斜卧青霉高效基因打靶系统的构建与转录调控因子CreA功能的研究
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摘要
斜卧青霉是一种丝状子囊真菌,可产生大量的植物细胞壁降解酶类。由于斜卧青霉能将可再生的纤维素生物质转化为可发酵的糖类,因此在木质纤维素物质水解和转化方面具有重要的应用价值,近年来已经被作为工业生产菌株用于生产纤维素、半纤维素酶类。为了提高纤维酶的产量,经典的诱变策略及培养条件的优化已应用于斜卧青霉菌株改造,如通过对斜卧青霉114-2菌株进行紫外线诱变筛选得到抗代谢物阻遏突变株JU-A10。尽管斜卧青霉菌株改良已经取得了明显的进展并克隆得到了部分与纤维素和半纤维素水解相关的基因,但斜卧青霉高产纤维素酶的分子机制还不清楚。最近,斜卧青霉基因组测序工作已基本完成。因此,开发与功能基因组学研究相关的工具为进一步开展斜卧青霉相关研究,具有重要的意义。
丝状真菌木聚糖酶的产生均受葡萄糖阻遏(碳源代谢产物阻遏)控制,例如曲霉和木霉,其编码纤维素酶和半纤维素酶的基因受到碳源的统一调控。纤维素和其它一些碳源可诱导纤维素酶和半纤维素酶基因的表达,而葡萄糖可抑制这些基因的表达。到目前为止,已分离获得了一些编码纤维素酶、半纤维素酶基因表达调控因子的基因,包括介导代谢产物阻遏的CREA基因。研究表明,纤维素酶和半纤维素酶编码基因的调控机制呈现出明显的相似性,但也具有许多不同之处。在相关研究最为深入的两种菌黑曲霉和瑞氏木霉中,这些基因的调控也表现出很多不一致的方面,但在分子水平对斜卧青霉纤维素酶基因表达调控尚没有报道。
本论文克隆了斜卧青霉与人的异源整合途径关键蛋白KU70同源的基因pku70,敲除斜卧青霉pku70基因获得了△pku70菌株,构建了斜卧青霉高效基因敲除平台,并在此基础上对斜卧青霉CreA调控因子的功能和纤维素酶调控机理进行了初步的分析。论文实验结果为进行斜卧青霉基因功能高通量分析以及菌株的定向改造奠定了良好的基础。
本论文的主要研究结果如下：
1斜卧青霉pku70基因的克隆与分析
根据已公布的ku70基因序列,设计简并引物,以斜卧青霉114-2染色体为模板进行扩增,获得了长1.36-kb的序列。将扩增得到的序列测序,发现与曲霉属ku70基因具有较高的同源性，初步认定为斜卧青霉ku70基因序列并命名为pku70。根据已扩增的pku70基因部分序列设计引物,采用SELF-PCR技术分别扩增,获得了长3.2-kb的上述pku70部分序列的5’端上游序列和长2.8-kb的3’端下游序列。将克隆得到的全长pku70基因序列提交NCBI进行核酸比对,分析结果表明斜卧青霉pku70基因编码区长2047-bp,含3个内含子,分别位于559-611、1459-1518和1623-1693位点之间。该pku70基因所编码蛋白质长620个氨基酸,分子量为68.9 kDa。BLASTP分析结果表明斜卧青霉PKU70蛋白质与Neosartoryn fischeri、A.clavatus、P.chrysogenum.A.sojae、A.terreus和A. nidulans的KU70蛋白分别具有72%、71%、70%、69%、68%和67%的相似度。将PKU70蛋白质序列进行保守结构域分析,分析结果表明PKU70蛋白质具有3个不同的结构域：KU70／KU80 N-末端结构域(第1-193位氨基酸),KU70核心结构域(第238-516位氨基酸)和C-末端结构域(第573-607位氨基酸)。
2斜卧青霉高效同源重组系统的构建
为了建立高效的基因打靶系统同时确定pku70在斜卧青霉NHEJ途径中的作用,进一步克隆了pku70上下游同源区序列。以ptrA为筛选标记,利用Double-jointPCR方法构建了pku70敲除盒。将获得的6.4-kb△pku70：：ptrA敲除盒转化斜卧青霉菌株114-2原生质体,获得了pku70基因被敲除的△pku70突变株PKU70-42。
由于KU蛋白(KU70／80)参与修补DNA双链断裂的NHEJ途径过程,因此对该基因的敲除是否影响△pku70菌株的营养生长、生孢率、对化学试剂的敏感性、渗透压耐受性以及纤维素酶产生等进行了检测。试验结果表明,△pku70突变株的生长速率、菌丝形态、分生孢子的产生以及孢子的萌发速率等均与出发菌株相同。△pku70突变株对生化试剂EMS、潮霉素B或者H2O2的敏感性与出发菌株114-2相比未表现出差别,对渗透压的敏感性与出发菌株1 14-2相比亦未出现明显的差异。
为验证pku70的敲除对同源重组效率的影响,随机选取斜卧青霉creA和xlnR基因进行基因敲除试验。对斜卧青霉creA和xlnR基因功能的研究目前还未见文献报道,其中,xlnR基因序列还未克隆。本文首先克隆得到斜卧青霉xlnR基因全序列以及creA基因编码区侧翼序列。根据克隆得到的creA和xlnR基因序列,构建了△creA::hph和△xlnR::hph敲除盒,并和构建好的敲除盒分别转化斜卧青霉114-2菌株以及△pku70突变株PKU70-42。以PKU70-42菌株为受体所进行的creA和xlnR基因敲除试验结果表明,pku70基因的敲除可明显提高外源转化片段在斜卧青霉中的基因同源重组效率,△creA::hph以及△xlnR:hph敲除盒在△pku70突变株中的同源重组效率均达到100%。△pku70突变株的获得对于高通量分析斜卧青霉基因功能具有重要的作用,高效同源重组系统的建立有利于利用分子手段进行斜卧青霉菌株的遗传改良以及功能基因组学的研究。同时,creA和xlnR基因的敲除对进一步研究斜卧青霉的碳源代谢机制也有重要的意义。
3斜卧青霉转化片段整合类型的分析
为研究外源转化片段在斜卧青霉染色体中的整合类型,用△pku70::ptrA,△creA::hph以及△xlnR::hph敲除盒分别转化114-2野生型菌株和△pku70突变株PKU70-42,对筛选得到的转化子进行Southern blot分析。分析结果表明：在PKU70-42菌株中,pku70基因的敲除造成外源转化片段不能进行异源整合而只能以同源重组的形式整合到细胞染色体中。而在野生型菌株114-2中,外源片段同时发生异源整合和同源整合的情况占转化子总数的6.2%(5／81),只发生异源整合的情况占转化子总数的32.1%(26／81),只发生同源整合的转化子占转化子总数的61.7%(50／81),其中存在多拷贝异源整合片段的情况占转化子总数的19.8%(16／81)。
4斜卧青霉△pku70突变株筛选标记的置换
为扩展△pku70菌株的利用范围,构建了△pku70::hph基因敲除盒。△pku70：：hph敲除盒与△pku70：：ptrA敲除盒携带有相同的pku70两侧同源区。将构建好的△pku70：：hph敲除盒转化斜卧青霉△pku 70：：ptrA突变株,在潮霉素平板上筛选转化子。将筛选获得的转化子分生孢子分别点接于不含任何抗生素的MM平板,含抗硫胺素的平板以及含有潮霉素的平板。转化子在抗硫胺素平板上不生长,而在含有潮霉素的平板上可以正常生长。该结果表明在这些转化子中均发生了外源转化片段的同源双交换。△pku70：：hph突变株的获得,不仅进一步验证了pku70的敲除可以明显提高斜卧青霉外源转化片段的同源重组效率,而且也扩展了△pku70突变株的适用范围,有利于利用△pku70突变株能进行高效基因打靶这一特点进行更加方便的遗传操作。
5斜卧青霉共转化的初步研究
为了检测在同一转化实验操作中,两种不同基因的敲除盒是否可以同时在相应基因位点发生同源双交换,我们构建了△acel：：dsRed2敲除盒,并将构建好的△acel：：dsRed2敲除盒与△pku70：：ptrA敲除盒以摩尔数1：1的比例共转化斜卧青霉1 14-2菌株。发现在获得的转化子中仅有7%的转化子发生了共转化。为了验证在高同源重组环境中是否有利于两种不同基因的共敲除,将△acel：：dsRed2敲除盒与△pku70：：ptrA敲除盒一起转化斜卧青霉△pku70：：hph菌株,结果未发现有共转化现象发生。随机挑取在抗硫胺素平板上生长的9株转化子,提取染色体DNA,进行Southern blot验证分析。试验结果表明,在所有选取的转化子中,转化的△pku 70：：ptrA敲除盒均在染色体pku70：：hph位点发生同源双交换,而在染色体的其位点未发现△pku70：：ptrA转化片段的异源重组情况。此结果初步表明,在斜卧青霉中,两种不同敲除盒线性片段的共转化效率很低,特别是在以△pku70突变株为受体菌时,难以进行两种敲除盒的共转化试验。因此我们认为在斜卧青霉中,很难或者不能用两种不同的基因敲除盒进行共敲除／共转化实验,其原因尚待探讨。
6斜卧青霉114-2creA功能的分析
为分析验证斜卧青霉creA基因的功能,在获得AcreA突变株后,进一步进行了基因回补试验。构建creA基因回补盒并将其转入AcreA突变株,筛选获得了creA回复菌株creA-4。通过对回补转化子creA-4,△creA突变株以及野生型菌株1 14-2进行分析发现creA的敲除很可能影响多条生长代谢途径从而使该突变株菌落的形态与野生型相比发生了明显的改变。CreA的缺失不仅导致纤维素酶相关基因葡萄糖阻遏效应的解除,而且使AcreA突变株在有葡萄糖存在的情况下仍具有较高的纤维素酶基因表达,进而在较大程度上提高了纤维素酶的产量,同时也说明CreA在斜卧青霉中对纤维素酶基因表达起负调控作用。以乳糖作为唯一碳源培养AcreA菌株的试验结果表明,creA基因的敲除不影响菌株对乳糖的利用,AcreA菌株在乳糖培养基上生长正常。在乳糖和纤维素底物共同存在的情况下首先利用乳糖。试验结果表明乳糖对纤维素酶基因表达没有诱导作用或者诱导作用不明显。在creA基因缺失的情况下,乳糖的存在不影响纤维素底物对纤维素酶的诱导作用。同时还发现,CreA的缺失对斜卧青霉术聚糖酶、内切葡聚糖酶以及滤纸酶活的提高均有明显的促进作用,但对外切葡聚糖纤维二糖水解酶和β-葡聚糖苷酶的产生影响不明显。
7斜卧青霉JU-A10抗代谢物阻遏的分析
斜卧青霉JU-A10是野生型菌株114-2经过多轮诱变得到的一株抗葡萄糖代谢阻遏的突变株。该菌在葡萄糖存在的情况下仍能高效地表达纤维素酶。经测序比对发现,在A10菌株creA基因的+1205位点缺失一碱基′C′,从而造成该基因的移码突变。为研究A10中突变的CreA调控因子的作用,分别对A10菌株的creA基因进行了敲除与基因回补纠错试验。研究结果表明,creA基因在+1205位点碱基“C”的缺失造成了CreA调控因子的部分功能失活,但该突变的调控因子仍具有一定的活性,因此所产生的对葡萄糖效应的解阻遏作用是不完全的。但在敲除了A10菌株的creA基因后所得到的A10△creA突变株中,葡萄糖阻遏效应被完全解除,其纤维素酶和半纤维素酶产量有所提高。在研究中我们发现,菌株JU-A10的乳糖代谢途径很可能发生了变化,这种变化非常明显地对该菌体的营养生长产生了抑制作用,并且很可能减少了乳糖代谢对CreA调控因子的依赖。
以上研究结果对在转录水平了解斜卧青霉葡萄糖阻遏机制、纤维素酶系基因表达调控机理,以及进一步的纤维素酶系优化提供了理论支持,并为进一步进行斜卧青霉菌株定向遗传改造提供了实验数据和奠定了一定的基础。
Penicillium decumbens is a filamentous ascomycete fungus isolated from soil and has capacity to produce large amounts of cellulases and hemicellulases. Due to its high efficiency in enzymatic conversion of renewable cellulosic biomass to fermentable sugars, P. decumbens is becoming particularly attractive for the study of hydrolysis of lignocellulose. To improve the production of biomass-hydrolyzing enzymes, classical mutagenesis strategies and cultivation conditions optimization have been applied for P. decumbens. For example, the strain P. decumbens A10, as a catabolite repression-resistant mutant, was obtained from P. decumbens 114-2 by UV mutagenesis. Recently, genome shuffling was used to further enhance cellulase production by repeating protoplast fusions. Although the strain improvement in this fungus appears to be well established and several genes involved in cellulose and hemicellulose hydrolysis have been cloned (GenBank:EU239661.1; EU239662.1, etc.), the molecular mechanism for the enhancement of cellulase activity is not known. Especially, the P. decumbens genome sequencing project will be completed shortly, thus allowing the development of genome-wide knockouts. Therefore, to develop new molecular tools and novel technologies for functional genomic studies in P. decumbens is becoming increasingly urgent.
Gene targeting is one of the most important approaches for comprehensive understanding of gene function. Using this strategy, target gene can be displaced with its deletion cassette, and a preexisting genomic mutation can also be corrected to wild-type with its endogenous allele. Unfortunately, the high frequency of illegitimate integration of transforming DNA has hampered gene targeting approaches in most filamentous fungi. In eukaryotes, the repair of DNA double-strand breaks (DSB) works through two main recombination pathways. One is the homologous recombination (HR) pathway, which involves interactions between homologous DNA sequences. Another is the nonhomologous end-joining (NHEJ) pathway, which involves direct ligation of DNA ends independent of sequence homology. The NHEJ pathway has been described as consisting of several components, including Ku heterodimer (Ku70/Ku80), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Xrcc4-DNA ligase IV complex. The model organism Saccharomyces cerevisiae preferentially utilizes the HR pathway in DSB repair although having NHEJ pathway. In contrast to S. cerevisiae, filamentous fungi mainly utilize the NHEJ pathway of DSB repair and exogenous DNA can be integrated anywhere in the genome. Inhibition of the NHEJ pathway in these fungi is hypothesized to force the transforming DNA to integrate into the genome via HR. Therefore, disruption of Ku70 homologue in P. decumbens is postulated to enhance the gene targeting efficiency and facilitate functional genomic studies.
The transcriptional repressor CreA is the main regulatory element that has been proposed to participate in cellulase and xylanase gene expression. CreA is a negatively acting protein that mediates carbon catabolite repression in Aspergilli and Trichoderma. Despite the fact that there is a similar repression mechanism in the Aspergilli and Trichoderma, some distinct modes of CreA regulation exit in cellulose and xylan degradation.
In this study, the pku70 gene homologous to A. nidulans ku70 was cloned and identified. The pku70 targeting cassette was obtained by PCR-based fusion technique, and the ability to undergo HR was assessed in pku70-defective strain compared to the wild-type parental strain at two independent loci. Subsequently, Functional analysis of CreA in wild-type strain 114-2 and derepression phenotype in mutant A10 were performed in P. decumbens.
1 Isolation and analysis of putative pku 70 gene
A 1.36-kb sequence with similarity to A. nidulans ku70 gene was amplified in P. decumbens, on the basis of the three different ortholog sequences of human Ku70 from Aspergillus. Based on the obtained partial sequence, the 3.2-kb and 2.8-kb up- and downstream regions of this sequence were obtained by SEFA PCR. BLAST analysis of these amplified sequences revealed that they contain a full-length open reading frame (ORF) consisting of a 2047-bp. This predicated ku 70 gene, named pku70, has three introns which are located at nucleotides 559 to 611,1459 to 1518 and 1623 to
The P. decumbens pku70 gene encodes a protein of 620 amino acids (aa) with a predicated molecular mass of 68.9 kDa. BLASTP analysis showed that the Pku70 protein shares 72,71,70,69,68 and 67% identity with the respective Ku70 proteins of Neosartorya fischeri, A. clavatus, P. chrysogenum, A. sojae, A. terreus and A. nidulans. The predicted Pku70 protein sequence was submitted to the Conserved Domain Search service. The analysis revealed that Pku70 protein containes three distinct domains: Ku70/Ku80 N-terminal (vWA-ku) domain (aa 1 to 193), Ku70-core domain (aa 238 to 516) and SAP-domain (aa 573 to 607). The N-terminal domain is not involved in DNA binding but may very likely mediate Ku heterodimer interactions with other proteins involved in DSB repair process. The Ku70-core domain is constructed from a core of sevenβ-strands arranged in an antiparallel fashion, and it provides an relatively flat surface for DNA binding. The SAP-domain is a putative DNA-binding motif involved in chromosomal organization.
2 Development of a highly efficient gene targeting system in Penicillium decumbens To establish highly efficient gene targeting system and confirm the function of pku70 involved in NHEJ pathway in P. decumbens,pku70 targeting cassette was constructed with a pyrithiamine resistance gene ptrA as selectable marker. The 6.4-kb△pku70::ptrA targeting cassette was transformed into P. decumbens wild-type strain 114-2. The coding sequence of the pku 70 locus was removed from the genome of the strain PKU70-42. We therefore concluded that a single gene replacement occurred at the resident pku70 locus, indicating that the pku70 gene had been disrupted in the strain PKU70-42.
Since Ku proteins (Ku70/80) are involved in NHEJ repair of DSBs in eukaryotes, the△pku70 strain was examined for phenotypic characterization with respect to vegetative growth, germination rate, sensitivity to mutagen toxicity and osmotic stress, as well as cellulose hydrolysis. Plate assay showed that the growth rate of the△pku70 strain PKU70-42 was identical to that of the wild-type strain on MM. No irregularities in mycelial morphology and vegetative growth were observed in the strain PKU70-42, and the sporulation and spore germination rates of the△pku70 strain were not affected. The sensitivity to mutagen toxicity was determined with plate assay using MM plates independently supplemented with different amounts of EMS, hygromycin B and H2O2. The strain PKU70-42 showed no sensitivity differences in comparison with the wild-type strain. Sensitivity to osmotic stress was examined with spot tests using conidiospore suspension. In all cases, sensitivities to osmotic stress were not significantly different between the wild-type strain and the△pku70 strain PKU70-42. Additionally, in order to effectively utilize△pku70 background for creating deletions in genes regarding cellulose hydrolysis, cellulase activity was assayed and no significant differences were detected between△pku70 and wild-type genetic backgrounds of P. decumbens. These results indicated that the Apku70 strain is a suitable host for gene function analysis.
The effect of the disruption of pku70 on homologous gene targeting was evaluated by the deletion of two randomly selected genes (creA and xlnR) in a Apku70 strain. The functions of both genes have not been elucidated in P. decumbens. The homolog of creA gene in A. nidulans encodes a negatively-acting protein, which mediates carbon catabolite repression, and the homolog of xlnR gene in Hypocrea jecorina encodes a transcriptional activator that governs cellulolytic and xylanolytic gene expression. Disruption of creA and xlnR genes by gene targeting may facilitate further study of the important regulatory machinery of carbon metabolism in P. decumbens.
The△creA::hph cassette contained 3.5-kb 5'and 3.0-kb 3'of the target creA gene and the△xlnR::hph cassette harbored 1.8-kb and 1.9-kb up- and downstream genomic sequences of xlnR. Deletion of the pku70 gene in P. decumbens clearly increased gene targeting frequencies. The frequency of gene targeting was 100% when flanking regions longer than 1.8-kb were used. In the parental strain, however, gene targeting frequencies were 33% (3/9) and 91% (32/35) at creA and xlnR loci, respectively. These results suggested that gene targeting in△pku 70 was effective at various loci.
3 Analysis of integration patterns P. decumbens
Previous studies have shown that ectopic integration events are usually more frequent than homologous integration when foreign DNA is integrated into the fungal genome. In the three independent gene-targeting experiments in P. decumbens wild-type strain, relatively high frequencies of homologous integration were found at xlnR, creA and pku70 loci. Possible factors influencing the homologous integration include the conformation of transforming DNA, length of homology and the recipent strain. Conformation of transforming DNA (circular or linear) can significantly affect gene targeting and transformation frequency. In addition, we discovered that complex integration patterns were existed only in a minority of the transformants, in which homologous integration of targeting cassette was accompanied by additional ectopic integration events. In most transformants foreign DNA was either ectopically or homologously integrated into the genome. Thus it seemed to be a strong incompatibility or competition between two main classes of recombination mechanisms for transforming DNA. On average, approximately 61.5% (16/26) of nontargeted cells incorporated two or multiple copies of targeting cassette integrated randomly into the genome, compared with 9.4% (5/53) of the targeted cells incorporated more than one copy of transforming DNA due to ectopic integration. These results indicated that the nontargeted cells were more likely to incorporate a second fragment by ectopic integration than were targeted cells in P. decumbens.
4 Marker exchanging in the△pku70::ptrA strain
To adequately validate the△pku70 genetic background, the P. decumbens△pku70::hph strain was constructed by exchanging selectable markers in Apku70::ptrA strain with hph expression cassettes. The△pku70::hph targeting cassette was created and transformed into the P. decumbens△pku70::ptrA strain PKU70-42 as recipient. Sixteen randomly selected transformants were subjected to PCR analysis and plate assay. All transformants obtained ultimately became resistant to hygromycin B and sensitive to pyrithiamine. These results indicated that allelic exchange between the two pku70 targeting cassettes successfully occurred at the pku70::ptrA locus by homologous integration in strain PKU70-42.
5 Analysis of cotransformation in P. decumbens
To determine whether two loci could be independently targeted within a single transformation experiment by using mixtures of two different targeting cassettes, the△pku70::ptrA and△ace1::dsRed2 cassettes were constructed and cotransformed into the wild-type and Apku70::hph strains, respectively. In the first case, cotransformants were found at a frequency of approximately one per thirteen PyrR transformants, and the△ace1::dsRed2 cassette was integrated ectopically at unknown sites in the genome of wild-type strain. The remainder of PyrR transformants showed no cotransforming△ace1::dsRed2 cassette anywhere. In parallel experiments with the same targeting cassettes, thirty-eight PyrR transformants were obtained using the△pku70::hph strain as recipient. However, all of them did not contain cotransforming△ace1::dsRed2 fragment. The analysis confirmed that the hph marker in△pku70::hph strain was replaced by the ptrA expression cassette without additional integration of△pku70::ptrA fragment into genome. Collectively, these data demonstrated that cotransformation with two linear targeting cassettes occurred at a relatively low frequency in P. decumbens, especially in△pku70 strain as recipient. We therefore concluded that simultaneous targeting of independent genes is not a frequent occurrence during cotransformation of two different targeting cassettes.
6 Functional analysis of CreA in P. decumbens
In order to analyze the function of the CreA protein, we produced total deletion of creA produced by using in vitro techniques and△creA strain was complemented by another creA allele. In P. decumbens strain 114-2, deletions of the creA gene lead to derepression of cellulase and xylanase production even in the medium containing the D-glucose. The results showed that the regulator CreA, as a carbon catabolite repressor, mediates the repression of genes encoding cellulase and hemicellulase. Our results also indicated that lactose has no obvious effect on induction of cellulase production.
7 Analysis of derepression phenotype in P. decumbens A10
The known mutation in creA confers a derepressed phenotype in P. decumbens A10. A mutant was constructed in which a total deletion of creA produced by using in vitro techniques in P. decumbens A10. We also produced a strain the creA was corrected by another creA allele cloned from wild-type strain 114-2. In the rectified strain, the phenotype for catabolite repression can be rescued by transformation with the wild-type creA allele. Information from plate analysis of creA mutants, combined with information derived from analysis of rectified strain, allow us to determine that CreA lead to partial derepression and retain some activity in P.decumbens A10, since a total deletion has much more derepression phenotype in medium containing glucose.
丝状真菌木聚糖酶的产生均受葡萄糖阻遏(碳源代谢产物阻遏)控制,例如曲霉和木霉,其编码纤维素酶和半纤维素酶的基因受到碳源的统一调控。纤维素和其它一些碳源可诱导纤维素酶和半纤维素酶基因的表达,而葡萄糖可抑制这些基因的表达。到目前为止,已分离获得了一些编码纤维素酶、半纤维素酶基因表达调控因子的基因,包括介导代谢产物阻遏的CREA基因。研究表明,纤维素酶和半纤维素酶编码基因的调控机制呈现出明显的相似性,但也具有许多不同之处。在相关研究最为深入的两种菌黑曲霉和瑞氏木霉中,这些基因的调控也表现出很多不一致的方面,但在分子水平对斜卧青霉纤维素酶基因表达调控尚没有报道。
本论文克隆了斜卧青霉与人的异源整合途径关键蛋白KU70同源的基因pku70,敲除斜卧青霉pku70基因获得了△pku70菌株,构建了斜卧青霉高效基因敲除平台,并在此基础上对斜卧青霉CreA调控因子的功能和纤维素酶调控机理进行了初步的分析。论文实验结果为进行斜卧青霉基因功能高通量分析以及菌株的定向改造奠定了良好的基础。
本论文的主要研究结果如下：
1斜卧青霉pku70基因的克隆与分析
根据已公布的ku70基因序列,设计简并引物,以斜卧青霉114-2染色体为模板进行扩增,获得了长1.36-kb的序列。将扩增得到的序列测序,发现与曲霉属ku70基因具有较高的同源性，初步认定为斜卧青霉ku70基因序列并命名为pku70。根据已扩增的pku70基因部分序列设计引物,采用SELF-PCR技术分别扩增,获得了长3.2-kb的上述pku70部分序列的5’端上游序列和长2.8-kb的3’端下游序列。将克隆得到的全长pku70基因序列提交NCBI进行核酸比对,分析结果表明斜卧青霉pku70基因编码区长2047-bp,含3个内含子,分别位于559-611、1459-1518和1623-1693位点之间。该pku70基因所编码蛋白质长620个氨基酸,分子量为68.9 kDa。BLASTP分析结果表明斜卧青霉PKU70蛋白质与Neosartoryn fischeri、A.clavatus、P.chrysogenum.A.sojae、A.terreus和A. nidulans的KU70蛋白分别具有72%、71%、70%、69%、68%和67%的相似度。将PKU70蛋白质序列进行保守结构域分析,分析结果表明PKU70蛋白质具有3个不同的结构域：KU70／KU80 N-末端结构域(第1-193位氨基酸),KU70核心结构域(第238-516位氨基酸)和C-末端结构域(第573-607位氨基酸)。
2斜卧青霉高效同源重组系统的构建
为了建立高效的基因打靶系统同时确定pku70在斜卧青霉NHEJ途径中的作用,进一步克隆了pku70上下游同源区序列。以ptrA为筛选标记,利用Double-jointPCR方法构建了pku70敲除盒。将获得的6.4-kb△pku70：：ptrA敲除盒转化斜卧青霉菌株114-2原生质体,获得了pku70基因被敲除的△pku70突变株PKU70-42。
由于KU蛋白(KU70／80)参与修补DNA双链断裂的NHEJ途径过程,因此对该基因的敲除是否影响△pku70菌株的营养生长、生孢率、对化学试剂的敏感性、渗透压耐受性以及纤维素酶产生等进行了检测。试验结果表明,△pku70突变株的生长速率、菌丝形态、分生孢子的产生以及孢子的萌发速率等均与出发菌株相同。△pku70突变株对生化试剂EMS、潮霉素B或者H2O2的敏感性与出发菌株114-2相比未表现出差别,对渗透压的敏感性与出发菌株1 14-2相比亦未出现明显的差异。
为验证pku70的敲除对同源重组效率的影响,随机选取斜卧青霉creA和xlnR基因进行基因敲除试验。对斜卧青霉creA和xlnR基因功能的研究目前还未见文献报道,其中,xlnR基因序列还未克隆。本文首先克隆得到斜卧青霉xlnR基因全序列以及creA基因编码区侧翼序列。根据克隆得到的creA和xlnR基因序列,构建了△creA::hph和△xlnR::hph敲除盒,并和构建好的敲除盒分别转化斜卧青霉114-2菌株以及△pku70突变株PKU70-42。以PKU70-42菌株为受体所进行的creA和xlnR基因敲除试验结果表明,pku70基因的敲除可明显提高外源转化片段在斜卧青霉中的基因同源重组效率,△creA::hph以及△xlnR:hph敲除盒在△pku70突变株中的同源重组效率均达到100%。△pku70突变株的获得对于高通量分析斜卧青霉基因功能具有重要的作用,高效同源重组系统的建立有利于利用分子手段进行斜卧青霉菌株的遗传改良以及功能基因组学的研究。同时,creA和xlnR基因的敲除对进一步研究斜卧青霉的碳源代谢机制也有重要的意义。
3斜卧青霉转化片段整合类型的分析
为研究外源转化片段在斜卧青霉染色体中的整合类型,用△pku70::ptrA,△creA::hph以及△xlnR::hph敲除盒分别转化114-2野生型菌株和△pku70突变株PKU70-42,对筛选得到的转化子进行Southern blot分析。分析结果表明：在PKU70-42菌株中,pku70基因的敲除造成外源转化片段不能进行异源整合而只能以同源重组的形式整合到细胞染色体中。而在野生型菌株114-2中,外源片段同时发生异源整合和同源整合的情况占转化子总数的6.2%(5／81),只发生异源整合的情况占转化子总数的32.1%(26／81),只发生同源整合的转化子占转化子总数的61.7%(50／81),其中存在多拷贝异源整合片段的情况占转化子总数的19.8%(16／81)。
4斜卧青霉△pku70突变株筛选标记的置换
为扩展△pku70菌株的利用范围,构建了△pku70::hph基因敲除盒。△pku70：：hph敲除盒与△pku70：：ptrA敲除盒携带有相同的pku70两侧同源区。将构建好的△pku70：：hph敲除盒转化斜卧青霉△pku 70：：ptrA突变株,在潮霉素平板上筛选转化子。将筛选获得的转化子分生孢子分别点接于不含任何抗生素的MM平板,含抗硫胺素的平板以及含有潮霉素的平板。转化子在抗硫胺素平板上不生长,而在含有潮霉素的平板上可以正常生长。该结果表明在这些转化子中均发生了外源转化片段的同源双交换。△pku70：：hph突变株的获得,不仅进一步验证了pku70的敲除可以明显提高斜卧青霉外源转化片段的同源重组效率,而且也扩展了△pku70突变株的适用范围,有利于利用△pku70突变株能进行高效基因打靶这一特点进行更加方便的遗传操作。
5斜卧青霉共转化的初步研究
为了检测在同一转化实验操作中,两种不同基因的敲除盒是否可以同时在相应基因位点发生同源双交换,我们构建了△acel：：dsRed2敲除盒,并将构建好的△acel：：dsRed2敲除盒与△pku70：：ptrA敲除盒以摩尔数1：1的比例共转化斜卧青霉1 14-2菌株。发现在获得的转化子中仅有7%的转化子发生了共转化。为了验证在高同源重组环境中是否有利于两种不同基因的共敲除,将△acel：：dsRed2敲除盒与△pku70：：ptrA敲除盒一起转化斜卧青霉△pku70：：hph菌株,结果未发现有共转化现象发生。随机挑取在抗硫胺素平板上生长的9株转化子,提取染色体DNA,进行Southern blot验证分析。试验结果表明,在所有选取的转化子中,转化的△pku 70：：ptrA敲除盒均在染色体pku70：：hph位点发生同源双交换,而在染色体的其位点未发现△pku70：：ptrA转化片段的异源重组情况。此结果初步表明,在斜卧青霉中,两种不同敲除盒线性片段的共转化效率很低,特别是在以△pku70突变株为受体菌时,难以进行两种敲除盒的共转化试验。因此我们认为在斜卧青霉中,很难或者不能用两种不同的基因敲除盒进行共敲除／共转化实验,其原因尚待探讨。
6斜卧青霉114-2creA功能的分析
为分析验证斜卧青霉creA基因的功能,在获得AcreA突变株后,进一步进行了基因回补试验。构建creA基因回补盒并将其转入AcreA突变株,筛选获得了creA回复菌株creA-4。通过对回补转化子creA-4,△creA突变株以及野生型菌株1 14-2进行分析发现creA的敲除很可能影响多条生长代谢途径从而使该突变株菌落的形态与野生型相比发生了明显的改变。CreA的缺失不仅导致纤维素酶相关基因葡萄糖阻遏效应的解除,而且使AcreA突变株在有葡萄糖存在的情况下仍具有较高的纤维素酶基因表达,进而在较大程度上提高了纤维素酶的产量,同时也说明CreA在斜卧青霉中对纤维素酶基因表达起负调控作用。以乳糖作为唯一碳源培养AcreA菌株的试验结果表明,creA基因的敲除不影响菌株对乳糖的利用,AcreA菌株在乳糖培养基上生长正常。在乳糖和纤维素底物共同存在的情况下首先利用乳糖。试验结果表明乳糖对纤维素酶基因表达没有诱导作用或者诱导作用不明显。在creA基因缺失的情况下,乳糖的存在不影响纤维素底物对纤维素酶的诱导作用。同时还发现,CreA的缺失对斜卧青霉术聚糖酶、内切葡聚糖酶以及滤纸酶活的提高均有明显的促进作用,但对外切葡聚糖纤维二糖水解酶和β-葡聚糖苷酶的产生影响不明显。
7斜卧青霉JU-A10抗代谢物阻遏的分析
斜卧青霉JU-A10是野生型菌株114-2经过多轮诱变得到的一株抗葡萄糖代谢阻遏的突变株。该菌在葡萄糖存在的情况下仍能高效地表达纤维素酶。经测序比对发现,在A10菌株creA基因的+1205位点缺失一碱基′C′,从而造成该基因的移码突变。为研究A10中突变的CreA调控因子的作用,分别对A10菌株的creA基因进行了敲除与基因回补纠错试验。研究结果表明,creA基因在+1205位点碱基“C”的缺失造成了CreA调控因子的部分功能失活,但该突变的调控因子仍具有一定的活性,因此所产生的对葡萄糖效应的解阻遏作用是不完全的。但在敲除了A10菌株的creA基因后所得到的A10△creA突变株中,葡萄糖阻遏效应被完全解除,其纤维素酶和半纤维素酶产量有所提高。在研究中我们发现,菌株JU-A10的乳糖代谢途径很可能发生了变化,这种变化非常明显地对该菌体的营养生长产生了抑制作用,并且很可能减少了乳糖代谢对CreA调控因子的依赖。
以上研究结果对在转录水平了解斜卧青霉葡萄糖阻遏机制、纤维素酶系基因表达调控机理,以及进一步的纤维素酶系优化提供了理论支持,并为进一步进行斜卧青霉菌株定向遗传改造提供了实验数据和奠定了一定的基础。
Penicillium decumbens is a filamentous ascomycete fungus isolated from soil and has capacity to produce large amounts of cellulases and hemicellulases. Due to its high efficiency in enzymatic conversion of renewable cellulosic biomass to fermentable sugars, P. decumbens is becoming particularly attractive for the study of hydrolysis of lignocellulose. To improve the production of biomass-hydrolyzing enzymes, classical mutagenesis strategies and cultivation conditions optimization have been applied for P. decumbens. For example, the strain P. decumbens A10, as a catabolite repression-resistant mutant, was obtained from P. decumbens 114-2 by UV mutagenesis. Recently, genome shuffling was used to further enhance cellulase production by repeating protoplast fusions. Although the strain improvement in this fungus appears to be well established and several genes involved in cellulose and hemicellulose hydrolysis have been cloned (GenBank:EU239661.1; EU239662.1, etc.), the molecular mechanism for the enhancement of cellulase activity is not known. Especially, the P. decumbens genome sequencing project will be completed shortly, thus allowing the development of genome-wide knockouts. Therefore, to develop new molecular tools and novel technologies for functional genomic studies in P. decumbens is becoming increasingly urgent.
Gene targeting is one of the most important approaches for comprehensive understanding of gene function. Using this strategy, target gene can be displaced with its deletion cassette, and a preexisting genomic mutation can also be corrected to wild-type with its endogenous allele. Unfortunately, the high frequency of illegitimate integration of transforming DNA has hampered gene targeting approaches in most filamentous fungi. In eukaryotes, the repair of DNA double-strand breaks (DSB) works through two main recombination pathways. One is the homologous recombination (HR) pathway, which involves interactions between homologous DNA sequences. Another is the nonhomologous end-joining (NHEJ) pathway, which involves direct ligation of DNA ends independent of sequence homology. The NHEJ pathway has been described as consisting of several components, including Ku heterodimer (Ku70/Ku80), DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and Xrcc4-DNA ligase IV complex. The model organism Saccharomyces cerevisiae preferentially utilizes the HR pathway in DSB repair although having NHEJ pathway. In contrast to S. cerevisiae, filamentous fungi mainly utilize the NHEJ pathway of DSB repair and exogenous DNA can be integrated anywhere in the genome. Inhibition of the NHEJ pathway in these fungi is hypothesized to force the transforming DNA to integrate into the genome via HR. Therefore, disruption of Ku70 homologue in P. decumbens is postulated to enhance the gene targeting efficiency and facilitate functional genomic studies.
The transcriptional repressor CreA is the main regulatory element that has been proposed to participate in cellulase and xylanase gene expression. CreA is a negatively acting protein that mediates carbon catabolite repression in Aspergilli and Trichoderma. Despite the fact that there is a similar repression mechanism in the Aspergilli and Trichoderma, some distinct modes of CreA regulation exit in cellulose and xylan degradation.
In this study, the pku70 gene homologous to A. nidulans ku70 was cloned and identified. The pku70 targeting cassette was obtained by PCR-based fusion technique, and the ability to undergo HR was assessed in pku70-defective strain compared to the wild-type parental strain at two independent loci. Subsequently, Functional analysis of CreA in wild-type strain 114-2 and derepression phenotype in mutant A10 were performed in P. decumbens.
1 Isolation and analysis of putative pku 70 gene
A 1.36-kb sequence with similarity to A. nidulans ku70 gene was amplified in P. decumbens, on the basis of the three different ortholog sequences of human Ku70 from Aspergillus. Based on the obtained partial sequence, the 3.2-kb and 2.8-kb up- and downstream regions of this sequence were obtained by SEFA PCR. BLAST analysis of these amplified sequences revealed that they contain a full-length open reading frame (ORF) consisting of a 2047-bp. This predicated ku 70 gene, named pku70, has three introns which are located at nucleotides 559 to 611,1459 to 1518 and 1623 to
The P. decumbens pku70 gene encodes a protein of 620 amino acids (aa) with a predicated molecular mass of 68.9 kDa. BLASTP analysis showed that the Pku70 protein shares 72,71,70,69,68 and 67% identity with the respective Ku70 proteins of Neosartorya fischeri, A. clavatus, P. chrysogenum, A. sojae, A. terreus and A. nidulans. The predicted Pku70 protein sequence was submitted to the Conserved Domain Search service. The analysis revealed that Pku70 protein containes three distinct domains: Ku70/Ku80 N-terminal (vWA-ku) domain (aa 1 to 193), Ku70-core domain (aa 238 to 516) and SAP-domain (aa 573 to 607). The N-terminal domain is not involved in DNA binding but may very likely mediate Ku heterodimer interactions with other proteins involved in DSB repair process. The Ku70-core domain is constructed from a core of sevenβ-strands arranged in an antiparallel fashion, and it provides an relatively flat surface for DNA binding. The SAP-domain is a putative DNA-binding motif involved in chromosomal organization.
2 Development of a highly efficient gene targeting system in Penicillium decumbens To establish highly efficient gene targeting system and confirm the function of pku70 involved in NHEJ pathway in P. decumbens,pku70 targeting cassette was constructed with a pyrithiamine resistance gene ptrA as selectable marker. The 6.4-kb△pku70::ptrA targeting cassette was transformed into P. decumbens wild-type strain 114-2. The coding sequence of the pku 70 locus was removed from the genome of the strain PKU70-42. We therefore concluded that a single gene replacement occurred at the resident pku70 locus, indicating that the pku70 gene had been disrupted in the strain PKU70-42.
Since Ku proteins (Ku70/80) are involved in NHEJ repair of DSBs in eukaryotes, the△pku70 strain was examined for phenotypic characterization with respect to vegetative growth, germination rate, sensitivity to mutagen toxicity and osmotic stress, as well as cellulose hydrolysis. Plate assay showed that the growth rate of the△pku70 strain PKU70-42 was identical to that of the wild-type strain on MM. No irregularities in mycelial morphology and vegetative growth were observed in the strain PKU70-42, and the sporulation and spore germination rates of the△pku70 strain were not affected. The sensitivity to mutagen toxicity was determined with plate assay using MM plates independently supplemented with different amounts of EMS, hygromycin B and H2O2. The strain PKU70-42 showed no sensitivity differences in comparison with the wild-type strain. Sensitivity to osmotic stress was examined with spot tests using conidiospore suspension. In all cases, sensitivities to osmotic stress were not significantly different between the wild-type strain and the△pku70 strain PKU70-42. Additionally, in order to effectively utilize△pku70 background for creating deletions in genes regarding cellulose hydrolysis, cellulase activity was assayed and no significant differences were detected between△pku70 and wild-type genetic backgrounds of P. decumbens. These results indicated that the Apku70 strain is a suitable host for gene function analysis.
The effect of the disruption of pku70 on homologous gene targeting was evaluated by the deletion of two randomly selected genes (creA and xlnR) in a Apku70 strain. The functions of both genes have not been elucidated in P. decumbens. The homolog of creA gene in A. nidulans encodes a negatively-acting protein, which mediates carbon catabolite repression, and the homolog of xlnR gene in Hypocrea jecorina encodes a transcriptional activator that governs cellulolytic and xylanolytic gene expression. Disruption of creA and xlnR genes by gene targeting may facilitate further study of the important regulatory machinery of carbon metabolism in P. decumbens.
The△creA::hph cassette contained 3.5-kb 5'and 3.0-kb 3'of the target creA gene and the△xlnR::hph cassette harbored 1.8-kb and 1.9-kb up- and downstream genomic sequences of xlnR. Deletion of the pku70 gene in P. decumbens clearly increased gene targeting frequencies. The frequency of gene targeting was 100% when flanking regions longer than 1.8-kb were used. In the parental strain, however, gene targeting frequencies were 33% (3/9) and 91% (32/35) at creA and xlnR loci, respectively. These results suggested that gene targeting in△pku 70 was effective at various loci.
3 Analysis of integration patterns P. decumbens
Previous studies have shown that ectopic integration events are usually more frequent than homologous integration when foreign DNA is integrated into the fungal genome. In the three independent gene-targeting experiments in P. decumbens wild-type strain, relatively high frequencies of homologous integration were found at xlnR, creA and pku70 loci. Possible factors influencing the homologous integration include the conformation of transforming DNA, length of homology and the recipent strain. Conformation of transforming DNA (circular or linear) can significantly affect gene targeting and transformation frequency. In addition, we discovered that complex integration patterns were existed only in a minority of the transformants, in which homologous integration of targeting cassette was accompanied by additional ectopic integration events. In most transformants foreign DNA was either ectopically or homologously integrated into the genome. Thus it seemed to be a strong incompatibility or competition between two main classes of recombination mechanisms for transforming DNA. On average, approximately 61.5% (16/26) of nontargeted cells incorporated two or multiple copies of targeting cassette integrated randomly into the genome, compared with 9.4% (5/53) of the targeted cells incorporated more than one copy of transforming DNA due to ectopic integration. These results indicated that the nontargeted cells were more likely to incorporate a second fragment by ectopic integration than were targeted cells in P. decumbens.
4 Marker exchanging in the△pku70::ptrA strain
To adequately validate the△pku70 genetic background, the P. decumbens△pku70::hph strain was constructed by exchanging selectable markers in Apku70::ptrA strain with hph expression cassettes. The△pku70::hph targeting cassette was created and transformed into the P. decumbens△pku70::ptrA strain PKU70-42 as recipient. Sixteen randomly selected transformants were subjected to PCR analysis and plate assay. All transformants obtained ultimately became resistant to hygromycin B and sensitive to pyrithiamine. These results indicated that allelic exchange between the two pku70 targeting cassettes successfully occurred at the pku70::ptrA locus by homologous integration in strain PKU70-42.
5 Analysis of cotransformation in P. decumbens
To determine whether two loci could be independently targeted within a single transformation experiment by using mixtures of two different targeting cassettes, the△pku70::ptrA and△ace1::dsRed2 cassettes were constructed and cotransformed into the wild-type and Apku70::hph strains, respectively. In the first case, cotransformants were found at a frequency of approximately one per thirteen PyrR transformants, and the△ace1::dsRed2 cassette was integrated ectopically at unknown sites in the genome of wild-type strain. The remainder of PyrR transformants showed no cotransforming△ace1::dsRed2 cassette anywhere. In parallel experiments with the same targeting cassettes, thirty-eight PyrR transformants were obtained using the△pku70::hph strain as recipient. However, all of them did not contain cotransforming△ace1::dsRed2 fragment. The analysis confirmed that the hph marker in△pku70::hph strain was replaced by the ptrA expression cassette without additional integration of△pku70::ptrA fragment into genome. Collectively, these data demonstrated that cotransformation with two linear targeting cassettes occurred at a relatively low frequency in P. decumbens, especially in△pku70 strain as recipient. We therefore concluded that simultaneous targeting of independent genes is not a frequent occurrence during cotransformation of two different targeting cassettes.
6 Functional analysis of CreA in P. decumbens
In order to analyze the function of the CreA protein, we produced total deletion of creA produced by using in vitro techniques and△creA strain was complemented by another creA allele. In P. decumbens strain 114-2, deletions of the creA gene lead to derepression of cellulase and xylanase production even in the medium containing the D-glucose. The results showed that the regulator CreA, as a carbon catabolite repressor, mediates the repression of genes encoding cellulase and hemicellulase. Our results also indicated that lactose has no obvious effect on induction of cellulase production.
7 Analysis of derepression phenotype in P. decumbens A10
The known mutation in creA confers a derepressed phenotype in P. decumbens A10. A mutant was constructed in which a total deletion of creA produced by using in vitro techniques in P. decumbens A10. We also produced a strain the creA was corrected by another creA allele cloned from wild-type strain 114-2. In the rectified strain, the phenotype for catabolite repression can be rescued by transformation with the wild-type creA allele. Information from plate analysis of creA mutants, combined with information derived from analysis of rectified strain, allow us to determine that CreA lead to partial derepression and retain some activity in P.decumbens A10, since a total deletion has much more derepression phenotype in medium containing glucose.
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