丝状真菌遗传转化体系的研究进展

随着现代测序技术的飞速发展,主要丝状真菌的基因组序列相继公布,对丝状真菌基因水平的研究也不断深入,发展高效的遗传转化技术是真菌基因功能研究以及定向改良菌种的前提。近年来,尽管研究者不断优化并开发新的遗传转化方法,然而在丝状真菌中仍然存在转化效率较低、阳性转化子筛选困难等问题。适宜的转化方法和筛选标记对丝状真菌的遗传转化有至关重要的作用。该文综述了丝状真菌遗传转化系统的研究进展,主要从受体细胞的处理、外源基因的整合与表达、常用转化方法和阳性转化子的筛选鉴定等方面进行概述,旨在为今后丝状真菌遗传转化体系的构建提供思路。     

根癌农杆菌介导的球毛壳菌遗传转化及T-DNA插入突变体的获得

根癌农杆菌介导的遗传转化系统是植物基因工程常用方法,目前已将这一转化系统应用到酵母、丝状真菌以及人类细胞的转化。利用这一转化系统,成功地实现了丝状真菌球毛壳菌（Chaetomium globosum）的遗传转化,转化率约为60～180个转化子/10.7个孢子 。通过对转化子的PCR检测和Southern 杂交分析表明,TDNA已整合进毛壳菌基因组中,而且在所检测的转化子中都是以单拷贝的形式整合,转化子都能够稳定遗传。根癌农杆菌介导的遗传转化具有转化率高、低拷贝、遗传稳定、操作简便等优点,因此有可能成为丝状真菌遗传转化和功能基因组研究的有力工具。     

丝状真菌遗传转化系统的最新研究进展

随着大量丝状真菌基因组序列的公布,丝状真菌基因功能研究已然成为现今分子生物学的热点领域。真菌的遗传转化是研究基因功能的重要手段,研究者不断优化和开发出新的、高效的遗传转化方法,以满足更多真菌物种研究的需要。本综述总结了丝状真菌的遗传转化体系的最新研究进展,如遗传转化方法、选择标记的类型和转化系统的应用。     

一种用于PCR扩增的丝状真菌DNA快速提取方法

丝状真菌在工业、农业、医药以及基础生物学研究中具有重要作用。利用遗传转化技术对丝状真菌进行菌株改良和基因功能分析, 也越来越受到重视。然而, 丝状真菌DNA提取方法繁琐、费时, 难以满足利用PCR技术高通量筛选转化子的需要。本文以曲霉菌为例建立了一种快速提取丝状真菌DNA的实验方法, 微波处理置于10 × TE buffer中的菌丝即可得到DNA。RAPD试验和PCR扩增证明, 该方法提取的DNA能够达到PCR扩增的要求。研究结果为高通量快速筛选丝状真菌转化子奠定了基础。     

微生物遗传转化筛选标记及其生物安全性的研究进展

在分子生物技术中,筛选标记基因是遗传转化载体所必备的基本元件之一,其主要功能是在基因操作中进行目标克隆的筛选,以及在应用过程中通过选择压力维持基因重组性状。抗药基因是微生物遗传转化中常用的筛选标记,大肠杆菌载体和一般穿梭载体中通常带有抗药基因。带有抗药基因的工程菌可以被广泛地应用于酶和有机化学品的发酵生产,因为工业发酵过程是在封闭系统中进行的,并且最终产品需要经过提炼。但是当人们需要用基因改良的菌株进行食品和饲料加工、环境修复、病虫害生物防治时,抗药基因类筛选标记应该被禁止使用。因此,发展生物安全性筛选标记成为遗传转化技术推广应用中的一个技术关键。本文介绍常用作筛选标记的抗药基因,以及针对抗药基因的安全性问题而发展的无选择标记的遗传转化技术及生物安全性筛选标记的基因工程技术。葡萄糖胺合成酶基因是近年发展起来的新型生物安全性筛选标记,它弥补了其他营养缺陷互补型和功能附加型筛选标记的缺陷,具有广阔的应用前景。     

反馈抑制不敏感邻氨基苯甲酸合成酶基因作为筛选标记基因用于大豆遗传转化研究

目前广泛采用的抗菌素或抗除草剂基因作为植物转化筛选标记基因可能带来转基因逃逸,因此寻找能够用于植物转化的来源于植物本身的筛选基因是解决这一问题的方法之一。通过从烟草中克隆的邻氨基苯甲酸合成酶基因(ASA2)作为筛选标记基因,并采用氨基酸的类似物5—甲基色氨酸为筛选剂,进行了农杆菌介导的大豆成熟胚尖转化研究。Southern杂交结果表明ASA2基因成功整合到大豆基因组,Northern杂交也显示该基因在转化大豆叶片中表达。HPLC检测转化大豆叶片游离色氨酸的含量比野生型要高59％～123％。PCR检测转化子1代结果显示转化基因通过孟德尔规律稳定遗传。这些结果表明反馈抑制不敏感ASA2基因可以作为筛选标记基因用于大豆遗传转化。同时也证实来源于一种植物(烟草)编码的邻氨基苯甲酸α—亚基能够与另一种植物(大豆)编码该酶的β—亚基结合形成具有完整活性的邻氨基苯甲酸合成酶。对ASA2基因作为一种新的植物转化筛选标记基因的优缺点进行了讨论。     

反馈抑制不敏感邻氨基苯甲酸合成酶基因作为筛选标记基因用于大豆遗传转化研究

目前广泛采用的抗菌素或抗除草剂基因作为植物转化筛选标记基因可能带来转基因逃逸 ,因此寻找能够用于植物转化的来源于植物本身的筛选基因是解决这一问题的方法之一。通过从烟草中克隆的邻氨基苯甲酸合成酶基因 (ASA2 )作为筛选标记基因 ,并采用氨基酸的类似物 5-甲基色氨酸为筛选剂 ,进行了农杆菌介导的大豆成熟胚尖转化研究。Southern杂交结果表明ASA2基因成功整合到大豆基因组 ,Northern杂交也显示该基因在转化大豆叶片中表达。HPLC检测转化大豆叶片游离色氨酸的含量比野生型要高 5 9%～ 12 3%。PCR检测转化子 1代结果显示转化基因通过孟德尔规律稳定遗传。这些结果表明反馈抑制不敏感ASA2基因可以作为筛选标记基因用于大豆遗传转化。同时也证实来源于一种植物 (烟草 )编码的邻氨基苯甲酸α 亚基能够与另一种植物 (大豆 )编码该酶的 β 亚基结合形成具有完整活性的邻氨基苯甲酸合成酶。对ASA2基因作为一种新的植物转化筛选标记基因的优缺点进行了讨论     

一种拟南芥基因高效编辑体系研究

CRISPR/Cas9基因编辑系统操作简单易行,无需引入外源基因,生物安全性高。但怎样快速筛选获得不含外源转化元件的基因编辑后代是一个关键技术问题。本研究创造性的将拟南芥种皮特异性启动子At2S3与荧光筛选标记基因mCherry组装进植物基因组定点编辑CRISPR载体pHDE中,以拟南芥as1为靶基因,构建一种通过荧光标记筛选、实现转化后代中Cas9 Free的基因高效编辑体系。结果表明,通过同源重组方法构建的带有筛选标记的CRISPR载体与设计相符,外源插入片段正确。挑选转化后种皮上带有红色荧光标记的阳性种子培育得到T₁代植株,经PCR验证,成功获得as1定点敲除的纯合突变植株,纯合子比率达到40%;挑选T₁代纯合突变上不带荧光的种子,培育得到的T₂代植株中,PCR检测不到Cas9片段,实现了编辑后代的Cas9 Free。本研究构建的一种带有可视化筛选标记的基因高效编辑体系,成功实现编辑后代中无外源插入的Cas9等转化元件,生物安全性高,为基因组定点编辑技术在植物遗传资源改良中的高效利用提供了借鉴与参考。     

高等植物叶绿体基因组的转化

介绍了高等植物叶绿体基因组转化技术的原理和优点,外源基因导入叶绿体基因组的方法,外源基因与叶绿体基因组的整合及其表达,常用的叶绿体基因组转化的筛选标记基因及其去除的研究进展.     

基因组编辑技术在精准医学中的应用

基因组编辑技术的飞速发展,尤其是近年来CRISPR/Cas9基因组编辑体系的出现,使得研究人员能高效地在细胞系和动物模型中对基因组进行精确编辑。基于基因组编辑技术的各种实验研究平台被相继开发,包括通过在细胞系中引入疾病相关突变位点建立疾病模型,通过高通量筛选寻找导致肿瘤耐药性的突变基因,通过体内原位靶向致病基因并修改突变进行基因治疗等。这些基因组编辑技术研究平台极大推动了精准医学研究领域的发展。本文对基因组编辑技术在精准医学领域的基础研究、转化应用、目前存在的问题以及未来发展的方向进行了讨论。     

质体基因工程中选择标记基因研究进展

与细胞核基因工程相比,质体基因工程能更安全、精确和高效地对外源基因进行表达,作为下一代转基因技术已广泛用于基础研究和生物技术应用领域。与细胞核基因工程一样,质体基因工程中也需要合适的选择标记基因用于转化子的筛选和同质化,但基于质体基因组的多拷贝性和母系遗传特点,转化子的同质化需要一个长期的筛选过程,这就决定了质体基因工程中选择标记基因的选择标准将不同于细胞核基因工程中广泛使用的现行标准。目前,质体基因工程的遗传转化操作中使用较多的是抗生素选择标记基因,出于安全性考虑,需要找到可替换、安全的选择标记基因或有效的标记基因删除方法。本文在对质体基因工程研究的相关文献分析基础之上,对主要使用的选择标记基因及其删除体系进行了综述,并对比了其优缺点,同时探讨了质体基因工程中所使用的报告基因,以期为现有选择标记基因及其删除体系的改进和开发提供一定参考,进一步推动质体基因工程,尤其是单子叶植物质体基因工程的发展。     

CRISPR/Cas9介导的基因组定点编辑技术在丝状真菌中的研究进展

CRISPR/Cas9技术是在特定的RNA引导下,利用特异的核酸酶实现对基因组进行编辑的新技术。自2013年该技术体系建立起来已成功应用于动物、植物及真菌中。本文简述了3种基于核酸酶的基因编辑技术及其应用,概述了CRISPR/Cas9系统的组成及其作用机理,总结了CRISPR/Cas9在模式真菌酿酒酵母及丝状真菌中的应用,并就在丝状真菌中应用该技术时sg RNA表达盒的设计、Cas9表达盒的优化、抗性标记的筛选、受体的选择等方面提出具体的研究方法。另外,针对该技术应用过程中出现的脱靶效应、Cas9核定位信号的添加、启动子的选择及多个靶基因的编辑等问题提出了建议与展望,希望能够为初次涉足该领域的科研人员提供理论参考和技术支持。     

Elimination of marker genes from transformed filamentous fungi by unselected transient transfection with a Cre-expressing plasmid

A convenient method to remove selectable markers from fungal transformants permits the markers to be used for sequential transformations, and should also reduce public concerns and regulatory impediments to applications involving environmental release of genetically modified fungi. We report a method for marker removal that requires no genetic selection. Protoplasts from Neotyphodium coenophialum, Neotyphodium uncinatum and Epichloë festucae transformants containing a hygromycin B phosphotransferase gene (hph) flanked by loxP sites in direct orientation were transiently transfected with a Cre-recombinase expression plasmid, and then cultured without selection. The marker was eliminated in 0.5–2% of the colonies, leaving a single loxP sequence and no other exogenous DNA in the genome. This approach was also applied to the yA gene of Aspergillus nidulans as a laboratory exercise to demonstrate multiple principles of transformation and genome manipulation. Thus, the Cre-expression plasmid and transient transfection approach was rapid, flexible and useful for diverse filamentous fungi.     

A new dominant selection marker for transformation of Pichia pastoris to soraphen A resistance

Despite the considerable progress molecular genetics of filamentous fungi has made during the past decade, there is still an urgent need for efficiently working selectable markers for fungal transformation. Using Pichia pastoris as a host, we describe the development of a new dominant selectable marker of prokaryotic origin. This system, termed sor(R), is based upon the resistance of the bacterial enzyme acetyl-CoA carboxylase (ACCase) to the macrocyclic polyketide soraphen A, a potent inhibitor of fungal ACCase produced by the myxobacterium Sorangium cellulosum. In this study, we firstly demonstrate that the integration of a single sor(R) cassette into the P. pastoris genome confers resistance to elevated concentrations of soraphen A. Furthermore, it has been shown that the versatility of this marker can be considerably increased by splitting the sor(R) cassette, especially when successive transformations are performed on the same strain. As pronounced sensitivity to soraphen A is the rule among filamentous fungi, we expect the sor(R) marker to be a widely applicable tool for fungal transformation.     

Genetic transformation of filamentous fungi

Many filamentous fungi of all taxa can now be subject to DNA-mediated transformation. Many dominant selectable markers are available and the range available is increasing as new genes are cloned. Transformation is especially valuable in cloning genes defined by mutations with selectable phenotypes and is allowing investigation of many problems in fungi with good genetic systems. Increasingly sophisticated techniques for inactivating genes, targetingin vitro generated mutations to specific loci, and altering gene expression and its regulation are being developed. These approaches are being used to investigate the wealth of basic and applied biological problems available in filamentous fungi.     

CRISPR/Cas9在丝状真菌基因组编辑中的应用

丝状真菌(filamentous fungi)通常指那些菌丝体较发达且不产生大型肉质子实体结构的真核微生物。丝状真菌不仅在自然界物质循环中发挥着重要作用,还与人类健康和工农业生产有着紧密的联系。然而,对丝状真菌进行遗传操作相对困难,极大地妨碍了丝状真菌的遗传学研究。成簇的规律间隔的短回文重复序列及其相关系统(clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9, CRISPR/Cas9)是近年来发现的一种存在于细菌和古菌中保守的获得性免疫防御机制。最近,CRISPR/Cas9被开发成为了一种方便灵活的基因组编辑技术。目前,该技术已经广泛应用在不同物种的基因组编辑中。本文概述了CRISPR/Cas9在丝状真菌基因组编辑中的应用进展,旨在为开展该领域的研究工作提供参考。     

Transformation of Aspergillus sp. and Trichoderma reesei using the pyrithiamine resistance gene (ptrA) of Aspergillus oryzae

A pyrithiamine (PT) resistance gene (ptrA) was cloned from a PT resistant mutant of Aspergillus oryzae and was useful as a dominant selectable marker for transformation of all A. oryzae wild type strain as well as A. nidulans. For further study, we examined whether or not ptrA could be used as the transformation marker in other species of filamentous fungi. Two types of plasmid, which contain ptrA as a selectable marker, were constructed, and the transformation experiments were done with them. One is an integrative plasmid, pPTRI, and another is the autonomously replicating plasmid pPTRII, which contains AMA1. PT-resistant transformants were obtained in the cases of A. kawachii, A. terreus, A. fumigatus, and Trichoderma reesei as hosts with pPTRI and pPTRII. Furthermore, a beta-glucuronidase (GUS) gene was introduced into A. kawachii and A. fumigatus using pPTRII. Almost all the transformants turned blue on GUS assay plates. These results indicate that ptrA can also be used for some other filamentous fungi besides A. oryzae and A. nidulans.     

Efficient genome editing in filamentous fungi via an improved CRISPR-Cas9 ribonucleoprotein method facilitated by chemical reagents

DNA double-strand break (DSB) repair induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing. Direct genome editing via Cas9-CRISPR gRNA (guide RNA) ribonucleoprotein (RNP) complexes assembled in vitro has also been successful in some fungi. However, the efficiency of direct RNP transformation into fungal protoplasts is currently too low. Here, we report an optimized genome editing approach for filamentous fungi based on RNPs facilitated by adding chemical reagents. We increased the transformation efficiency of RNPs significantly by adding Triton X-100 and prolonging the incubation time, and the editing efficiency reached 100% in Trichoderma reesei and Cordyceps militaris. The optimized RNP-based method also achieved efficient (56.52%) homologous recombination integration with short homology arms (20 bp) and gene disruption (7.37%) that excludes any foreign DNA (selection marker) in T. reesei. In particular, after adding reagents related to mitosis and cell division, the further optimized protocol showed an increased ratio of edited homokaryotic transformants (from 0% to 40.0% for inositol and 71.43% for benomyl) from Aspergillus oryzae, which contains multinucleate spores and protoplasts. Furthermore, the multi-target engineering efficiency of the optimized RNP transformation method was similar to those of methods based on in vivo expression of Cas9. This newly established genome editing system based on RNPs may be widely applicable to construction of genome-edited fungi for the food and medical industries, and has good prospects for commercialization.     

Efficient PCR-based gene targeting with a recyclable marker for Aspergillus nidulans

The rapid accumulation of genomic sequences from a large number of eukaryotes, including numerous filamentous fungi, has created a tremendous scientific potential, which can only be realized if precise site-directed genome modifications, like gene deletions, promoter replacements, in-frame GFP fusions and specific point mutations can be made rapidly and reliably. The development of gene-targeting techniques in filamentous fungi and other higher eukaryotes has been hampered because foreign DNA is predominantly integrated randomly into the genome. For Aspergillus nidulans, we have developed a flexible method for gene-targeting employing a bipartite gene-targeting substrate. This substrate is made solely by PCR, which obviates the need for bacterial subcloning steps. The method reduces the number of false positives and can be used to produce virtually any genome alteration. A major advance of the method is that it allows multiple subsequent genome manipulations to be performed as the selectable marker is recycled.     

Tools for chloroplast transformation in Chlamydomonas: expression vectors and a new dominant selectable marker

Reverse-genetic studies of chloroplast genes in the green alga Chlamydomonas reinhardtii have been hampered by the paucity of suitable selectable markers for chloroplast transformation. We have constructed a series of vectors for the targeted insertion and expression of foreign genes in the Chlamydomonas chloroplast genome. Using these vectors we have developed a novel selectable marker based on the bacterial gene aphA-6, which encodes an aminoglycoside phosphotransferase. The aphA-6 marker allows direct selection for transformants on medium containing either kanamycin or amikacin. The marker can be used to inactivate or modify specific chloroplast genes, and can be used as a reporter of gene expression. The availability of this marker now makes possible the serial transformation of the chloroplast genome of Chlamydomonas. Received: 26 October 1999 / Accepted: 28 December 1999