2024-09-19 RESEARCH ARTICLE
2024-09-19研究文章

Transcriptome analysis of Ochratoxin A (OTA) producing Aspergillus westerdijkiae fc-1 under varying osmotic pressure
不同渗透压条件下赭曲霉毒素A产生菌Aspergillus westerdijkiaefc-1的转录组分析

Yanling Ma^1,#, Muyuan Zhuang^1,#, Tanvir Ahmad^1,*, Mingxuan Li^1,2 , Guangyou Tan¹, Yingyao Deng¹, Yang Liu^1,*
马艳玲^1，#，庄穆元^1，#，Tanvir Ahmad^1，*，李明轩^1，2，谭广友¹，邓颖耀¹，刘扬^1，*

¹School of Food Science and Engineering, Foshan University/ National Technical Center (Foshan) for Quality Control of Famous and Special Agricultural Products /Guangdong Key Laboratory of Food Intelligent Manufacturing, Foshan, China
¹佛山学院食品科学与工程学院/国家名特优农产品质量控制技术中心（佛山）/广东省食品智能制造重点实验室

²Guangdong Provincial Key Laboratory of Protein Function and Regulation in Agricultural Organisms, College of Life Sciences, South China Agricultural University, Guangzhou, Guangdong, China
²华南农业大学生命科学学院农业生物蛋白质功能与调控广东省重点实验室，广东广州

^# Yanling Ma and Muyuan Zhuang have contributed equally to this work
^#马艳玲和庄慕媛为这项工作做出了同样的贡献

Corresponding authors:
通讯作者：

Yang Liu liuyang@fosu.edu.cn
刘阳liuyang@fosu.edu.cn

Tanvir Ahmad tanvir@fosu.edu.cn
Tanvir Ahmadtanvir@fosu.edu.cn

School of Food Science and Engineering, Foshan University/ National Technical Center (Foshan) for Quality Control of Famous and Special Agricultural Products /Guangdong Key Laboratory of Food Intelligent Manufacturing, Foshan 528231, China
佛山学院食品科学与工程学院/国家名特优农产品质量控制技术中心（佛山）/广东省食品智能制造重点实验室，广东佛山528231

ABSTRACT
A抽象

Ochratoxin A (OTA) is a highly toxic secondary metabolite produced by the Aspergillus species. These fungal species are widely found in nature and can contaminate various food products. This study analyzed the transcriptome of the Aspergillus westerdijkiae fc-1 strain under NaCl concentrations of 0, 20, and 100 g/L using RNA-Seq technology. The aim of the study was to examine gene transcriptional changes in metabolic-pathways associated with osmotic stress and OTA production. The study revealed substantial changes in metabolic-pathways associated with carbohydrates, cellular communication, and hydrolase activity under 20 g/L NaCl. The HOG1 gene, associated with osmotic pressure regulation was down-regulated by 78.06%. In contrast, OTA biosynthesis genes otaA, otaB, and otaC were up-regulated by 3.26 fold, 1.99 fold, and 2.06 fold respectively. Conversely, the otaD gene was down-regulated by 43.50%. Under 100 g/L NaCl conditions, pathways related to ion transport, peptide metabolism, ribosomal function, and transmembrane transporter protein activities were significantly up-regulated. In this context, the HOG1 gene was up-regulated by 28.32% and OTA biosynthesis genes otaA, otaB, otaC, and otaD showed up-regulation of 27.06%, 36.80%, 19.59%, and 5.72 fold respectively. Metabolic-pathways play a significant role in the regulation of osmotic stress in the A. westerdijkiae fc-1 strain. Expression of the HOG1 gene varies under different NaCl conditions (0, 20, 100 g/L). There is a negative correlation between the HOG1 gene and expression of OTA biosynthesis genes; otaA, otaB, and otaC. Simultaneously, there is a positive correlation with the expression of the otaD gene. Understanding the osmotic pressure regulation mechanism of OTA could aid in developing effective strategies to prevent and control OTA contamination in food.
赭曲霉毒素A（OTA）是由曲霉属产生的一种高毒性次级代谢产物。这些真菌物种广泛存在于自然界中，可以污染各种食品。本研究利用RNA-Seq技术分析了Aspergillus westerdijkiaefc-1菌株在0、20和100 g/L NaCl浓度下的转录组。本研究的目的是检测与渗透胁迫和OTA产生相关的代谢途径中的基因转录变化。该研究揭示了20 g/L NaCl下与碳水化合物，细胞通讯和水解酶活性相关的代谢途径的实质性变化。与渗透压调节相关的HOG 1基因表达下调78.06%。 相比之下，OTA生物合成基因otaA、otaB和otaC分别上调3.26倍、1.99倍和2.06倍。相反，otaD基因下调43.50%。在100 g/L NaCl条件下，与离子转运、肽代谢、核糖体功能和跨膜转运蛋白活性相关的途径显著上调。在此背景下，HOG 1基因上调了28.32%，OTA生物合成基因otaA、otaB、otaC和otaD分别上调了27.06%、36.80%、19.59%和5. 7.2倍。 代谢途径在渗透胁迫的调控中起重要作用。韦斯特代克氏菌FC-1菌株。HOG1基因的表达在不同的NaCl条件下（0、20、100 g/L）变化。HOG1基因与OTA生物合成基因otaA、otaB和otaC的表达呈负相关。同时，与otaD基因的表达呈正相关。了解OTA的渗透压调节机制有助于制定有效的策略来预防和控制食品中的OTA污染。

KEYWORDS: OTA; Aspergillus westerdijkiae; NaCl; RNA-Seq; HOG signaling cascade.
关键词：OTA;西氏曲霉; NaCl; RNA-Seq;HOG信号级联。

1. Introduction
1.介绍

Aspergillus species are the most common fungi that produce OTA, a toxin commonly found in various types of foods and feed products, posing a significant and serious health threat to humans and animals (Hesseltine et al. 1972; Cabañes et al. 2010; Navale et al. 2021). The International Agency for Research on Cancer (IARC) has classified OTA as a Group 2B potential human carcinogen. OTA causes immunotoxicity, neurotoxicity, and genotoxicity (Anli and Alkis 2010; Pyo et al. 2021). Aspergillus westerdijkiae is known to be one of the most significant producers of OTA. It commonly contaminates food products, including beverages, grapes, wheat, coffee beans, and dry pickles. Therefore, it is essential to prevent and control OTA-producing strains in foods (Cabañas et al. 2008; Vipotnik et al. 2017; Altafini et al. 2019; Chen et al. 2022; Wei et al. 2022).
曲霉属是产生OTA的最常见真菌，OTA是一种常见于各种类型食品和饲料产品中的毒素，对人类和动物构成重大和严重的健康威胁（Hesseltine等人，1972; Cabañes等人，2010; Navale等人，2021）。国际癌症研究机构（IARC）已将OTA列为2B组潜在人类致癌物。OTA可引起免疫毒性、神经毒性和遗传毒性（Anli和Alkis 2010; Pyo等人2021）。已知Aspergillus westerdijkiae是OTA的最重要的生产者之一。它通常污染食品，包括饮料，葡萄，小麦，咖啡豆和干泡菜。因此，预防和控制食品中产生OTA的菌株至关重要（Cabañas et al. 2008; Vipotnik et al. 2017; Altafini et al. 2019; Chen et al. 2020 年 ; Wei et al. 2022.

The researchers have found various types of genes, including the polyketide synthase (PKS) encoding gene otaA, the non-ribosomal peptide synthase (NRPS) encoding gene otaB, the P450 oxidase encoding gene otaC, and the halogens encoding gene otaD are associated with OTA biosynthesis (Hussein et al. 2024). In the OTA biosynthesis process, the PKS and otaD genes are responsible for initiating and completing the OTA synthesis, respectively (Geisen et al. 2004; Gallo et al. 2013; Ferrara et al. 2016). New genes related to OTA production known as AoOTApks were discovered and found to be positively correlated (Wang et al. 2015). A notable correlation was found between the expression profile of AcOTApks and OTA production kinetics (Gallo et al. 2014). The OTA biosynthesis-pathway remains incompletely understood, even though lots of studies have been conducted in various OTA-producing fungal isolates and specific genes have been identified. These genes include PKS, needed for biosynthesis of dihydro-coumarin precursors, and non-ribosomal-peptide synthetase (NRPS) (O’Callaghan et al. 2013; Gerin et al. 2016). Recent findings have shown that OTA is produced through a series of steps. First, 7-methylmalonic is synthesized by PKS using acetyl-coenzyme A and malonyl-coenzyme A. Then, 7-methylmalonic acid is oxidized to OTB by cytochrome monooxygenase (otaC). OTB then reacts with L-β-phenylalanine to produce OTB through an amide bond, facilitated by the NRPS-otaB enzyme. Finally, OTB is chlorinated to produce OTA by the halogenase enzyme (otaD) (O’Callaghan et al. 2003; Wang et al. 2018).
研究人员已经发现各种类型的基因，包括聚酮合酶（PKS）编码基因otaA、非核糖体肽合酶（NRPS）编码基因otaB、P450氧化酶编码基因otaC和卤素编码基因otaD与OTA生物合成相关（Hussein et al. 2024）。在OTA生物合成过程中，PKS和otaD基因分别负责启动和完成OTA合成（Geisen et al. 2004; Gallo等人2013;费拉拉等人2016）。发现了与OTA产生相关的新基因（称为AoOTApks），并发现其呈正相关（Wang et al. 2015）。发现AcOTApks的表达谱与OTA产生动力学之间存在显著相关性（Gallo et al.2014）。尽管人们对产OTA的真菌进行了大量的研究，并鉴定了特定的基因，但OTA的生物合成途径仍不完全清楚。这些基因包括二氢香豆素前体生物合成所需的PKS和非核糖体肽合成酶（NRPS）（O 'Callaghan et al. 2013; Gerin et al. 2016）。最近的研究表明，OTA是通过一系列步骤产生的。 首先，使用乙酰辅酶A和丙二酰辅酶A通过PKS合成7-甲基丙二酸。然后，7-甲基丙二酸被细胞色素单加氧酶（otaC）氧化成OTB。然后，OTB与L-β-苯丙氨酸反应，通过NRPS-otaB酶促进的酰胺键产生OTB。最后，OTB通过卤化酶（otaD）氯化产生OTA（O 'Callaghan等人，2003; Wang等人，2018）。

However, the production of fungal mycelium growth and OTA biosynthesis are primarily influenced by environmental factors. Osmolarity is considered one of the most important environmental factors, and OTA contamination is also widespread in dry cured foods with high NaCl content (Wang et al. 2023). It seems that OTA biosynthesis is associated with NaCl rich substrates. Osmotic pressure changes induced by NaCl are often transmitted to the transcriptional level through the high osmolarity glycerol mitogen-activated-protein-kinase (HOG-MAPK) signaling pathway, which is the primary pathway involved in responding to osmotic stress (Schmidt-Heydt et al. 2011; Stoll et al. 2013). For Penicillium nordicum and Penicillium verrucosum strains, the biosynthesis of OTA was correlated with the phosphorylation of HOG MAP kinase induced by NaCl (Stoll et al. 2013; Wang et al. 2016). The AwHOG1 gene deletion significantly impacted A. westerdijkiae colony morphology, mycelial growth, sporulation, OTA accumulation, and infective capacity. It also increased the mutant's sensitivity to high osmotic pressure (Wang et al. 2023) The osmotic pressure environmental factor responded to the activation of the HOG pathway, and different NaCl conditions had a significant effect on OTA production (Schmidt-Heydt et al. 2012). Furthermore, other metabolic pathways, such as carbon metabolism, ribosomes, cellular communication, and peptide metabolic processes, are closely related to the production of fungus mycelium growth and biosynthesis of mycotoxins (Corrochano et al. 2016; Wei et al. 2022).
然而，真菌菌丝体生长和OTA生物合成的生产主要受环境因素的影响。渗透压被认为是最重要的环境因素之一，OTA污染也广泛存在于NaCl含量高的干腌食品中（Wang等人，2023）。OTA的生物合成似乎与富含NaCl的底物有关。NaCl诱导的渗透压变化通常通过高渗透压甘油促分裂原活化蛋白激酶（HOG-MAPK）信号传导途径传递至转录水平，该途径是参与响应渗透胁迫的主要途径（Schmidt-Heydt et al. 2011; Stoll et al. 2013）。 对于北欧青霉和疣状青霉菌株，OTA的生物合成与NaCl诱导的HOG MAP激酶磷酸化相关（Stoll et al. 2013; Wang et al. 2016）。AwHOG1基因缺失显著影响A. westerdijkiae菌落形态、菌丝生长、孢子形成、OTA积累和感染能力。它还增加了突变体对高渗透压的敏感性（Wang et al. 2023）。渗透压环境因素对HOG途径的激活有反应，不同的NaCl条件对OTA产生有显著影响（Schmidt-Heydt et al. 2012）。 此外，其他代谢途径，如碳代谢、核糖体、细胞通讯和肽代谢过程，与真菌菌丝体生长的产生和真菌毒素的生物合成密切相关（Corrochano et al. 2016; Wei et al. 2022）。

RNA sequencing (RNA-seq) is extensively used to study osmotic pressure regulatory pathways. It is an effective tool for analyzing transcriptomes and has been magnificently employed to analyze differential gene expression in fungal metabolic pathways and transcriptomes under different NaCl conditions (Novodvorska et al. 2013). In this study, RNA-Seq technology was used to understand the role of genes involved in OTA synthesis, secondary metabolic processes, HOG pathway, and other metabolic pathways in the A. westerdijkiae fc-1 strain under different NaCl concentrations. The exposure of this regulatory mechanism can help to develop effective control strategies to reduce food contamination by OTA. The study conducted transcriptome analysis to examine how different levels of NaCl affect gene expression and metabolic pathways particularly HOG pathway in the A. westerdijkiae fc-1 strain. By analyzing changes in the expression of HOG1 and otaA-D biosynthesis genes along with qRT-PCR results, this study aimed to understand the factors influencing the growth and OTA production in A. westerdijkiae fc-1. The findings will be valuable for developing effective strategies to minimize OTA contamination in food.
RNA测序（RNA-seq）被广泛用于研究渗透压调节途径。它是分析转录组的有效工具，并已被广泛用于分析不同NaCl条件下真菌代谢途径和转录组中的差异基因表达（Novodvorska et al. 2013）。本研究利用RNA-Seq技术对OTA合成、次级代谢过程、HOG途径等代谢途径相关基因在A. Westerdijkiaefc-1菌株对NaCl胁迫的响应。这一监管机制的曝光，可以帮助制定有效的控制策略，以减少食物污染的OTA。该研究通过转录组分析来研究不同水平的NaCl如何影响基因表达和代谢途径，特别是HOG途径。韦斯特代克氏菌FC-1菌株。通过分析HOG 1和OTA A-D生物合成基因沿着的表达变化以及qRT-PCR结果，了解影响A. westerdijkiaefc-1。研究结果将有助于制定有效的策略，以减少OTA污染的食品。

2. Materials and methods
2.材料和方法

2.1. Fungal strain and culture medium
2.1.真菌菌株和培养基

In this study, A. westerdijkiae fc-1 (Wei et al. 2022) was activated and cultured on potato dextrose agar medium (PDA) and incubated at 28 °C for 7 d. The spores were scraped from a PDA plate with a sterilized cotton swab and then resuspended in sterile distilled water. The pore suspension (10⁷ spores/mL) was kept at –80 °C with 15% glycerol solution (Wang et al. 2020).
在本研究中，以A.将westerdijkiae fc-1（Wei等人，2022）活化并在马铃薯葡萄糖琼脂培养基（PDA）上培养，并在28 ℃下孵育7天。用无菌棉签从PDA平板上刮下孢子，然后重悬于无菌蒸馏水中。将孔悬浮液（10个孢子/mL）与15%甘油溶液一起保持在-80 °C下（Wang等人，2020）。

2.2. Extraction of RNA, construction of libraries, and sequencing using Illumina technology
2.2. RNA的提取、文库的构建和使用Illumina技术的测序

A 100 μL of spore suspension was inoculated into PDA culture medium and incubated at 28 °C for 3 d. Subsequently, 100 mL PDB solutions with NaCl concentrations of 0, 20, and 100 g/L were prepared and transferred into 250 mL conical flasks in triplicate. After sterilization, the NaCl-containing PDB was inoculated with fungal mycelium and incubated at 160 rpm at 28 °C for 3 d. The mycelium was collected and transferred into 100 mL centrifuge tubes, then centrifuged at 10,000 r/min for 5 min. The supernatant was filtered and spores were collected for total RNA extraction under 0, 20, and 100 g/L NaCl conditions with three replications for each condition resulting in a total of 9 samples for sequencing analysis (Table 2). Poly(A) mRNA was enriched using oligo(dT) magnetic beads, fragmented with divalent cations in the NEB Fragmentation Buffer, and the library was constructed following the NEB standard protocol (Parkhomchuk et al. 2009). The first strand of cDNA was generated using fragmented mRNA as a template and random oligonucleotides as primers in the M-MuLV reverse transcriptase system. Afterward, the RNA strand was degraded with RNaseH, and the second strand of cDNA was synthesized with dNTPs in the DNA polymerase I system. The purified double-stranded cDNA underwent end-repair and A-tailing attachment of sequencing adapters. cDNA fragments around 250–300 bp were selected using AMPure XP beads, followed by PCR amplification. The PCR products were purified again with AMPure XP beads, resulting in the final libraries. The NEBNext® Ultra™ RNA Library Prep Kit for Illumina® was used for library construction. Afterward, the Qubit 2.0 Fluorometer provided a preliminary quantification, and the library was diluted to 1.5 ng/µL. The insert size was then assessed using an Agilent 2100 bioanalyzer. Once the insert size met expectations, qRT-PCR was employed to accurately quantify the library's effective concentration, ensuring it exceeded 2 nmol/L for quality assurance.
将100 μL孢子悬浮液接种到PDA培养基中，并在28 ℃下孵育3 d。随后，制备NaCl浓度为0、20和100 g/L的100 mL PDB溶液，并一式三份转移至250 mL锥形瓶中。灭菌后，用真菌菌丝体接种含NaCl的PDB，并在28 °C下以160 rpm孵育3 d。收集菌丝体，转移至100 mL离心管中，10,000 r/min离心5 min，过滤上清液，收集孢子，在0、20和100 g/L NaCl条件下提取总RNA，每个条件重复3次，共9份样品用于测序分析（表2）。 使用寡聚（dT）磁珠富集Poly（A）mRNA，在NEB片段化缓冲液中用二价阳离子片段化，并按照NEB标准方案构建文库（Parkhomchuk等人，2009）。在M-MuLV逆转录酶系统中使用片段化mRNA作为模板和随机寡核苷酸作为引物产生cDNA的第一链。然后，用RNaseH降解RNA链，并在DNA聚合酶I系统中用dNTPs合成cDNA的第二链。纯化的双链cDNA进行末端修复和测序接头的A加尾连接。使用AMPure XP珠粒选择约250-300 bp的cDNA片段，然后进行PCR扩增。用AMPure XP珠再次纯化PCR产物，得到最终文库。用于Illumina®的NEBNext® Ultra™ RNA文库制备试剂盒用于文库构建。之后，量子比特2。0荧光计提供初步定量，并将文库稀释至1.5 ng/µL。然后使用Agilent 2100生物分析仪评估插入物尺寸。一旦插入片段大小符合预期，则采用qRT-PCR来准确定量文库的有效浓度，确保其超过2 nmol/L以保证质量。

2.3. Gene Ontology (GO) function annotation of differently expressed genes (DEGs) and kyoto encyclopedia of genes and genomes (KEGG) pathway differential gene analysis
2.3.差异表达基因（DEG）的基因本体论（GO）功能注释和京都基因与基因组百科全书（KEGG）途径差异基因分析

Cluster-Profiler software (Lamarre et al. 2008) was used for GO and KEGG enrichment analysis on the differential gene sets. Enrichment analysis was carried out using the hyper-geometric distribution principle. It involved using a set of differential genes obtained from the significant differential analysis and annotated to the GO or KEGG database as the differential gene set. The background gene set comprised all genes subjected to significant differential analysis and annotated to the GO or KEGG database. For GO functional enrichment, a padj value of less than 0.05 was set as the threshold for significance. The top 30 most significant terms from the GO enrichment analysis were selected for histogram visualization, or all available terms were used if fewer than 30 were identified. These histograms were organized into categories of biological processes, cellular components, and molecular functions and also illustrated the up-regulation and down-regulation of differential genes. KEGG pathway enrichment analysis was performed using Cluster-Profiler software, with a padj value of less than 0.05 as the threshold for significance. This analysis focused on differentially expressed genes in the A. westerdijkiae FC-1 strain under three NaCl concentrations (0, 20, 100 g/L). Similarly, the top 30 most significant terms from the GO enrichment analysis were selected for histogram visualization; if fewer than 30 terms were available, all terms were included. The results were summarized by major categories such as biological processes, cellular components, and molecular functions, and histograms were created to illustrate both up-regulation and down-regulation of differential genes.
使用Profiler软件（Lamarre等人，2008）对差异基因集进行GO和KEGG富集分析。利用超几何分布原理进行富集分析。它涉及使用从显著差异分析中获得的一组差异基因，并将其注释到GO或KEGG数据库中作为差异基因集。背景基因集包括进行显著差异分析并注释到GO或KEGG数据库的所有基因。对于GO功能富集，将小于0.05的padj值设定为显著性阈值。选择来自GO富集分析的前30个最显著的术语用于直方图可视化，或者如果识别出少于30个，则使用所有可用的术语。 这些直方图被组织成生物学过程、细胞组分和分子功能的类别，并且还说明了差异基因的上调和下调。KEGG途径富集分析使用Profiler软件进行，padj值小于0.05作为显著性阈值。本分析集中在A. WesterdijkiaeFC-1菌株在3种NaCl浓度（0、20、100 g/L）下的生长曲线。类似地，选择来自GO富集分析的前30个最重要的术语用于直方图可视化;如果可用的术语少于30个，则包括所有术语。结果按生物学过程、细胞组分和分子功能等主要类别进行总结，并创建直方图来说明差异基因的上调和下调。

2.4. Gene expression studies
2.4.基因表达研究

A. westerdijkiae fc-1 strain cultured in a PDB medium with 0, 20, and 100 g/L concentrations of NaCl at 28 °C, and gene expression levels of otaA, otaB, otaC, otaD, and HOG1 related to OTA synthesis were detected on the 3 d of post inoculation (dpi). Primers designed for target gene detection are shown in Table 1. The RNA concentration of the strain under 0, 20, and 100 g/L NaCl conditions was measured to be 56.8, 120.1, and 397.4 ng/µL respectively by using an ultra-micro UV spectrophotometer (QuawellQ3000). cDNA was synthesized from RNA using M-MLV reverse transcriptase. Gene expressions under specific conditions were evaluated using qRT-PCR. Relative gene expression levels were calculated with CFX Manager software (Bio-Rad Laboratories) using the 2-ΔΔCT method and third parallel replicates (Bai et al. 2017).
A.将Westerdijkiaefc-1菌株在具有0、20和100 g/L浓度的NaCl的PDB培养基中在28 °C下培养，并在接种后3 d（dpi）检测与OTA合成相关的otaA、otaB、otaC、otaD和HOG 1的基因表达水平。设计用于靶基因检测的引物如表1所示。通过使用超微紫外分光光度计（UltravellQ 3000）测量，在0、20和100 g/L NaCl条件下菌株的RNA浓度分别为56.8、120.1和397.4 ng/µL。使用M-MLV逆转录酶从RNA合成cDNA。使用qRT-PCR评价特定条件下的基因表达。 使用CFX Manager软件（Bio-Rad Laboratories），使用2-ΔΔCT方法和第三次平行重复测定计算相对基因表达水平（Bai et al. 2017）。

3. Results
3.结果

3.1. Analysis of RNA sequencing and differential gene expression
3.1. RNA测序和差异基因表达分析

The raw sequencing data included some reads with adapters and low-quality sequences. To ensure data reliability, these reads were filtered out, removing adapter-containing reads, reads with undetermined bases (N), and low-quality reads (where Qphred ≤ 20 bases constituted more than 50% of the read length). After filtering, sequencing errors and GC content distribution were checked, resulting in high-quality clean reads for further analysis. As shown in Table 2, the Q30 standard was met by over 91% of the bases, with GC content ranging from 35% to 65%. After de novo assembly, 389,403,562 genes were obtained, with an average length of 643.2 bp, indicating high sequencing quality suitable for further analysis.
原始测序数据包括一些具有衔接子和低质量序列的读段。为了确保数据可靠性，过滤掉这些读段，去除含有衔接子的读段、具有未确定碱基（N）的读段和低质量读段（其中Qphred ≤ 20个碱基构成读段长度的50%以上）。过滤后，检查测序错误和GC含量分布，得到高质量的干净读数用于进一步分析。如表2所示，超过91%的碱符合Q30标准，GC含量范围为35%至65%。经从头组装后，共获得389，403，562个基因，平均长度为643.2bp，测序质量高，适合进一步分析。

Co-expression Venn diagrams display the genes uniquely expressed in each treatment group and indicate the number of genes co-expressed in two or more groups (Figure 1). A total of 11,391 differentially expressed genes (DEG; FC > |2|, FDR 0.05) were identified and functionally analyzed by comparing the conditions of 0 g/L, 20 g/L, and 100 g/L NaCl. The lowest number of genes was found in the 0 g/L NaCl condition (Set1), the second highest in the 100 g/L NaCl condition (Set3), and the highest in the 20 g/L NaCl condition (Set2). A total of 9,210 genes were compared among the 0 g/L, 20 g/L, and 100 g/L NaCl conditions (Set7). The highest number of differentiated genes was 579 in the 0 g/L NaCl condition compared with the 20 g/L NaCl condition (Set4), the second highest number was 292 in the 20 g/L NaCl condition compared with the 100 g/L NaCl condition (Set6), and the highest number was 579 in the 0 g/L NaCl condition compared with the 100 g/L NaCl condition (Set6). The comparison of the 0 g/L NaCl condition with the 100 g/L NaCl condition had the lowest number of differential genes at 268 (Set5). The volcano plot can visualize the distribution of differential genes in each comparison combination, as shown in Figure 2. In the experimental group with a salt concentration of 20 g/L NaCl, there were 13,228 DEGs compared to the control group with 0 g/L NaCl. Among these, 740 DEGs were up-regulated, and 575 DEGs were down-regulated (Figure 2a). In the experimental group with a salt concentration of 100 g/L NaCl, there were 12,941 DEGs compared to the control group (0 g/L NaCl). Within this group, 560 DEGs were up-regulated and 736 DEGs were down-regulated (Figure 2b). When comparing the experimental group with a salt concentration of 100 g/L NaCl to the control group with 20 g/L NaCl, there were a total of 13,040 DEGs. Among these, 1,015 DEGs were up-regulated and 1,324 DEGs were down-regulated (Figure 2c). 重试    错误原因

3.2. Differential gene function analysis
3.2.差异基因功能分析

GO (Gene Ontology) is a comprehensive database that describes gene function, cellular components, and molecular function. At a NaCl concentration of 20 g/L compared to a NaCl concentration of 0 g/L, the most significant numbers of genes enriched in biological processes were in carbohydrate metabolic processes, cellular communication, and signaling. There were no differentially expressed genes and no significant changes in cellular composition. The most significant gene enrichment in molecular function was in hydrolase activity and isomerase activity (Figure 3a). While a NaCl concentration of 100 g/L compared to a NaCl concentration of 0 g/L, the genes of high to low significance in biological processes were ion transport and ion transmembrane transport. In cellular composition, significance was high in cytoplasm, ribosomes, ribosomal protein complexes, and mitochondria. In molecular function, the significance in descending order is ribosomal protein complex, structural molecular activity, transport activity, and transmembrane transporter protein activity (Figure 3b). The significance of a NaCl concentration of 100 g/L compared to a NaCl concentration of 20 g/L was observed in various biological processes. In descending order, the significance was observed in the organic nitrogen compound biosynthesis process, translation, peptide metabolism process, peptide biosynthesis process, and cytosolic amide metabolism process. In terms of cellular composition, the significance from higher to lower was associated with ribosomes, cytoplasm, and protein complexes. In molecular function, higher significance was seen in structural molecular activity and ribosomal structural components (Figure 3c). 重试    错误原因

3.3. Differential gene KEGG function analysis 重试    错误原因

From the KEGG enrichment results, the 20 most significant KEGG pathways were selected to be plotted in a scatter plot for presentation. If there were fewer than 20, all the pathways were plotted, as shown in Figure 4. When comparing a NaCl concentration of 20 g/L to 0 g/L, the number of differentially enriched genes within KEGG-enriched pathways was identified. These pathways were listed in descending order of the number of genes they contain, including the Carbon Metabolism pathway, the Glycolysis pathway, the Glyoxylate and Dicarboxylic Acid Metabolism pathway, the Galactose Metabolism pathway, the MAPK Signaling pathway. Furthermore, in descending order of their significance, the pathways observed were Glycolysis, Carbon Metabolism, Fructose and Mannose Metabolism, the Pentose Phosphate pathway, and the MAPK Signaling pathway (Figure 4a). When the NaCl concentration was 100 g/L compared to 0 g/L, the ribosomal pathway, oxidative phosphorylation pathway, and carbon metabolism pathway showed the highest enrichment of differential genes in KEGG-enriched pathways (Figure 4b). When the concentration of NaCl was 100 g/L compared with NaCl 20 g/L, the KEGG-enriched pathways in which the number of differentially enriched genes was higher and significant were the ribosomal pathway, carbon metabolism pathway, oxidative phosphorylation pathway, pyruvate metabolism pathway, and amino acid biosynthesis pathway (Figure 4c). Comparing NaCl concentrations, Glycolysis, Carbon Metabolism, and MAPK Signaling were significant at 20 g/L, while the ribosomal, oxidative phosphorylation, and carbon metabolism pathways were highly enriched at 100 g/L. 重试    错误原因

3.4. HOG-MAPK pathway differential gene analysis 重试    错误原因

The results of the KEGG pathway-enriched HOG-MAPK pathway are shown in Figure 5. Among them, KEGG nodes containing up-regulated genes are labeled in red, and KEGG nodes containing down-regulated genes are labeled in green. Comparison of the experimental group (20 g/L NaCl) with the control group (0 g/L NaCl) showed Figure 5a that one gene encoding for an article (Ste50) was upregulated and four genes encoding for phosphotransfer protein genes (Ypd1), mitogen-activated protein kinase genes (HOG1), protein tyrosine phosphatase genes (Ptp2, Ptp3). Comparison of experimental group (100 g/L NaCl) with control group (0 g/L NaCl) showed that four coding genes of HOG-MAPK pathway were up-regulated in Figure 5b were glycerol synthase gene (Gpd1), small chromosome maintenance protein 1 gene (Mcm1), catalase gene (Ctt1), and HOG1 in which mitogen-activated protein kinase gene encoding gene was significantly up-regulated. Two coding genes were down-regulated for MAP3K (Ste11) and type 2C protein phosphatase gene (Ptc1). Comparison of the experimental group (100 g/L NaCl) with the control group (20 g/L NaCl) showed that 4 coding genes in the HOG-MAPK pathway were up-regulated in Figure 5c as Gpd1, Mcm1, Ctt1, and HOG1 respectively, among which Ctt1 and HOG1 genes were significantly up-regulated. Two coding genes were down-regulated Ptc1 and tyrosine-protein kinase gene (Swe1). 重试    错误原因

3.5. OTA biosynthetic genes and HOG1 gene expression under different osmolarity conditions
3.5.不同渗透压条件下OTA生物合成基因和HOG 1基因的表达

The qRT-PCR analysis revealed the expression of OTA biosynthesis genes (otaA-D) and the HOG1 gene. The results indicated that the transcript expression level of the HOG1 gene decreased by 78.06%. When the NaCl concentration was 20 g/L compared to 0 g/L NaCl. When the NaCl concentration was 100 g/L, the transcript expression level of the HOG1 gene increased by 28.32% compared to 0 g/L NaCl. Additionally, the OTA synthesis genes (otaA, otaB, otaC, and otaD) showed increases of 3.26-fold, 1.99-fold, 2.06-fold, and 0.41-fold, respectively, at 20 g/L NaCl compared to 0 g/L NaCl. The up-regulation of the otaD gene was particularly notable, showing a 6.49-fold increase under the 100 g/L NaCl culture condition compared to the 0 g/L NaCl condition (Figure 6).
qRT-PCR分析显示OTA生物合成基因（otaA-D）和HOG 1基因的表达。结果表明，HOG 1基因转录本的表达水平下降了78.06%。当NaCl浓度为20 g/L时，与0 g/L NaCl相比。当NaCl浓度为100 g/L时，HOG 1基因的转录表达量比0 g/L NaCl时增加了28.32%。此外，OTA合成基因（otaA、otaB、otaC和otaD）在20 g/L NaCl下与0 g/L NaCl相比分别显示出3.26倍、1.99倍、2.06倍和0.41倍的增加。otaD基因的上调尤其显著，显示出6.49-与0 g/L NaCl条件相比，在100 g/L NaCl培养条件下的细胞增殖倍数增加（图6）。

4. Discussion
4.讨论

In this study, RNA-Seq technology was used to investigate genes that are expressed differently and conducted a KEGG pathway analysis of the A. westerdijkiae fc-1 strain under conditions with 0, 20, and 100 g/L of NaCl. Similarly, genes involved in glycerol accumulation, sugar metabolism, organic acid storage, pigment production, and asexual spore formation in A. westerdijkiae are regulated differently in response to salt stress (Liu et al. 2017). Significant changes in genes involved in the process of sugar metabolism in A. westerdijkiae fc-1 under 20 g/L NaCl versus 0 g/L NaCl concentration and in the HOG pathway (tyrosine protein phosphatase, Ptp2,3) involved in the regulation of meiosis and sporulation were observed as a result of the consistency of present findings. A study reported that genes differentially expressed by Aspergillus montevidensis ZYD4 under high salt concentration conditions are involved in regulating amino acid, metabolism, ion transport, saturated fatty acid synthesis, fatty acid β-oxidation, oxidative stress tolerance, and soluble sugar accumulation electron transport (Ding et al. 2019). Consistent with the present study's findings, A. westerdijkiae fc-1 exhibited significant changes in genes related to ion transport, ion transmembrane translocation and those encoding proteins involved in carbohydrate and lipid metabolism in the HOG pathway under 100 g/L NaCl compared to 0 g/L NaCl conditions. The goal was to achieve a more comprehensive and accurate understanding of the pathways involved in OTA production and their regulatory mechanisms.
本研究利用RNA-Seq技术对不同表达的基因进行了研究，并对A. Westerdijkiaefc-1菌株在0、20和100 g/L NaCl的条件下进行。与此类似，在A. Westerdijkiae在盐胁迫下的调节方式不同（Liu et al. 2017）。糖代谢相关基因的显著变化。 Westerdijkiaefc-1在20 g/L NaCl和0 g/L NaCl浓度下的差异以及参与减数分裂和孢子形成调节的HOG途径（酪氨酸蛋白磷酸酶，Ptp 2，3）中的差异与本研究结果一致。一项研究报告，在高盐浓度条件下，Montevidensis ZYD 4差异表达的基因参与调节氨基酸、代谢、离子转运、饱和脂肪酸合成、脂肪酸β-氧化、氧化应激耐受性和可溶性糖积累电子传递（Ding et al. 2019）。与本研究的结果一致，A. Westerdijkiaefc-1在100 g/L NaCl胁迫下HOG途径中离子转运、离子跨膜转运相关基因及糖脂代谢相关蛋白的表达发生了显著变化。其目标是实现对OTA产生及其调控机制所涉及的途径的更全面和准确的理解。

The change in NaCl concentration resulted in substantial transcriptional changes in an enormous number of genes. GO functional classification and KEGG enrichment analysis of DEGs revealed significant changes in carbohydrate metabolism pathways when the NaCl concentration reached 20 g/L compared to 0 g/L. This concentration may be more suitable for A. westerdijkiae fc-1, as it appears to stimulate OTA biosynthesis. The enzyme otaD helps maintain intracellular chloride ion homeostasis by excreting chloride-bound OTA molecules. This suggests that Aspergillus species involved to produce OTA can sense and react to NaCl concentration changes in their environment to regulate OTA production (Wei et al. 2022; Wang et al. 2023). The increased number of cellular communication and signaling-enriched differential genes may be the result of the A. westerdijkiae receiving NaCl as an environmental signal to regulate its mycelium growth, and metabolism (Corrochano et al. 2016). Up-regulated DEGs, including genes for hydrolases and isoenzymes play a hydrolytic role in sugar metabolism, generating malonate and acetic acid as feedstock for PKS and meeting the nutritional requirements of the fungus (Rafiei et al. 2021; Nam 2022). Upregulation expression of genes related to the glyoxylate and dicarboxylic acid metabolic pathways could be a crucial factor in the accumulation of metabolites in fungi (Lorenz and Fink 2001; Ostachowska et al. 2017) .
NaCl浓度的变化导致了大量基因的转录变化。DEG的GO功能分类和KEGG富集分析显示，与0 g/L相比，当NaCl浓度达到20 g/L时，碳水化合物代谢途径发生显着变化。该浓度可能更适合于A. westerdijkiaefc-1，因为它似乎刺激OTA生物合成。酶otaD通过分泌氯离子结合的OTA分子来帮助维持细胞内氯离子稳态。这表明参与产生OTA的曲霉属物种可以感知其环境中的NaCl浓度变化并对其做出反应，以调节OTA的产生（Wei等人，2022; Wang等人，2023）。 细胞通讯和信号丰富的差异基因数量的增加可能是A. westerdijkiae接受NaCl作为环境信号来调节其菌丝体生长和代谢（Corrochano等人，2016）。上调的DEG（包括水解酶和同工酶的基因）在糖代谢中发挥水解作用，生成丙二酸和乙酸作为PKS的原料，并满足真菌的营养需求（Rafiei等人，2021; Nam，2022）。乙醛酸和二羧酸代谢途径相关基因表达上调可能是真菌中代谢物蓄积的关键因素（Lorenz and Fink 2001; Ostachowska et al. 2017）。

The GO functional classification and KEGG enrichment study of DEGs revealed significant changes in differentially expressed genes related to ion transport and ion transmembrane transporter activity when the NaCl concentration reached 100 g/L compared to 0 g/L. This change is likely attributed to the substantial increase in Na⁺ and Cl^- concentrations. The elevated ion levels may have accelerated the rate of ion transport and enhanced transporter protein activity (Wang et al. 2023). The substantial rise in the number of differential genes in cellular components; mitochondria, ribosomes and cytoplasmic differential genes may be due to high osmotic stress disruption in A. westerdijkiae fc-1 strain. The upregulation of oxidative phosphorylation pathway differential genes may be because the active transport of ions requires oxidative phosphorylation for ATP synthesis (Lavín et al. 2013). The increase in ribosomal pathway differential genes may be an alteration in the efficiency of synthesized proteins (Mullis et al. 2020). When NaCl concentration reached 100 g/L compared to 20 g/L, GO functional classification and KEGG enrichment pathway analysis in up-regulated genes showed significant elevation of ribosomal and amino acid metabolic pathways. Amino acids have been reported to be important components of proteins and secondary metabolites in different fungal species (Steyer and Todd 2023). The higher enrichment of carbon metabolism and pyruvate metabolism pathway genes may be the function of A. westerdijkiae fc-1 strain to carry out the synthesis of secondary metabolites as well as its aerobic respiration (Chroumpi et al. 2020).
GO功能分类和KEGG富集DEG的研究表明，与0 g/L相比，当NaCl浓度达到100 g/L时，与离子转运和离子跨膜转运蛋白活性相关的差异表达基因发生显著变化。这一变化可能归因于Na⁺和Cl^-浓度的大幅增加。升高的离子水平可能加速了离子转运速率并增强了转运蛋白活性（Wang等人，2023）。细胞组分中差异基因数量的大量增加，线粒体、核糖体和细胞质差异基因可能是由于高渗胁迫对A.韦斯特代克氏菌FC-1菌株。 氧化磷酸化途径差异基因的上调可能是因为离子的主动转运需要氧化磷酸化来合成ATP（Lavín et al. 2013）。核糖体途径差异基因的增加可能是合成蛋白质效率的改变（Mullis et al. 2020）。当NaCl浓度达到100 g/L时，GO功能分类和上调基因中的KEGG富集途径分析显示核糖体和氨基酸代谢途径显著升高。据报道，氨基酸是不同真菌种属中蛋白质和次级代谢产物的重要组分（Steyer和托德2023）。碳代谢和丙酮酸代谢途径基因的高度富集可能是A. westerdijkiaefc-1菌株进行次级代谢产物的合成及其有氧呼吸（Chroumpi等人，2020）。

Recent studies have demonstrated that the MAPK cascade, a central element of the HOG-MAPK pathway, is highly conserved. This cascade is composed of a tri-kinase module, including MAP3K, MAP2K and MAPK (Ma and Li 2013). In response to osmotic stress, two independent upstream osmotic sensing mechanisms, the Sln1 and Sho1 branches, are activated. These mechanisms trigger specific MAP3Ks, Ssk2/22 and Ste11, respectively. Both MAP3Ks colocalize on the common Pbs2 MAP2K. Once activated, Pbs2 phosphorylates the HOG1 MAPK, which then initiates a series of adaptive responses (Dexter et al. 2015; de Nadal and Posas 2022). The mucin-like transmembrane proteins Hkr1 and Msb2 act as potential osmo-sensors in the SHO1 branch and found that Hkr1 and Msb2 alone form a complex with Sho1, inducing intracellular signaling from Sho1 under high external osmotic stress (Tatebayashi et al. 2007). Additionally, Sho1 homo-oligomers which are four-transmembrane permeability sensors interact with the transmembrane co-permeability sensors Hkr1 and Msb2, as well as the membrane-anchoring protein Opy2, through their transmembrane structural domains. This interaction activates the Ste20-Ste11-Pbs2-HOG1 kinase cascade (Takayama et al. 2019). 重试    错误原因

However, genes in the HOG-MAPK pathway changed significantly under different osmolarity conditions (Ma et al. 2024). The results of the HOG-MAPK pathway differential gene analysis showed that the gene encoding Ste50 was up-regulated in the A. westerdijkiae fc-1 strain at 20 g/L NaCl compared to 0 g/L NaCl. A report suggests that in Saccharomyces cerevisiae, Ste50 plays a role in cellular signaling between activated G proteins and MAP3K Ste11, especially during periods of increased signaling activity in S. cerevisiae (Ramezani-Rad 2003). MAP3K Ste11 is involved in transmitting signals between the G protein and itself, possibly during times of increased signaling activity in this yeast (Ikunaga et al. 2011). The genes coding for Ypd1, HOG1, Ptp2, and Ptp3 were down-regulated, and the deletion of Ptp2, and Ptp3, which are reported to be involved in the regulation of meiosis and sporulation, resulted in a defect in sporulation possibly when the concentration of 20 g/L NaCl was not optimal concentration for spore formation in A. westerdijkiae fc-1 strain (Zhan et al. 2000). The down-regulation of genes encoding Ypd1 and HOG1 may be due to the fact that a concentration of 20 g/L NaCl is within normal growth conditions. The three-component phosphorelay system, consisting of the histidine kinase Sln1, the transfer protein Ypd1, and the response regulator Ssk1, likely inhibits the HOG pathway under these conditions. This inhibition occurs through the phosphorylation of Ssk1, which suppresses HOG pathway activity. These findings suggest that the HOG pathway may not be activated at this NaCl concentration (Dexter et al. 2015).
然而，HOG-MAPK途径中的基因在不同渗透压条件下发生显著变化（Maet al. 2024）。HOG-MAPK通路差异基因分析结果表明，编码Ste 50的基因在A. westerdijkiaefc-1菌株在20 g/L NaCl下的生长曲线与0 g/L NaCl下的生长曲线比较。一份报告表明，在酿酒酵母中，Ste 50在活化的G蛋白和MAP 3 K Ste 11之间的细胞信号传导中起作用，特别是在S.酿酒酵母（Ramezani-Rad 2003）。 MAP 3 K Ste 11参与G蛋白和自身之间的信号传递，可能是在该酵母中信号活性增加的时候（Ikunaga et al. 2011）。编码Ypd 1、HOG 1、Ptp2和Ptp 3的基因被下调，并且据报道参与减数分裂和孢子形成调节的Ptp 2和Ptp 3的缺失可能导致孢子形成缺陷。当浓度为20 g/L时，可能导致孢子形成缺陷。westerdijkiaefc-1菌株（Zhan等人，2000）。 编码Ypd 1和HOG 1的基因的下调可能是由于20 g/L NaCl浓度在正常生长条件下的事实。由组氨酸激酶Sln 1、转移蛋白Ypd 1和反应调节剂Ssk 1组成的三组分磷酸化系统可能在这些条件下抑制HOG途径。这种抑制通过Ssk 1的磷酸化发生，其抑制HOG通路活性。这些结果表明，在该NaCl浓度下，HOG通路可能不会被激活（Dexter等人，2015）。

Genes encoding for Gpd1, Mcm1, Ctt1, and HOG1 were up-regulated in A. westerdijkiae fc-1 strain at 100 g/L compared with 0 g/L NaCl, with the gene encoding for Ctt1 being significantly up-regulated. The up-regulation of the gene encoding for HOG1 may be the result of activation of the HOG pathway under 100 g/L NaCl conditions. The Gpd1 (3-phosphoglycerol dehydrogenase) encoding gene has been reported to be involved in lipid metabolism, carbohydrate and provides protection against osmotic and hypoxic stress in S. cerevisiae possibly indicating activation of the HOG-pathway in response to high osmotic pressure environment for self-protection at a concentration of 100 g/L NaCl (Alarcon et al. 2012). The Mcm1 encoding gene can protect against impaired cellular integrity, cell wall structural distortion, and altered cell wall composition may indicate that the expression of the strain was upregulated at 100 g/L NaCl concentration to maintain its cellular integrity (Zhao et al. 2019). Ctt1 encoding genes have an important role in cross-protection against osmotic stress (Schüller et al. 1994). Genes encoding for Ste11 and Ptc1 were downregulated, as Ste11 was activated during the high osmotic glycerol pathway activation is stabilized during the period. Since the steady-state amount of Ste11 does not change significantly during pheromone induction, it can be hypothesized that the pheromone induction process in A. westerdijkiae fc-1 strain is inhibited at 100 g/L NaCl concentration (Ramezani-Rad 2003). It has been reported that the deletion of the Ptc1 iso-coding gene leads to delayed mitochondrial transport but does not block it completely thus it is hypothesized that the down-regulation of the Ptc1 gene may be responsible for the inhibitory effect of mitochondrial transport of the strain at 100 g/L NaCl thus affecting its growth (Roeder et al. 1998). When 100 g/L was compared with 20 g/L NaCl. The results of the HOG-MAPK pathway differential gene analysis were consistent with the expression of the majority of the encoded genes at 100 g/L versus 0 g/L NaCl. The difference was that the Swe1-encoded genes were down-regulated. In contrast, the Ste11 genes were not significantly changed, and the Δswe1 mutant was reported to be smaller than wild-type cells, which led to the speculation that the down-regulation of the Swe1-encoded genes might delay the cell cycle and reduce the cell size (McNulty and Lew 2005).
Gpd1、Mcm 1、Ctt1和HOG 1基因在A. Westerdijkiaefc-1菌株在100 g/L NaCl浓度下与0 g/L NaCl相比，Ctt 1基因表达显著上调。HOG 1编码基因的上调可能是在100 g/L NaCl条件下HOG途径激活的结果。Gpd1（3-phosp hog lycerol dehydrogenase，3-phosp hoglycerol dehydrogenase）编码基因参与脂质代谢和碳水化合物代谢，并对渗透压和低氧胁迫提供保护。 酿酒酵母的HOG -通路可能表明HOG-通路响应于高渗透压环境而激活，以在100 g/L NaCl浓度下进行自我保护（Alarcon等人，2012）。Mcm1编码基因可以保护细胞完整性受损、细胞壁结构变形和细胞壁组成改变，这可能表明菌株的表达在100 g/L NaCl浓度下上调，以维持其细胞完整性（Zhao等人，2019）。Ctt 1编码基因在抗渗透胁迫的交叉保护中具有重要作用（Schüller et al. 1994）。 编码Ste 11和Ptc1的基因下调，因为Ste 11在高渗甘油途径激活期间被激活，在此期间激活稳定。由于Ste 11的稳态量在信息素诱导过程中没有显著变化，因此可以假设A. Westerdijkiaefc-1菌株在100 g/L NaCl浓度下受到抑制（Ramezani-Rad 2003）。 据报道，Ptc 1同种编码基因的缺失导致线粒体转运延迟，但并未完全阻断，因此假设Ptc 1基因的下调可能是100 g/L NaCl下菌株线粒体转运抑制效应的原因，从而影响其生长（Roeder等人，1998）。当100 g/L与20 g/L NaCl比较时。HOG-MAPK途径差异基因分析的结果与100 g/L NaCl与0 g/L NaCl下大多数编码基因的表达一致。不同的是，Swe 1编码的基因被下调。 相比之下，Ste 11基因没有显著变化，并且据报道Δ swe 1突变体比野生型细胞小，这导致推测Swe 1编码基因的下调可能会延迟细胞周期并减小细胞大小（McNexty和Lew 2005）。

The expression of OTA biosynthesis genes (
OTA生物合成基因的表达（otaA-D
奥塔A-D) and
）和HOG1 genes of
基因 A.westerdijkiae fc-1 strain was correlated with OTA biosynthesis with different concentrations of NaCl as detected by qRT-PCR
qRT-PCR检测到fc-1菌株在不同NaCl浓度下与OTA生物合成相关(Wang et al. 2020)
(Wang等，2020年）. Changes in the expression of OTA biosynthesis genes (
OTA生物合成基因表达的变化（otaA-D
奥塔A-D) play a crucial role in regulating OTA production. At a NaCl concentration of 20 g/L, the
）在规管OTA生产方面发挥关键作用。在NaCl浓度为20 g/L时，HOG1 gene was down-regulated in the
基因表达下调，A. westerdijkiae
A.韦斯特代克埃 fc-1 strain leading to a significant increase in the expression of the
fc-1菌株的表达显著增加，otaA-C
奥塔A-C biosynthesis genes. This increase was positively correlated which can be associated with higher OTA production as compared to the control group (0 g/L NaCl). These results suggest that
生物合成基因与对照组（0 g/L NaCl）相比，这种增加呈正相关，这可能与更高的OTA产量有关。这些结果表明A. westerdijkiae
A.韦斯特迪基亚 fc-1 is more efficient at synthesizing OTA under elevated NaCl concentrations. Further transcriptome analysis revealed significant changes in pathways related to carbohydrate metabolism, hydrolytic enzyme synthesis, and the metabolism of acetaldehyde and dicarboxylic acids indicating that these metabolic processes may also be closely linked to OTA production. At a higher NaCl concentration of 100 g/L, the
fc-1在升高的NaCl浓度下更有效地合成OTA。进一步的转录组分析显示，与碳水化合物代谢、水解酶合成以及乙醛和二羧酸代谢相关的途径发生了显着变化，这表明这些代谢过程也可能与OTA的产生密切相关。在100 g/L的较高NaCl浓度下，HOG1 gene was up-regulated, which resulted in a marked increase in the expression of the
基因表达上调，这导致了otaD biosynthesis gene. However, the expression of
生物合成基因的表达水平otaA-C
奥塔A-C was only slightly increased and showed a negative correlation which can be associated with OTA production. This pattern suggests that when the
仅略有增加，并显示出负相关性，这可能与OTA的生产。这种模式表明，当HOG
生猪 pathway is activated, possibly as a protective mechanism, OTA production may be significantly inhibited. These findings highlight key genes and pathways involved in OTA biosynthesis and offer a broader context for understanding the molecular mechanisms underlying OTA production in
途径被激活，可能作为一种保护机制，OTA的产生可能被显著抑制。这些发现突出了OTA生物合成中涉及的关键基因和途径，并为理解OTA产生的分子机制提供了更广泛的背景。A. westerdijkiae
A.韦斯特迪基亚 fc-1 under specific environmental conditions.
在特定环境条件下的FC-1。When the concentration of NaCl was 100 g/L compared to 20 g/L, the expression of the
当NaCl浓度为100 g/L与20 g/L相比时，HOG1 gene was significantly up-regulated but the expression of OTA synthesis genes (
基因的表达显著上调，但OTA合成基因的表达（otaA-C
奥塔A-C) was lower than that of 20 g/L (
）低于20 g/L（otaA-C
奥塔A-C), which may indicate that the expression of OTA biosynthesis genes (
），这可能表明OTA生物合成基因（otaA-D
奥塔A-D) and
）和HOG1 genes are not positively correlated
基因之间没有正相关(Stoll et al. 2013)
（Stoll等人，2013年）. otaD genes in the OTA synthesis pathway are responsible for introducing chloride ions at the chlorination step
OTA合成途径中的基因负责在氯化步骤中引入氯离子(Ferrara et al. 2016)
（费拉拉等人，2016）. The up-regulation of expression may be due to the high Cl
.表达的上调可能是由于高Cl^- concentration at this point to maintain partial intracellular Cl
在这一点上的浓度，以维持部分细胞内Cl^- homeostasis
稳态(Wang et al. 2018)
(Wang等，2018年）. In this study, transcriptome analysis of key metabolic pathways and differential genes in the
在这项研究中，转录组分析的关键代谢途径和差异基因，A. westerdijkiae
A.韦斯特迪基亚 fc-1 strain under varying osmotic pressure conditions was combined with OTA production data to gain a precise understanding of the molecular pathways involved in OTA production. The findings can be applied to develop strategies for controlling OTA contamination in the food and agricultural industries. This research provides a theoretical foundation for future studies on specific genes and metabolic pathways and can contribute to the development of molecular markers for monitoring OTA contamination levels. The broader implications of these results include improving food safety and understanding the environmental factors that influence OTA biosynthesis.
fc-1菌株在不同的渗透压条件下与OTA生产数据相结合，以获得涉及OTA生产的分子途径的精确理解。研究结果可用于制定控制食品和农业行业OTA污染的策略。本研究为进一步研究特定基因和代谢途径提供了理论基础，并有助于开发用于监测OTA污染水平的分子标记物。这些结果的更广泛的影响包括改善食品安全和了解影响OTA生物合成的环境因素。

5. Conclusions
5.结论s

This research employed RNA sequencing technology to identify differentially expressed genes in A. westerdijkiae fc-1 strain under varying osmotic pressure conditions. Through an in-depth analysis, particularly of the HOG pathway, significant pathways were uncovered. A connection was also established between the altered gene expression within the HOG pathway and OTA production. These findings lay the groundwork for developing strategies to reduce OTA contamination in foodstuffs.
本研究利用RNA测序技术对A. WesterdijkiaeFC-1菌株在不同渗透压条件下的渗透压。通过深入分析，特别是HOG途径，发现了重要的途径。在HOG途径内改变的基因表达和OTA产生之间也建立了联系。这些发现为制定减少食品中OTA污染的策略奠定了基础。

Disclosure statement
披露声明

No potential conflict of interest was reported by the author(s).
作者未报告潜在的利益冲突。

Author Contributions:
作者贡献：

Methodology, Y.M. and M.Z.; formal analysis, G.T. and Y.D.; validation, Y.M, T.A. and M.L.; writing and original draft preparation, M.Z, and Y.M.; writing, review and editing, Y.L., T.A, Y.M., and M.Z.; project administration, T.A. and Y.L.; funding acquisition, Y.L.
方法，Y.M.和M.Z.;形式分析，G.T.和Y.D.;验证，Y.M，T.A.和M.L.;写作和初稿准备，M.Z.和Y.M.;写作、评论和编辑，Y.L.，T.A. Y.M.和M.Z.;项目管理，助教和Y.L.;融资收购，Y.L。

Funding
资金

The work was supported by Guangdong Basic and Applied Basic Research Foundation (2022A1515010037), and the National Key Research and Development Program of China (2022YFE0139500).
本课题得到了广东省基础与应用基础研究基金（2022 A1515010037）和国家重点研究发展计划（2022 YFE 0139500）的资助。

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Table 1. Primers were used to amplify otaA, otaB, otaC, otaD and HOG1 through qRT-PCR.
表1. 使用引物通过qRT-PCR扩增otaA、otaB、otaC、otaD和HOG 1。

Table 2. Transcriptome sequencing summary of clean reads, error rate, Q30, and GC content for Aspergillus westerdijkiae fc-1 strain under different NaCl conditions.
表2. 不同NaCl条件下西氏曲霉fc-1菌株的干净读数、错误率、Q30和GC含量的转录组测序总结。

<sup>*Q > 30% is the percentage of bases with a mass value greater than 30 in the total bases with an error rate < 0.1%; **GC (%) is the percentage of G and C bases in the total bases in the filtered reads count.
* Q>30%是总碱基中质量值大于30的碱基的百分比，错误率<0.1%; **GC（%）是过滤的读段计数中总碱基中G和C碱基的百分比。</sup>

Figure 1. Wayne plots of DEG (FC >|2|), comparing differential genes under (0, 20, 100) g/L NaCl condition.
图1. DEG（FC %3 E）的韦恩图|2|在（0、20、100）g/LNaCl条件下比较差异基因。

Figure 2. Volcano plots of differential gene expression under different osmotic pressure conditions. (a) 20 g/L NaCl vs. 0 g/L NaCl; (b) 100 g/L NaCl vs. 0 g/L NaCl; (c) 100 g/L NaCl vs. 20 g/L NaCl. The horizontal axis represents gene fold-changes (log2 fold change), and the vertical axis shows the significance of gene expression differences (-log10 padj). Red dots represent up-regulated genes, and green dots represent down-regulated genes.
图2.不同渗透压条件下差异基因表达的火山图。(a)20 g/L NaCl对0 g/L NaCl;（B）100 g/L NaCl对0 g/L NaCl;（c）100 g/L NaCl对20 g/L NaCl。水平轴代表基因倍数变化（log2fold变化），垂直轴显示基因表达差异的显著性（-log 10 padj）。红点代表上调基因，绿色点代表下调基因。

Figure 3. GO functional analysis of differential genes. (a) GO function classification under 20 g/L NaCl vs. 0 g/L NaCl; (b) 100 g/L NaCl vs. 0 g/L NaCl; (c) 100 g/L NaCl vs. 20 g/L NaCl. The horizontal axis represents GO terms, and the vertical axis indicates the significance of GO term enrichment. Different colors represent BP (biological process), CC (cellular component), and MF (molecular function). GO enrichment is considered significant when padj < 0.05.
图3.差异基因的GO功能分析。（a）20 g/L NaCl对0 g/L NaCl下的GO功能分类;（B）100 g/L NaCl对0 g/L NaCl下的GO功能分类;（c）100 g/L NaCl对20 g/L NaCl下的GO功能分类。横轴表示GO项，纵轴表示GO项富集的显著性。不同的颜色代表BP（生物过程），CC（细胞成分）和MF（分子功能）。当padj % 3C 0.05时，认为GO富集显著。

Figure 4. KEGG functional analysis of differential genes. (a) KEGG-enriched pathways for 20 g/L NaCl vs. 0 g/L NaCl; (b) 100 g/L NaCl vs. 0 g/L NaCl; (c) 100 g/L NaCl vs. 20 g/L NaCl. The horizontal axis shows the ratio of annotated differential genes, and the vertical axis indicates the KEGG pathway. Dot size reflects the number of annotated genes, while color (red to purple) indicates enrichment significance.
图4. 差异基因的KEGG功能分析。（a）20 g/L NaCl与0 g/L NaCl的KEGG富集途径;（B）100 g/L NaCl与0 g/L NaCl;（c）100 g/L NaCl与20 g/L NaCl。横轴表示注释的差异基因的比率，纵轴表示KEGG途径。点大小反映了注释基因的数量，而颜色（红色至紫色）表示富集显著性。

Figure 5. Differential gene analysis of the HOG-MAPK pathway. (a) Gene changes under 20 g/L NaCl vs. 0 g/L NaCl; (b) 100 g/L NaCl vs. 0 g/L NaCl; (c) 100 g/L NaCl vs. 20 g/L NaCl. Up-regulated genes are in red, down-regulated genes are in green, with color intensity indicating significance.
图5. HOG-MAPK通路的差异基因分析。（a）20 g/L NaCl对0 g/L NaCl下的基因变化;（B）100 g/L NaCl对0 g/L NaCl下的基因变化;（c）100 g/L NaCl对20 g/L NaCl下的基因变化。上调基因为红色，下调基因为绿色，颜色强度表示显著性。

Figure 6. Expression of Hog1 and OTA biosynthesis genes (otaA-D) under different NaCl concentrations. (a) Lowercase letters a,b,c denote the expression of Hog1 gene in A. westerdijkiae fc-1 strain at (100,0,20) g/L NaCl, respectively. (b) Lowercase letters a,b,c denote the expression of otaA gene in A. westerdijkiae fc-1 strain under (20,100,0) g/L NaCl conditions, respectively. (c) Lowercase letters a,b,c denote the expression of otaB gene in A. westerdijkiae fc-1 strain under (20,100,0) g/L NaCl, respectively. (d) Lowercase letters a,b,c denote the expression of otaC gene in A. westerdijkiae fc-1 strain at (20, 100, 0 ) g/L NaCl, respectively. (e) Lowercase letters a,b,c denote the expression of otaD gene in A. westerdijkiae fc-1 strain at (100, 20, 0 ) g/L NaCl, respectively. Different letters represent significant differences at P < 0.05.
图6. Hog 1和OTA生物合成基因（otaA-D）在不同NaCl浓度下的表达。(a)小写字母a、B、c表示Hog 1基因在A中的表达。Westerdijkiae fc-1菌株在（100、0、20）g/L NaCl条件下的耐盐性差异显著。(b)小写字母a、B、c表示otaA基因在A中的表达。Westerdijkiae fc-1菌株在（20，100，0）g/L NaCl条件下的生长曲线。(c)小写字母a、B、c表示A中ota B基因的表达。Westerdijkiae fc-1菌株在（20，100，0）g/L NaCl胁迫下的耐盐性。(d)小写字母a、B、c表示otaC基因在A中的表达。Westerdijkiae fc-1菌株在（20、100、0）g/L NaCl条件下的耐盐性。(e)小写字母a、B、c表示otaD基因在A中的表达。Westerdijkiae fc-1菌株在（100、20、0）g/L NaCl中的生长曲线。不同字母表示P < 0.05的显著差异。