酵母基因中断技术

酵母基因中断技术是研究酵母基因功能的重要手段,自80年代初诞生以来经历了不断的改进和发展.PCR介导的酵母基因中断技术,大大简化了操作,实现了酵母基因的精确缺失;酵母基因的多重中断技术,可在酵母内实现多个基因的中断;可进行大规模基因中断和功能分析的酵母基因中断技术,适应了在酵母全基因组测序完成的情况下进行功能基因组学研究的要求.酵母基因中断技术对人类基因功能研究也有很大启示作用.     

酵母基因中断技术研究进展

综述酵母基因中断技术（gene disruption）自创立以来的不断改进与发展。随着分子生物学技术的更新,酵母基因中断技术已经实现了目标基因的精确缺失、多重中断、无痕中断等,已成为功能基因组学研究和酵母分子育种研究的重要手段之一。     

基因敲除技术研究进展

基因中断技术是研究基因功能和实现基因的精确缺失重要手段.原核中断系统最近发展较快的是利用λ噬菌体的Red系统在细茵中进行的基因敲除技术,但多在大肠杆菌中进行;真核中断系统属PCK介导的酵母基因中断技术和RNAi干涉技术发展最快.这几种中断技术都大大简化了实验操作,缩短了实验时间,在研究生物基因组中未知基因及蛋白质的功能等领域具有诱人的应用前景.     

酵母菌Y12667 ERG11基因删除及验证研究

采用同源重组的方法删除酵母茵Y12667内源ERG11基因.并用PCR、Western、细胞计数和GC-MS的方法分别在基因水平、蛋白水平、细胞水平和功能代谢水平进行验证.结果表明,本研究成功删除了酵母茵Y12667内源ERG11基因,该基因删除茵能稳定表达外源性白念珠茵ERG11基因的同源蛋白.因此,该基因删除茵可作为下一步靶酶蛋白功能研究的基因工程表达操作茵.     

人源抗狂犬病毒糖蛋白稳定型抗体精氨酸密码子突变株的构建及其在巴斯德毕赤酵母中的分泌表达

目的：对人源抗狂犬病毒糖蛋白（GPRV）单链二硫键稳定抗体（ScdsFv）进行精氨酸密码子修饰，实现其在酵母中的分泌表达，并检测其生物学活性。方法：参照巴斯德毕赤酵母偏好密码子，对抗GPRV ScdsFv原核表达基因进行密码子修饰，并通过点基因融合技术构建ScdsFv重组酵母表达基因，连接pPIC9K构建重组表达质粒pPIC9K-ScdsFv，电转化毕赤酵母GS115，经筛选后进行甲醇诱导表达。结果：SDS-PAGE及Western blot检测到重组表达质粒在30℃经甲醇诱导表达的蛋白相对分子质量为30000；荧光抗体试验验证ScdsFv能靶向结合GPRV；MTT试验说明ScdsFv能中和狂犬病毒，具有一定的细胞保护作用。结论：重组ScdsFv在酵母系统中可以有效表达。具有较好的生物学活性。     

植物逆境胁迫耐受性功能基因组研究进展

为了更加高效地利用基因工程技术提高植物对逆境胁迫的耐受性,需要在全基因组水平上对植物逆境胁迫耐受性的复杂机制进行整合性研究.植物逆境胁迫耐受性功能基因组的研究可概括为：利用胁迫特异性的表达序列标签(EST)及cDNA微阵列（或基因芯片）技术筛选与胁迫相关的候选基因,然后利用反向遗传学等技术对候选基因的功能进行研究,利用酵母双杂交、正向遗传学等技术对基因及基因产物间的相互关系进行研究.通过这些研究可以全面地了解植物对胁迫（渗透、干旱、极端温度）响应的复杂机制和相互作用以及相应的信号转导途径,从而为更加高效地利用基因工程技术提高植物对逆境胁迫的耐受性奠定基础.     

毕赤酵母细胞工厂工程化改造与应用

毕赤酵母作为细胞工厂在小分子代谢产物发酵和蛋白制品生物合成中扮演着重要角色，具有极其重要的工业应用价值。随着CRISPR/Cas9等新型编辑工具的开发和应用，对毕赤酵母细胞工厂进行多基因高效率的工程化改造已成为可能。本文首先对毕赤酵母工程化改造的遗传操作技术和目标方向进行了归纳总结，其次介绍了毕赤酵母作为细胞工厂的应用现状，同时探讨了毕赤酵母细胞工厂的优点及缺陷，并对其发展方向作出展望；以期为未来的毕赤酵母工程化改造研究提供参考和启示，推动毕赤酵母细胞工厂在生物产业中的创新应用。     

厚藤cDNA文库的构建和重金属镉耐受相关基因的筛选

厚藤（Ipomoea pes-caprae（L.） Sweet）是一种具有重要生态、观赏及药用价值的沙滩植物，对重金属镉（Cd）具有一定的富集能力，可作为Cd污染滨海地区的修复植物进行引种栽植和利用。本研究通过Gateway技术构建厚藤的cDNA文库，将该文库质粒转化酵母对Cd敏感的突变株ycf1△，采用全长cDNA过表达基因捕获系统（FOX）筛选厚藤重金属Cd胁迫耐受相关基因，并采用酵母互补实验进行基因的功能验证。本研究获得了2个能够恢复ycf1△对Cd敏感表型的重组质粒，经测序分析，该重组质粒包含的cDNA全长序列分别对应厚藤植物螯合肽合成酶基因（phytochelatin synthase）和金属硫蛋白基因（metallothionein），分别将其命名为IpPCS和IpMT，通过功能分析，初步认定该基因为编码Cd耐受和解毒相关蛋白的候选基因。     

酱香型白酒发酵过程中核心酵母的鉴别及其功能

【背景】酵母是酱香型白酒发酵过程中最重要的微生物,但酵母群落中核心酵母的种类和功能尚不清晰。【目的】通过探索酱香型白酒发酵微生物群落中核心酵母的种类和功能,为揭示酱香型白酒酿造机制以及提升白酒品质提供理论支撑。【方法】联合使用未培养(内转录间隔区扩增子和宏转录组高通量测序技术)和可培养(菌种筛选和模拟发酵实验)技术对酱香型白酒发酵过程中核心酵母的结构和功能进行定性和定量分析。【结果】内转录间隔区扩增子和宏转录组测序结果显示,酱香型白酒发酵过程中涉及10个属的酵母微生物,其中在发酵过程中平均相对丰度大于0.2%的酵母有13种,有2种核心酵母分别为库德威兹氏毕赤酵母(Pichia kudriavzevii)和粟酒裂殖酵母(Schizosaccharomyces pombe)。在发酵过程中,P. kudriavzevii占总量的89%以上,Schi. pombe表达了占总量21%以上的功能基因。模拟发酵实验结果显示P. kudriavzevii在酵母群落中发挥耐受乳酸积累的作用,而Schi. pombe在酵母群落中发挥耐受乙醇积累的作用,这两种作用保证了酱香型白酒发酵过程中酵母群落的结构和功能的稳定性。【结论】P. kudriavzevii和Schi. pombe是酱香型白酒发酵过程中的优势酵母,这两种酵母在生产中发挥了各自不同的作用。本研究进一步加深了核心酵母对于整个生产过程贡献的认识,有助于研究者认识到白酒发酵过程中对核心微生物进行调控的重要性。     

产甘油假丝酵母胞浆3-磷酸甘油脱氢酶基因（CgGPD）功能分析

【目的】从高产甘油生产菌株产甘油假丝酵母（Candida glycerinogenes）基因组中克隆了NAD+依赖3-磷酸甘油脱氢酶编码基因（CgGPD），但是该基因及其上游调控序列具体的功能还是未知的。本文研究了CgGPD基因及其上游调控序列的功能。【方法】本文以酿酒酵母（Saccharomyces cerevisiae）及其渗透压敏感型突变株为宿主，构建3种不同的酵母表达载体导入酵母细胞，研究了不同酵母转化子在渗透压胁迫条件下CgGPD基因表达对细胞的耐高渗透压胁迫应答及其细胞的甘油合成能力的影响。【结果】实验结果表明无论是以来源于S. cerevisiae 的TPI启动子还是来源于CgGPD基因的启动子，过量表达CgGPD基因的转化子均能够显著加速葡萄糖消耗速度和提高甘油合成能力，在gpd1/gpd2突变株中表达CgGPD基因能够消除细胞对外界高渗透压的敏感性，同时转化子胞内甘油大量积累。【结论】CgGPD基因在野生型酵母S. cerevisiae W303-1A表达显著提高细胞的甘油合成能力，在gpd/1gpd2突变株中能够互补GPD1基因的功能，CgGPD基因表达受渗透压诱导 调控。     

Genome editing of different strains of Aureobasidium melanogenum using an efficient Cre/loxp site-specific recombination system

It has been well known that different strains of Aureobasidium spp. can produce commercial pullulan, polymalate, liamocin, intracellular lipids, gluconic acid, siderophore, melanin and various enzymes. In order to fully elucidate their synthetic pathways and regulation, it is necessary to have an efficient gene editing system for genetic modification of Aureobasidium spp. In this study, an efficient Cre/loxp site-specific recombination system (pAMGDloxp-1, pAMEXlox-1 and pAMCRE1) was constructed. It was found that they could be successfully used to sequentially delete and express many genes in different strains of A. melanogenum. After each round of gene disruption and expression, over 0.5 positive cells per 1000 competent cells and over 49.8 positive transformants per 1.0 μg DNA were achieved. After each round of the antibiotics gene excision by using the Cre-loxp site-specific recombination, over 95.4 % of the antibiotics-resistant cells became sensitive to both hygromycin B and nourseothricin again. This demonstrated that the Cre/loxp site-specific recombination system constructed in this study can efficiently be used to simultaneously delete and express many genes in different strains of A. melanogenum. These systems are promising approaches for the easily modifying genomics of the yeast-like fungal strains with enhanced metabolic pathways through multicopy gene deletion and expression.     

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan^r resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5⁺ from Schizosaccharomyces pombe and the dominant resistance marker ble^r from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP–marker gene–loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble^r as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.     

黑曲霉htmA基因的分离鉴定及其功能分析

本研究通过分析比较黑曲霉基因组与人、哺乳动物和酿酒酵母基因组序列同源性,首次分离鉴定了黑曲霉htmA基因,该基因长3459bp,编码1083个氨基酸。已知真核生物的htmA基因编码一种类α-甘露糖苷酶I的非必须蛋白HTMA,在内质网中参与降解非正确折叠的糖蛋白,htmA基因的破坏会延迟非正确折叠糖蛋白的降解。为分析htmA基因在黑曲霉中的功能,运用同源重组技术敲除黑曲霉基因组中的htmA基因,获得htmA基因缺失突变菌株,并进行了缺失株外源漆酶分泌能力的检测。结果表明黑曲霉htmA基因的破坏延缓了外源漆酶的降解,由此推测黑曲霉htmA基因编码蛋白HTMA具有与酵母、哺乳动物HTM1P/EDEM类似的功能作用。     

The importance of being essential: EMBRYO-DEFECTIVE genes in Arabidopsis

With the completion of the sequence of the first bacterial genomes, scientists have been able to address the question: How many genes are required for cell viability? In attempting to reply to this question, the concept of the minimal gene set was developed and validated by systematic gene disruption. In a similar manner, whole genome comparisons and systematic Knock-Out have been performed in eukaryotes and have led to the identification to date of the set of essential genes in yeast and C. elegans. In the plant kingdom, the sequence of the Arabidopsis genome together with large-scale functional genomics programs now allow us to address the question of essentiality in Arabidopsis. These concerted efforts have resulted in the identification to date of up to 219 genes essential for seed development (EMBRYO-DEFECTIVE, EMB, genes). With this basic knowledge, we can start a valid comparison of essentiality in Arabidopsis and in other eukaryotes based on functional categories and orthologous relationships. Furthermore, the function of the EMB genes in the particular context of eukaryote evolution driven by whole genome duplications and selective gene loss will be discussed.     

A Method for Gene Disruption That Allows Repeated Use of USR3 Selection in the Construction of Multiply Disrupted Yeast Strains

In this paper, we describe a 3.8-kb molecular construct that we have used to disrupt yeast genes. The construct consists of a functional yeast URA3 gene flanked by 1.1-kb direct repeats of a bacterial sequence. It is straightforward to insert the 3.8-kb segment into a cloned target gene of interest and then introduce the resulting disruption into the yeast genome by integrative transformation. An appropriate DNA fragment containing the disruption plus flanking homology can be obtained by restriction enzyme digestion. After introducing such fragments into yeast by transformation, stable integrants can be isolated by selection for Ura+. The important feature of this construct that makes it especially useful is that recombination between the flanking direct repeats occurs at a high frequency (10(-4)) in vegetatively grown cultures. After excision, only one copy of the repeat sequence remains behind. Thus in the resulting strain, the Ura+ selection can be used again, either to disrupt a second gene in similar fashion or for another purpose.     

Mutations in the CDP-choline pathway for phospholipid biosynthesis bypass the requirement for an essential phospholipid transfer protein

SEC14p is the yeast phosphatidylinositol (PI)/phosphatidylcholine (PC) transfer protein, and it effects an essential stimulation of yeast Golgi secretory function. We now report that the SEC14p localizes to the yeast Golgi and that the SEC14p requirement can be specifically and efficiently bypassed by mutations in any one of at least six genes. One of these suppressor genes was the structural gene for yeast choline kinase (CKI), disruption of which rendered the cell independent of the normally essential SEC14p requirement. The antagonistic action of the CKI gene product on SEC14p function revealed a previously unsuspected influence of biosynthetic activities of the CDP-choline pathway for PC biosynthesis on yeast Golgi function and indicated that SEC14p controls the phospholipid content of yeast Golgi membranes in vivo.     

Streptococcus pneumoniae as a genomics platform for broad-spectrum antibiotic discovery

Streptococcus pneumoniae is a useful tool for the discovery of broad-spectrum antibiotics because of its genetic malleability and importance as a pathogen. Recent publications of complete chromosomal DNA sequences for S. pneumoniae facilitate rapid and effective use of genomics-based technology to identify essential genes encoding new targets for antibacterial drugs. These methods include computational comparative genomics, gene disruption studies to determine essentiality or identify essential genes, and gene expression analysis using microarrays and gel-based proteomics. We review how genomics has transformed the use of the pneumococcus for the pursuit of new antibiotics, and made it the best species for the identification and validation of new antibiotic targets.     

Learning Gene Networks under SNP Perturbations Using eQTL Datasets

The standard approach for identifying gene networks is based on experimental perturbations of gene regulatory systems such as gene knock-out experiments, followed by a genome-wide profiling of differential gene expressions. However, this approach is significantly limited in that it is not possible to perturb more than one or two genes simultaneously to discover complex gene interactions or to distinguish between direct and indirect downstream regulations of the differentially-expressed genes. As an alternative, genetical genomics study has been proposed to treat naturally-occurring genetic variants as potential perturbants of gene regulatory system and to recover gene networks via analysis of population gene-expression and genotype data. Despite many advantages of genetical genomics data analysis, the computational challenge that the effects of multifactorial genetic perturbations should be decoded simultaneously from data has prevented a widespread application of genetical genomics analysis. In this article, we propose a statistical framework for learning gene networks that overcomes the limitations of experimental perturbation methods and addresses the challenges of genetical genomics analysis. We introduce a new statistical model, called a sparse conditional Gaussian graphical model, and describe an efficient learning algorithm that simultaneously decodes the perturbations of gene regulatory system by a large number of SNPs to identify a gene network along with expression quantitative trait loci (eQTLs) that perturb this network. While our statistical model captures direct genetic perturbations of gene network, by performing inference on the probabilistic graphical model, we obtain detailed characterizations of how the direct SNP perturbation effects propagate through the gene network to perturb other genes indirectly. We demonstrate our statistical method using HapMap-simulated and yeast eQTL datasets. In particular, the yeast gene network identified computationally by our method under SNP perturbations is well supported by the results from experimental perturbation studies related to DNA replication stress response.     

Double-joint PCR: a PCR-based molecular tool for gene manipulations in filamentous fungi

Gene replacement via homologous double crossover in filamentous fungi requires relatively long (preferentially >0.5 kb) flanking regions of the target gene. For this reason, gene replacement cassettes are usually constructed through multiple cloning steps. To facilitate gene function studies in filamentous fungi avoiding tedious cloning steps, we have developed a PCR-assisted DNA assembly procedure and applied it to delete genes in filamentous fungi. While the principle of this procedure is essentially the same as other recently reported PCR-based tools, our technique has been effectively used to delete 31 genes in three fungal species. Moreover, this PCR-based method was used to fuse more than 10 genes to a controllable promoter. In this report, a detailed protocol for this easy to follow procedure and examples of genes deleted or over-expressed are presented. In conjunction with the availability of genome sequences, the application of this technique should facilitate functional characterization of genes in filamentous fungi. To stream line the analysis of the transformants a relatively simple procedure for genomic DNA or total RNA isolation achieving approximately 100 samples/person/day is also presented.