冠突曲霉是茯砖茶上的优势菌,可通过渗透压调控获得纯的无性和有性阶段,是研究真菌产孢的好材料.flbD基因广泛存在于丝状真菌中,是调控丝状真菌无性孢子形成的关键基因.为了探究flbD基因在冠突曲霉(Aspergillus cristatus)发育阶段的功能,以冠突曲霉为试验材料,构建flbD基因敲除载体,利用根癌农杆菌介导转化,筛选得到flbD基因敲除菌株.通过对△flbD敲除株和野生型菌株的比较观察发现:野生型的分生孢子数量是敲除株的4.7倍,而子囊孢子数量相差不大;在不同渗透压培养基上,△flbD突变株的菌丝生长速率与野生型的没有明显差异,但是突变株会产生大量"棉花"状的气生菌丝;在高渗条件下,△flbD突变株分生孢子的产生有延迟现象.运用基因敲除技术检验了flbD基因在冠突曲霉的功能,结果显示flbD对冠突曲霉的无性产孢起正调控作用,敲除flbD基因将减少无性孢子的形成.此研究对开展冠突曲霉无性产孢的研究具有重要借鉴价值.

As the dominant fungus during the fermentation of Fuzhuan brick tea, Aspergillus cristatus is easily induced to undergo a sexual cycle under low-salt stress. However, the underlying regulatory mechanism of sexual reproduction is unclear. Here, we report a P53-like transcription factor AcndtA, which encodes an NDT80 DNA binding protein and regulates fungal reproduction, pigmentation and the stress response. Both insertion and deletion mutants of AcndtA exhibited a complete blockade of cleistothecium formation, and overexpressing AcndtA strains (OE: AcndtA) exhibited significantly reduced cleistothecium production, indicating that AcndtA plays a vital role in sexual development. Osmotic stress tests showed that overexpression of AcndtA had a negative impact on growth and conidia production. Additionally, AcndtA insertion, deletion and overexpression mutants exhibited reduced pigment formation. All the above developmental defects were reversed by the re-introduction of the AcndtA gene in ΔAcndtA. Moreover, the growth of AcndtA mutants in carbon-limited medium was better than that of the WT and OE: AcndtA strains, indicating that AcndtA is involved in carbon metabolism. Transcriptional profiling data showed that AcndtA regulated the expression of several genes related to development, osmotic stress and carbon metabolism.

为研究信息素前体基因ppgA在冠突曲霉(Aspergillus cristatus)有性产孢中的功能,本研究利用同源重组原理对冠突曲霉高效基因敲除菌株AKu70中ppgA基因进行敲除.形态学观察发现,在MYA培养基中,ppgA基因敲除株前期生长较△Ku70菌株快.培养6d后,敲除株形成较厚的菌落,浅黄色.此外,敲除株菌落中产生褐色色素的时间较晚,且明显减少.在1 mol/L NaCl条件下,敲除株仅菌落中央形成稀疏成熟闭囊壳,闭囊壳数量减少约15倍,子囊孢子成熟期延长;敲除株菌落边缘闭囊壳不成熟,无子囊孢子产生.在3 mol/L NaC1条件下,敲除株菌落白色,生长缓慢.这些结果表明,ppgA基因在冠突曲霉的有性产孢过程中发挥重要作用,该研究为进一步探讨冠突曲霉有性产孢机制的研究提供了理论参考和数据支持.

Filamentous fungi reproduce sexually or asexually, and the developmental processes are strictly regulated by a variety of transcription factors. In this study, we characterized a zinc finger transcription factor, called AcrpnR, in Aspergillus cristatus (GME2916). The ∆AcrpnR strain exhibited decreased asexual reproduction and increased cleistothecium production. The complementation strain showed restoration of these phenotypic differences. Overexpression of AcrpnR resulted in enhanced asexual development and delayed and inhibited sexual reproduction, suggesting that AcrpnR is required for proper asexual and sexual development in A. cristatus. In addition, AcrpnR positively regulated the expression of genes of the central regulatory pathway of conidiation and negatively regulated the expression of sex-related genes. Overall, these results demonstrate that AcrpnR is essential for maintaining a balance between asexual and sexual development.

本实验室前期在高渗条件下筛选得到8个转录因子,其中ACZ (SI65-00458)基因表达量最高,推测ACZ基因在冠突曲霉中参与响应渗透压及调控孢子的产生.因而本研究利用同源重组原理,构建了ACZ基因敲除载体,通过农杆菌介导转化,筛选获得了ACZ敲除菌株,将敲除株分生孢子液接种在含不同浓度NaCl的MYA培养基上,以野生型冠突曲霉为对照,观察敲除株与野生型菌株的区别.研究发现:培养基中不加NaCl时色素有明显变化,野生型为黄色,敲除株为褐黄色;在低渗透压下,菌落边缘不规则;在高渗透压下,敲除株产生的分生孢子数量为野生型的4倍多;无论是在27℃还是在37℃培养,敲除株在低渗及高渗条件下,菌落直径都较野生型小,菌丝较稀疏.表明敲除ACZ基因会影响冠突曲霉菌丝生长、色素的合成及孢子的产生.

为了研究冠突曲霉(Aspergillus cristatus) Fig基因的功能,本研究采用同源重组的方法获得敲除株.并对敲除株进行功能验证.结果 显示对Fig基因敲除株培养观察并与野生型菌株相比发现,在含有不同浓度NaCl的MYA培养基中,仍能产生成熟的子囊孢子和分生孢子,但分生孢子数量是野生型的1/4,子囊孢子数量是野生型的1/5,且敲除株菌落几乎没有渗出液,无皱褶,菌落表面干燥,色素产生量急剧下降,这些结果表明Fig基因对冠突曲霉产孢起正调控,本实验为冠突曲霉发育调控机制的研究提供了一定的技术基础.

The metabolites of mycelial samples of ΔveA mutant and wild-type strain E4 of Aspergillus cristatus with 48 hour incubation period were identified by gas chromatography-mass spectrometry (GC-MS).A total of 99 metabolites were identified,including 41 significantly differential metabolites.Among them,20 and 21 metabolites were much increased in the wild-type E4 and ΔveA,respectively.Statistical analysis revealed there were 623 individual metabolite having correlation,of which 313 were positive and 310 negative.These metabolites include organic acids,amino acids,carbohydrates,alcohols and oxylipin,while organic acids and amino acids predominate.This result lays the foundation for further study on the correlation between metabolite and sporulation in A.cristotus.

Response surface method and single-factor experiment were carried out to optimize the formulation of culture media of Aspergillus cristatus to obtain a maximum spore concentration. By three single-factor de-signs, the best amount of moisture content, NaCl concentration and inoculum amount are 10ml, 5% and 240μl, respectively. According to the Box-Behnken design( BBD) in Design Expert, the optimal liquid media formulation was determined by response surface analysis as 10g of sifted bran consisting of 9.79 ml water, 6. 13% NaCl and 246.48 μl spore suspension which concentration was about 108 spores/ml. After 7 days of cul-ture at 28℃, the maximum spore concentration was approximately 3.51×107 spores/ml. This research could facilitate the popularization of artificial inoculation, providing reference for future study on the preparation and optimization of liquid media for the culture of " Golden flower" fungus.

veA belongs to the velvet regulatory system that regulates the development and secondary metabolism of many fungi. To identify the function of veA in Aspergillus cristatus, veA deletion mutants were constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation. Deletion of veA led to increased conidial production and reduced sexual sporulation. The regulatory role of veA in A. cristatus was not light-dependent, and this differed from its role in other Aspergilli. Furthermore, veA deletion mutants were more sensitive to environmental stressors, including salt, osmotic pressure, temperature and pH. In contrast, deletion of veA resulted in increased resistance to oxidative stress. veA also affected aerial vegetative growth. Transcriptomic analysis of the veA-null mutant and wild type indicated that most asexual and sexual development genes were upregulated and downregulated, respectively. These findings confirmed that veA has a positive effect on sexual development but represses conidial formation. Overall, these results suggested that the veA gene plays a critical role in maintaining a developmental balance between asexual and sexual sporulation and is involved in vegetative growth and environmental stress response in A. cristatus.

In this study,the complete eDNA sequence ofzfpA gene ofAspergillus cristatus was cloned by RACE (rapid amplification of eDNA ends) technology and sequence analysis was performed by bioinformatics methods.The full-length sequence ofzfpA was 1 443 bp with a 924 bp ORF and had no intron sequence from 312 to 1 235 bp.It was predicted to have 307 predicted amino acids.It shared 71％ similarity with AN 1 zinc finger protein of A.oryzae (XP_001826567).The expression characteristics in different development stages ofzfpA gene was analyzed by SYBR Green I real time PCR.zfpA expression was the lowest in asexual stage and the highest in sexual stage of which the expression was up-regulated by 4 times compared with asexual stage.This work laid the foundation for studying the sporulation regulatory mechanism ofzfpA gene in A.cristatus.

本研究对贵州地区茯砖茶中的"金花菌"进行了分离鉴定。通过PCR扩增及测序技术获得转录间隔区序列及部分核糖体大亚基（ITS+LSU）、β-微管蛋白（β-tubulin）、钙调蛋白（Ca M）和RNA聚合酶Ⅱ（RPB2）4个基因的序列,然后构建多基因系统树,并对其形态特征进行观察,将其鉴定为冠突曲霉Aspergillus cristatus。

In order to investigate the mechanism of sexual sporulation regulation ofAspergillus cristatus,we selected a mutant strain of sexual sporulation from T-DNA random insertional mutant library.Southern blotting analysis showed that T-DNA insert was a single copy.The flanking sequence of T-DNA insertion site was amplified by TaiI-PCR technique,and the mutant gene was isolated.Sequence analysis revealed that the mutant gene was located on genomic scaffold 7 of Aspergillus cristatus and the open reading frame was 2 511 bp in length,which contained 4 introns.We predicted that it encoded 666 amino acids and had a conserved domain of NDT80_PhoG super family.BLASTp showed that the predicted protein has the highest similarity with Eurotium rub rum p53-1ike transcription factor (EYE95652.1) and the identity was 89％.Microscopic observation found that the insertion of T-DNA had an effect on the female reproductive structure formation.The research laid a foundation for further study on the molecular function and possible regulatory pathways of this gene during sexual sporulation.

CRISPR/Cas9,a simple and efficient tool for genome editing,is widely used in genomes of various cells and organisms.It uses Cas9 nuclease which is guided by a RNA sequence to cleave and modify genome at the specific sites.Ago,another kind of endonuclease,have shown a good application potential in recent studies.This review mainly summarized the structure foundation,mechanism and progress of CRISPR/Cas9 system,modified gene editing mechanism and the application of gene editing technology.

Global regulators play an important role in the secondary metabolic regulation and morphological development of filamentous fungi.LaeA is a key global regulator of filamentous fungi first found in 2004.In this paper,we reviewed the function and mechanism of action of LaeA in the development and secondary metabolic regulation of filamentous fungi,summarized the homologous genes of laeA that have been found in filamentous fungi,discussed the current existing problems and forecast the application prospect.