc1和CW117Δlc1-lc2,验证了漆酶基因(lc1和lc2)对AFB1体内降解作用。【结果】体外实验显示,重组酶rLC1具有AFB1降解活性,氧化性辅剂ABTS、AS或SA可显著地提高rLC1降解活性,但rLC2未显示降解活性。突变株CW117Δlc1和CW117Δlc1-lc2对AFB1仍显示了较高的降解活性,且在大多数降解时间点与野生株CW117无显著差异。【结论】微嗜酸寡养单胞菌CW117菌株中,LC1在体外显示了AFB1的降解活性,且降解活性可以被氧化性辅助因子增强,LC2未显示体外降解活性;体内试验发现,漆酶基因lc1和lc2对菌株CW117降解AFB1的贡献较小,该菌株还存在其他降解途径。     

Kinds of mutations formed when a shuttle vector containing adducts of benzo[a]pyrene-7,8-diol-9,10-epoxide replicates in COS7 cells.

We have investigated the kinds of mutations induced when a shuttle vector containing covalently bound residues of the (+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE) replicates in the monkey kidney cell line COS7. The target for detecting mutations was the 200-base pair gene for a tyrosine suppressor tRNA (supF), inserted at the EcoRI site in shuttle vector p3AC (Sarkar et al., Mol. Cell. Biol. 4:2227-2230, 1984). When introduced by transformation, a functioning supF gene in progeny plasmid recovered from COS7 cells allows suppression of a lacZ amber mutation in the indicator Escherichia coli host. Treatment of p3AC with BPDE caused a linear increase in the number of BPDE residues bound per plasmid. Untreated plasmids and plasmids containing 6.6 BPDE residues were transfected into COS7 cells, and the progeny were assayed for mutations in the supF gene. The frequency of mutants generated during replication of the BPDE-treated plasmids was not higher than that from untreated plasmids, but the two populations differed markedly in the kinds of mutations they contained. Gel electrophoresis analysis of the size alterations of 77 mutant plasmids obtained with untreated DNA and 45 obtained with BPDE-treated DNA showed that the majority of the mutant progeny of untreated plasmids exhibited gross alterations, principally large deletions. In contrast, the majority of the mutants generated during replication of the BPDE-treated plasmids contained only minor alterations, principally point mutations. Sequence analysis of progeny of untreated plasmids containing putative point mutations showed insertions and deletions of bases and a broad spectrum of base substitutions; in those from BPDE-treated plasmids, all base substitutions involved guanosine . cystosine pairs.