根癌农杆菌在丝状真菌转化中的应用

原生质体转化法是丝状真菌转化的传统方法,但其过程繁琐且转化效率低。根癌农杆菌介导的转化方法原本是进行植物遗传转化的标准方法,但近年来发现该方法还可用于丝状真菌的转化。根癌农杆菌介导的丝状真菌转化具有操作简便、转化效率高、重复性好等优点,从而可以解决丝状真菌转化难的问题。在本文中,就其转化机制、特点和转化条件优化等方面的最新研究进展进行了综述。     

元基因组学及其在转化医学中的应用

微生物群落遍布于人体的每个角落,与人共生并对人体健康产生重要和深刻的影响。与人类共生的全部微生物的基因组总和称为“元基因组”或“人类第二基因组”。研究人体微生物群落及相关元基因组数据,对转化医学领域的基础研究和临床应用具有重要的价值。通过对生物医学相关的高通量元基因组数据进行分析,不仅能为基础医学研究向医学临床应用转化提供新思路和新方法,而且具有广阔的应用前景。基于新一代测序技术产生的数据,元基因组分析技术和方法能够弥补以往人体微生物先培养后鉴定方法的缺陷,同时能有效鉴定和分析微生物群落的组成及功能,从而进一步探究和揭示微生物群落与机体生理状态之间的关系,为解决许多医学领域的难题提供了全新的切入角度和思维方法。文章系统介绍了元基因组研究的现状,包括元基因组的方法概念和研究进展,并以元基因组在医学研究中的应用为着眼点,综述了元基因组在转化医学方面的研究进展,进一步阐述了元基因组研究在转化医学应用领域中具有的重要地位。     

超声波介导的微生物细胞转化

随着分子生物学的发展, 微生物遗传改造越来越广泛地应用在微生物育种、临床医学、环境保护等方面。其中, DNA转化技术经常是高效遗传改造的瓶颈之一。应用超声波将目的基因导入微生物细胞的技术具有原位、多尺度、活体、高通量、低成本等优点, 因此发展较为迅速。其原理是超声波可以通过声学气穴现象产生一系列的非热能效应, 而声学气穴微泡可产生短暂的细胞膜透化作用。本文综述了超声波转化的基本原理及其在微生物细胞转化中的发展现状, 并结合本实验室应用超声波转化法转化革兰氏阳性菌等研究进展, 分析了其特色、优势及现存挑战。     

变形链球菌电击转化条件的优化研究

目的为增强对变形链球菌的电击转化效率,探索常用的胞壁弱化剂甘氨酸在电击转化中的加入模式。方法以氨苄青霉素抗性的pGL3 basic质粒作为外源DNA,通过电击转化导入变形链球菌参考株UA159内,并在选择性培养基上筛选阳性转化克隆,以优化筛选出最佳的甘氨酸加入浓度与模式,同时比较了不同电击方案转化效率的差异。结果在变形链球菌对数生长期后加入终浓度为10%的甘氨酸可有效地增强电击转化的效率;而不同的电转电压的选择对于转化效率的影响,在本实验中差异未见显著性。结论研究证实了甘氨酸作为胞壁弱化剂可增强对变形链球菌的转化效率,并优化了对变形链球菌的电击转化方案。     

根癌农杆菌介导真菌遗传转化的研究及应用

根癌农杆菌介导转化法（Agrobacterium tumefaciens-mediated transformation,ATMT）具有转化效率高、遗传稳定、适用范围广等诸多优点,已成为真菌遗传转化研究中的强有力手段,在真菌基因资源开发、真菌性疾病研究和外源蛋白表达研究中发挥巨大作用。本文概述了根癌农杆菌转化法在真菌转化中的研究进展、技术优缺点、转化机制、实验方法和应用现状,着重介绍影响其转化效率的因素并对优化方法进行探讨,展望了该技术在真菌基因资源发掘、基因编辑等方面的应用前景,为今后真菌的遗传转化研究提供参考。     

微生物除臭剂发酵工艺条件优化及硫素转化的研究

利用酵母菌、乳酸菌、醋酸菌三种可食性微生物复配发酵制备微生物除臭剂,研究微生物复配比、发酵时间、发酵温度、接种量四个因素对H_2S去除率的影响。以单因素实验为基础,利用Box-Behnken响应面法优化最佳发酵条件,进一步研究硫元素转化及含量动态变化。结果表明酵母菌、乳酸菌、醋酸菌质量比为1∶2∶2时,各因素对H_2S去除率的影响由高到低依次为发酵温度发酵时间接种量,最优发酵条件为发酵时间48. 5 h、发酵温度30℃、接种量12. 75%,H_2S的去除率可达到71. 84%;实验组与对照组的硫元素转化及含量动态变化相比,实验组的SO_4~(2-)含量显著较高(P0. 05),H_2S释放量显著较低(P0. 05)说明该微生物除臭剂可以调节硫元素转化,有效抑制H_2S产生。     

鸭疫里默氏杆菌自然转化的发现及机制研究进展

自然转化(natural transformation)是微生物水平基因转移的一种重要机制,其在遗传多样性的产生或修复DNA损伤等方面发挥着重要作用,并且与耐药基因、毒力因子的扩散息息相关。鸭疫里默氏杆菌(Riemerellaanatipestifer,RA)是威克斯菌科中第一个被发现可以发生自然转化的细菌,本文作者利用此特点建立了多种基因编辑的方法,促进了对其遗传多样性和致病机理的研究进程。通过系统性地研究影响鸭疫里默氏杆菌自然转化的因素,鉴定出了参与该菌自然转化的营养物质;通过筛选转座子插入突变体文库,鉴定出了参与该过程的必需基因;最终,在该菌发现了一种新型的自然转化系统。本文结合其他细菌自然转化研究进展,针对以上研究结果进行综述,以期对该菌的自然转化机制有更深入的理解,也为更进一步探明该菌的耐药和毒力基因获得机制提供参考。     

农杆菌介导的马尔尼菲青霉基因转化技术的建立及优化

目的建立农杆菌介导的马尔尼菲青霉(PM)基因转化技术,并对该技术条件进行优化。方法以二元质粒p DHt/SK为载体,通过农杆菌介导将pyr G基因插入马尔尼菲青霉尿嘧啶缺陷株SPM4(pyr G-,nia D-)中,在不含尿嘧啶的培养基中筛选阳性转化子。运用PCR验证重组子。进一步对影响转化效率的农杆菌类型、共培养浓度、转化媒介、共培养温度、共培养时间、乙酰丁香酮(AS)等六个条件进行优化。结果 PCR验证pyr G基因成功的插入SPM4中,所得到转化子可稳定传代,通过条件优化,得到转化子约300个/106个细胞。选用AGL-1,以农杆菌共培养浓度为OD600=0.8,AS浓度为200μmol/L,无膜IM固体共培养基为介质,25℃共培养48 h为最适转化条件。结论成功建立了农杆菌介导PM基因转化技术,简化并优化了转化条件,该方法可用于PM基因功能研究。     

不同诱导方法对农杆菌介导的木霉菌遗传转化效率的影响

[目的]比较不同诱导方法对根癌农杆菌EHA105介导的哈茨木霉T88遗传转化体系转化效率的影响,以确定最佳的诱导方法.[方法]通过对复铺培养基法、转膜培养法和液体共培养法转化效率的比较来确定3种方法的优劣.[结果]复铺培养基法转化效率约为10个转化子/107个木霉孢子,转膜培养法转化效率约为20个转化子/107个木霉孢子,液体共培养法转化效率达到100个转化子/107个木霉孢子.[结论]在农杆菌介导的木霉菌遗传转化体系中,液体共培养法的转化效率最高,是常用的复铺培养基法及转膜培养法转化效率的5-10倍.     

两相体系中菌体转化法制备2-苯乙醇的研究

目的:利用酿酒酵母(Saccharomyces cerevisiae CICIMY008 6)菌体在油酸-水两 相体系中转化L-苯丙氨酸生成2-苯乙醇,以期解除产物抑制的同时降低萃取相油酸对转化的 不利影响,提高2-苯乙醇产量.方法:对2-苯乙醇的生成与菌体生长的关系进行考察,以确 定菌体转化法的可行性;通过单因素试验和正交设计试验获得转化培养基最佳配方;对菌体 转化条件进行优化.结果:向装液量为25mL/250mL转化培养基中加入0.6g 酵母湿菌体,30℃ 、100r/min条件下转化,9h加入等体积油酸,催化27h,产物浓度达4.55g/L.结 论:2-苯乙醇的制备可以使用菌体转化法,该法可在一定程度上克服两相转化体系中油酸的毒性影响.     

Transformation of primary rat kidney cells by DNA fragments of weakly oncogenic adenoviruses.

Primary cultures of baby rat kidney (BRK) cells were transformed by intact DNA and DNA fragments of weakly oncogenic human adenovirus types 3 and 7. The smallest fragment found to contain transforming activity was the left-terminal 4% endo R.HindIII fragment (for both adenovirus type 3 and 7 DNAs). The efficiency of transformation of this fragment was low, and no permanent cell line could be established. Left-terminal fragments ranging from 84 to 4,5% of the viral genome could all transform BRK cells with the same efficiency as intact viral DNA. A number of adenovirus type 7 DNA fragment-transformed lines were established and were found to contain persistent viral DNA sequences and adenovirus subgroup B-specific T antigen. Consequently, the transforming functions of adenovirus types 3 and 7 are located at the extreme left-hand end of the genome, and the minimum size for a DNA fragment with transforming activity is 1.0 X 10(6) daltons. These results do not rule out the possibility that viral genes located outside the transforming region may also influence transformation.     

Strategies for the development of bacterial transformation systems

An effective transformation system is a prerequisite for facile genetic manipulation of bacteria. Bacteria may be naturally competent for transformation or may be treated with various agents, such as Tris buffers or divalent metal ions, to induce competence. Transformation can also be accomplished by electroporation, or by fusion of protoplasts with PEG in the presence of transforming DNA. Unfortunately, the mechanism by which cells become permeable to DNA and the process by which DNA enters the cells is frequently unknown. In order to establish a transformation system for an untransformable bacterium, recipient strains and transforming DNA must be carefully selected. Since it is impossible to predict in advance which method of transformation will be successful with a particular bacterial strain, several techniques are usually evaluated. This review describes a number of factors that appear to be critical for developing a transformation system and presents a strategy for experimentation with novel bacteria.     

Transformation of primary rat kidney cells by fragments of simian virus 40 DNA.

Linear simian virus 40 (SV40) DNA molecules of genome length and DNA fragments smaller than genome length when prepared with restriction endonucleases and tested for transforming activity on primary cultures of baby rat kidney cells. The linear molecules of genome length (prepared with endonucleases R-EcoRI, R-BamHI, and R-HpaII or R-HapII), a 74% fragment (EcoRI/HpaII or HapII-A), and a 59% fragment (BamHI/HapII-A) could all transform rat kidney cells with the same efficiency as circular SV40 DNA. All transformed lines tested contained the SV40-specific T-antigen in 90 to 100% of the cells, which was taken as evidence that the transformation was SV40 specific. The DNA fragments with transforming activity contained the entire early region of SV40 DNA. Endo R-HpaI, which introduced one break in the early region, apparently inactivated the transforming capacity of SV40 DNA, since no transformation was observed with any of the three HpaI fragments tested. Attempts were made to rescue infectious virus from some of the transformed lines by fusion with permissive BSC-1 cells. Infectious virus was only recovered from the cells transformed by circular form I DNA. No infectious virus could be isolated from any of the other types of transformed cells.     

Homologous recombination between single-stranded DNA and chromosomal genes in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae strains was examined by using the URA3 and TRP1 genes cloned into M13 vectors in the absence of sequences capable of promoting autonomous replication. These constructs transform S. cerevisiae cells to prototrophy by homologous recombination with the resident mutant gene. Single-stranded DNA was found to transform S. cerevisiae cells at efficiencies greater than that of double-stranded DNA. No conversion of single-stranded transforming DNA into duplex forms could be detected during the transformation process, and we conclude that single-stranded DNA may participate directly in recombination with chromosomal sequences. Transformation with single-stranded DNA gave rise to both gene conversion and reciprocal exchange events. Cotransformation with competing heterologous single-stranded DNA specifically inhibited transformation by single-stranded DNA, suggesting that one of the components in the transformation-recombination process has a preferential affinity for single-stranded DNA.     

Transfection of Fibroblasts by Cloned Abelson Murine Leukemia Virus DNA and Recovery of Transmissible Virus by Recombination with Helper Virus

A cloned, permuted DNA copy of the Abelson murine leukemia virus (A-MuLV) genome was capable of eliciting the morphological transformation of NIH/3T3 fibroblasts when applied to cells in a calcium phosphate precipitate. The efficiency of the process was extremely low, yielding approximately one transformant per microgram of DNA under conditions which give 10(4) transfectants per microgram of other DNAs (e.g., Moloney sarcoma virus proviral DNA). The DNA was able to induce foci, even though the 3' end of the genome was not present. The transforming gene was thus localized to the 5' portion of the genome. The transformed cells all produced viral RNA and the virus-specific P90 protein. Transmissible virus could be rescued from these cells at very low frequencies by superinfection with helper virus; the rescued A-MuLV virus had variable 3' ends apparently derived by recombination with the helper. Dimerization of the permuted A-MuLV cloned genome to reconstruct a complete provirus did not improve transformation efficiency. Virus could be rescued from these transformants, however, at a high efficiency. Cotransfection of the permuted A-MuLV DNA with proviral M-MuLV DNA yielded a significant increase in the efficiency of transformation and cotransfection of dimeric A-MuLV and proviral M-MuLV resulted in a high-efficiency transformation yielding several thousand more transformants per microgram than A-MuLV DNA alone. We propose that helper virus efficiently rescues A-MuLV from transiently transfected cells which would not otherwise have grown into foci. We hypothesize that multiple copies of A-MuLV DNA introduced into cells by transfection are toxic to cells. In support of this hypothesis, we have shown that A-MuLV DNA sequences can inhibit the stable transformation of cells by other selectable DNAs.     

Transformation of Streptococcus pneumoniae relies on DprA- and RecA-dependent protection of incoming DNA single strands

Seventy-five years after the discovery of transformation with Streptococcus pneumoniae, it is remarkable how little we know of the proteins that interact with incoming single strands in the early processing of transforming DNA. In this work, we used as donor DNA in transformation a radioactively labelled homologous fragment to examine the fate of the single-stranded (ssDNA) products of uptake in cells mutant for DprA or RecA, two proteins essential for transformation. Fifteen minutes after uptake, the labelling of specific chromosomal restriction fragments that demonstrated homologous integration in the wild type was not detected in dprA or recA cells, indicating that in the mutants incoming ssDNA could not be processed into recombinants. Investigation of the fate of donor label 1 min after uptake revealed that incoming ssDNA was immediately degraded in the absence of DprA or RecA. Our results demonstrate that incoming ssDNA requires active protection prior to the RecA-driven search for homology and that both DprA and RecA are needed for this protection.     

Requirement for the C-terminal region of middle T-antigen in cellular transformation by polyoma virus

Deletions of polyoma virus DNA around the region that codes for the C-terminus of the viral middle T-antigen were created using a transforming fragment (BamH I/EcoR I) of viral DNA cloned in the plasmid vector pAT153. These species were recloned and assayed for their ability to transform Rat-1 cells in culture. Our results showed that whereas the DNA sequence between the presumed translational termination codon for the viral middle T-antigen and the single viral EcoR I site could be removed with no apparent effect on transformation, the removal of the termination codon itself or any amino acid coding sequences of this protein caused a drastic decrease in the transforming ability of the DNA. Transfection of Rat-1 cells with plasmids that contained viral DNA with deletions which corresponded to the last fourteen or more amino acids of the middle T-antigen never gave rise to cellular transformation.     

Transformation by human adenoviruses

When, approximately 10 years ago, it was shown that the functions essential for cell transformation were localized in a small region of the adenovirus genome, a DNA segment which at that time was thought to be capable of encoding two or three average-sized proteins at most, it seemed reasonable to hope that an understanding of the mechanisms by which adenoviruses transform cells might be quickly achieved. While such optimism might be forgiven, it was quite clearly naive in the extreme. As a consequence of mRNA splicing and the use of overlapping reading frames the number of proteins encoded within E1 is 2-3-times greater than would have been predicted a decade ago, and post-translational modifications may add another dimension of complexity. In fact it has taken nearly all of the past decade just to identify the proteins encoded in E1 and to characterize them in the most rudimentary way. However, we have now entered a period in which new information is accumulating at an extremely rapid rate as a result of several major technical and fundamental advances. Chief among these are the use of recombinant DNA techniques, particularly site-directed mutagenesis, which combined with methods for introducing mutations made in cloned sequences back into infectious virus, clearly represents a powerful approach to studying the functions of transforming proteins. In addition, the ability to express transforming proteins in bacteria and to produce large amounts of highly purified proteins which previously were only just detectable in infected and transformed cells is a major breakthrough. Advances in immunological techniques, particularly the development of monoclonal antibodies and antisera against synthetic peptides, have enormously simplified the task of detecting and characterizing E1 proteins. Finally, recent results suggesting that adenovirus transforming proteins may be functionally and structurally similar to other oncogenes brings a new perspective to the study of oncogenic transformation. Have all the proteins involved in transformation by adenoviruses been identified? It seems probable that all those virally coded proteins which play a major role are now known but of course minor players in the cast could still be waiting in the wings. We have pointed out that viral functions encoded outside region E1 may have some importance at least in initiation of transformation by virions and have speculated on the possibility that one or more of these may be involved in the integration of viral DNA into the host cell chromosome.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic transformation of Saccharomyces cerevisiae with single-stranded circular DNA vectors

We asked if single-stranded vector DNA molecules could be used to reintroduce cloned DNA sequences into a eukaryotic cell and cause genetic transformation typical of that observed using double-stranded DNA vectors. DNA was presented to Saccharomyces cerevisiae following a standard transformation protocol, genetic transformants were isolated, and the physical state of the transforming DNA sequence was determined. We found that single-stranded DNA molecules transformed yeast cells 10- to 30-fold more efficiently than double-stranded molecules of identical sequence. More cells were competent for transformation by the single-stranded molecules. Single-stranded circular (ssc) DNA molecules carrying the yeast 2 μ plasmid-replicator sequence were converted to autonomously replicating double-stranded circular (dsc) molecules, suggesting their efficient utilization as templates for DNA synthesis in the cell. Single-stranded DNA molecules carrying 2 μ plasmid non-replicator sequences recombined with the endogenous multicopy 2 μ plasmid DNA. This recombination yielded either the simple molecular adduct expected from homologous recombination (40% of the transformants examined) or aberrant recombination products carrying incomplete transforming DNA sequences, endogenous 2 μ plasmid DNA sequences, or both (60% of the transformants examined). These aberrant recombination products suggest the frequent use of a recombination pathway that trims one or both of the substrate DNA molecules. Similar aberrant recombination products were detected in 30% of the transformants in cotransformation experiments employing single-stranded and double-stranded DNA molecules, one carrying the 2 μ plasmid replicator sequence and the other the selectable genetic marker. We conclude that single-stranded DNA molecules are useful vectors for the genetic transformation of a eukaryotic cell. They offer the advantage of high transformation efficiency, and yield the same intracellular DNA species obtained upon transformation with double-stranded DNA molecules. In addition, single-stranded DNA molecules can participate in a recombination pathway that trims one or both DNA recombination substrates, a pathway not detected, at least at the same frequency, when transforming with double-stranded DNA molecules     

In vitro methylation of specific regions of the cloned Moloney sarcoma virus genome inhibits its transforming activity.

The transforming activity of cloned Moloney sarcoma virus (MSV) proviral DNA was inhibited by in vitro methylation of the DNA at cytosine residues, using HpaII and HhaI methylases before transfection into NIH 3T3 cells. The inhibition of transforming activity due to HpaII methylation was reversed by treatment of the transfected cells with 5-azacytidine, a specific inhibitor of methylation. Analysis of the genomic DNA from the transformed cells which resulted from the transfection of methylated MSV DNA revealed that the integrated MSV proviral DNA was sensitive to HpaII digestion in all cell lines examined, suggesting that loss of methyl groups was necessary for transformation. When cells were infected with Moloney murine leukemia virus at various times after transfection with methylated MSV DNA, the amount of transforming virus produced indicated that the loss of methyl groups occurred within 24 h. Methylation of MSV DNA at HhaI sites was as inhibitory to transforming activity as methylation at HpaII sites. In addition, methylation at both HpaII and HhaI sites did not further reduce the transforming activity of the DNA. These results suggested that; whereas methylation of specific sites on the provirus may not be essential for inhibiting the transforming activity of MSV DNA, methylation of specific regions may be necessary. Thus, by cotransfection of plasmids containing only specific regions of the MSV provirus, it was determined that methylation of the v-mos gene was more inhibitory to transformation than methylation of the viral long terminal repeat.