The Mechanism of Arginine Regulating the Quality of Artificially Cultivated <i>Ophiocordyceps sinensis</i>

DENG Xiaoshu; XU Jinzhi; TAN Fayin; LUO Changshu; WANG Jia; GUO Lian'an; YOU Huajian; LIU Xiang; CHEN Shijiang

doi:10.13718/j.cnki.xdzk.2025.11.005

2025 Volume 47 Issue 11

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DENG Xiaoshu, XU Jinzhi, TAN Fayin, et al. The Mechanism of Arginine Regulating the Quality of Artificially Cultivated Ophiocordyceps sinensis[J]. Journal of Southwest University Natural Science Edition, 2025, 47(11): 62-72. doi: 10.13718/j.cnki.xdzk.2025.11.005

Citation:

DENG Xiaoshu, XU Jinzhi, TAN Fayin, et al. The Mechanism of Arginine Regulating the Quality of Artificially Cultivated Ophiocordyceps sinensis[J]. Journal of Southwest University Natural Science Edition, 2025, 47(11): 62-72. doi: 10.13718/j.cnki.xdzk.2025.11.005

The Mechanism of Arginine Regulating the Quality of Artificially Cultivated Ophiocordyceps sinensis

1.
Chongqing Academy of Chinese Materia Medica, Chongqing 400065, China
2.
Chongqing University of Chinese Medicine, Bishan Chongqing 402760, China

More Information

Corresponding author: CHEN Shijiang
Received Date: 28/04/2025
Available Online: 20/11/2025
MSC: S567.35

Abstract

The objectives of this study is to analyze the underlying mechanism of quality changes in artificially cultivated Ophiocordyceps sinensis and provide a theoretical guidance for improving its appearance quality. Using arginine treated mycoparasite complex (MC, TMC), sclerotium (ST, TST), and fruiting body 0-1 cm (FB, TFB) of O. sinensis as experimental materials, phenotype observation, comparison of the histological structures, and physiological-biochemical indexes were conducted. The results showed that arginine treatment significantly affected the appearance quality of O. sinensis, with notable differences in the activities of reactive oxygen metabolism-related enzymes (PPO, SOD, POD, CAT) and changes in H₂O₂ content. Additionally, RNA-seq was performed to reveal the impact of arginine on the quality of O. sinensis and identify the critical genes. During the development of O. sinensis, TMC-vs-MC, TST-vs-ST, and TFB-vs-FB showed significant changes in the expression of 733 genes (385 up-regulated, 348 down-regulated), 815 genes (435 up-regulated, 380 down-regulated), and 158 genes (95 up-regulated, 63 down-regulated), respectively. Among these, 162 genes exhibited consistently significant changes. KEGG enrichment analysis showed that those DE genes mainly enriched in the pathways of tyrosine metabolism (ko00350), arginine and proline metabolism (ko00330), DNA replication (ko03030), tryptophan metabolism (ko00380), Phenylalanine metabolism (ko00360), and Phenylpropanoid biosynthesis (ko00940).
- Ophiocordyceps sinensis,
- arginine,
- reactive oxygen species,
- color change

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冬虫夏草(Ophiocordyceps sinensis，以下简称虫草)是名贵中药材，具有独特的药用价值，含多种活性成分，如核苷、多糖、甾醇、蛋白质及氨基酸等，备受消费者的青睐^[1-3]。由于过度采挖，天然冬虫夏草资源日渐匮乏，市场上的需求缺口越来越大^[4]。随着室内人工培殖虫草技术的进步，能培殖并诱导出有子实体的虫草，但在外观品质和商品规格等级上，人工培殖的虫草与天然虫草仍存在差异，影响了人工培殖虫草的商品价值^[5-6]。

现有研究表明，昆虫体色分为色素色和结构色^[5-6]，其中，黑色素是昆虫体色多样性形成过程中至关重要的色素。黑色素通过酚氧化酶激活系统合成，与先天免疫、硬化和色素沉积密切相关^[7]。真菌颜色变化主要由褐变引起，如食药用菌双孢蘑菇、金针菇、猴头菌等，其中，酶促褐变起主导作用。酶促褐变形成的3个关键因素是底物(酚类物质)、酶和活性氧(ROS)^[8-10]。张芳^[11]的研究表明，双孢蘑菇发生褐变主要是通过影响活性氧代谢及酚类物质代谢进行调控。本课题组前期利用组织细胞学、转录组学、蛋白组学等对人工培育虫草进行了品质机制研究，发现虫草僵虫颜色变化与活性氧代谢、免疫耐受力代谢相关^[12]，精氨酸在虫草品质变化过程中起着重要作用^[13]。龚佳惠等^[14]研究发现，0.1 mmol/L精氨酸处理能够抑制绿芦笋丙二醛含量及木质素的生成，提高了抗氧化能力，维持了绿芦笋的品质；1 mmol/L精氨酸处理能够提高抗氧化系统活性，提高了石榴果实的抗冷性，延缓了果实冷害症状的发生^[15]。用不同浓度的精氨酸处理白蘑菇研究发现，精氨酸能够影响白蘑菇的贮藏品质^[16]。精氨酸酶参与了茉莉酸甲酯调控的双孢蘑菇采后子实体的品质维持^[17]。对番茄不同成熟期果实内源精氨酸代谢的研究发现，精氨酸在果实成熟过程中具有独特的生理作用^[18]。大量研究表明，精氨酸对改善品质具有重要作用。

本研究通过精氨酸处理不同发育时期的虫草，利用苏木精—伊红染色法(HE)结合光学显微镜观察组织细胞学变化，采用试剂盒检测不同发育时期样本的丙二醛(MDA)、过氧化氢(H₂O₂)含量以及多酚氧化酶(PPO)、过氧化物酶(POD)、过氧化氢酶(CAT)、超氧化物歧化酶(SOD)的活性变化；通过Illumina测序平台对3个时期的冬虫夏草进行转录组测序分析，挖掘与颜色变化相关的关键基因，并从活性氧代谢角度分析精氨酸在调控虫草品质中的功能，旨在为进一步解析虫草虫体颜色变化的成因奠定分子学基础。

1. 材料与方法

1.1. 供试材料

5龄感菌虫(MC)、僵虫(ST)、子座长度0~1 cm冬虫夏草(FB)，由重庆市中药研究院冬虫夏草研究所实验室人工培殖提供，饲养温度为(8±1) ℃，湿度为50%±5%，无光照。选取虫体大小、颜色、发育时期均一致的样本，采用10 mmol/L精氨酸溶液^[16]浸泡处理MC、ST、FB 30 min，处理后5龄感菌虫(TMC)、僵虫(TST)与MC、ST一起培养1个月，子座长度0~1 cm冬虫夏草(TFB)与FB培养至采收期。常规人工培殖冬虫夏草在人工模拟天然冬虫夏草产区环境进行培殖。

1.2. 试验方法

1.2.1. 组织细胞学鉴定

取不同发育时期样本[5龄感菌虫(MC、TMC)、僵虫(ST、TST)、子座长度0~1 cm冬虫夏草(FB、TFB)]于2%戊二醛固定液中固定24 h，经梯度酒精(70%、75%、85%、90%和100%酒精各30 min)脱水，再放入酒精与二甲苯混合液(体积比1∶1)中浸泡15 min，随后放入二甲苯中浸泡30 min。待样品完全透明后，将其放入二甲苯与石蜡混合液中浸泡10 min，最后放入软蜡和硬蜡中各30 min，冷却凝固。将石蜡包埋的样品经石蜡切片机进行切片，每个组织切片厚度为5~7 μm，放于载玻片制片，37 ℃过夜烤片，HE染色，树胶封片，光学显微镜下观察样品的组织细胞学结构并拍照。

1.2.2. 待测液提取

将-80 ℃保存的待测组织置于已用液氮预冷过的研钵中，加入液氮研磨成粉，准确称取0.1 g粉末，加入900 μL PBS缓冲液进行匀浆，8 500 r/min 4 ℃离心10 min，取上清液用于酶活性及相关含量分析，置冰上待测。

1.2.3. 活性氧代谢、MDA及H₂O₂含量测定

多酚氧化酶(PPO)试剂盒(微量法，PPO-1-Y)、超氧化物歧化酶(SOD)试剂盒(NBT法，SOD-1-Y)、过氧化氢酶(CAT)试剂盒(钼酸铵比色法，CAT-1-W)、过氧化物酶(POD)试剂盒(微量法，POD-1-Y)、丙二醛(MDA)试剂盒(微量法，MDA-1-Y)及过氧化氢(H₂O₂)试剂盒(微量法，H₂O₂-1-Y)，参照说明书进行检测，由苏州科铭生物技术有限公司提供。

1.2.4. Total RNA提取与高通量测序

Total RNA提取采用mirVana^TM miRNA Isolation Kit试剂盒(Ambion，美国)，检测RNA样品的纯度、浓度和完整性，按照TruSeq Stranded mRNA LT Sample Prep Kit试剂盒操作说明书进行cDNA文库构建，使用Aglient 2100 Bioanalyzer生物分析仪进行质检，质检合格后于Illumina HiSeq^TM 2500高通量测序平台对文库进行测序。

1.2.5. 序列筛选与组装

转录组测序后产生的原始数据(Raw reads)使用Trimmomatic软件进行质控并去除接头，在此基础上过滤掉低质量碱基以及N碱基，最终得到高质量的Clean reads。将Clean reads使用hisat2比对到物种的参考基因组，冬虫夏草菌参考基因组来源于NCBI数据库(https://www.ncbi.nlm.nih.gov/assembly/GCA_000448365.1/)，软件参数为默认参数，通过基因组比对率评估样本的情况。

1.2.6. Unigene功能注释和分类

对得到的Unigene进行基因功能注释，利用GO(基因本位论数据库，http://geneontology.org/)和KEGG(京都基因与基因组百科全书数据库，http://www.kegg.jp/)进行GO分类和注释、KEGG代谢通路分析，进而获得所有Unigene的功能注释信息。

1.2.7. 差异表达基因的筛选

利用DESeq软件对差异表达基因进行分析，结合Benjamini-Hochberg方法对原有假设检验得到的显著性p值进行校正，p＜0.05为差异表达基因筛选的关键指标。以错误发现率FDR＜0.05且|log₂FC|＞1(Fold Change，FC)作为筛选标准，并对筛选到的差异表达基因进行GO富集及KEGG通路富集分析。

1.2.8. qRT-PCR分析

为了验证转录组数据的可靠性，采用真菌18 S核糖体RNA作为内参基因，随机选取6个差异表达基因进行qRT-PCR验证，设计qRT-PCR特异引物(表 1)，利用反转录试剂盒HiScript^® Ⅱ 1st Strand cDNA Synthesis Kit(Vazyme，中国)将RNA反转录得到cDNA，采用SYBR Green Master Mix荧光定量试剂盒(Vazyme，中国)在荧光定量PCR仪上进行定量检测，每个样品3个重复，采用2^-ΔΔCt法计算差异基因的相对表达量^[19]。

1.3. 数据分析

采用Excel 2020进行数据整理，运用GraphPad Prism软件对数据进行显著性分析与作图。统计分析采用t检验，数据以 x±s表示。

3. 讨论与结论

3.1. 讨论

为明确人工培殖的虫草颜色变化品质机制，本研究对精氨酸处理不同发育时期虫草的表型及细胞学特征进行了分析。结果表明，精氨酸通过调控活性氧代谢相关酶活性(尤其是过氧化氢酶)，进而影响虫草虫体颜色的变化。经查阅文献资料发现，无论是探究昆虫颜色变化，还是探究食药用菌颜色变化涉及的酶促褐变，都与活性氧代谢密切相关^[22-25]。

差异表达基因筛选分析发现，TMC-vs-MC共有733个基因、TST-vs-ST共有815个、TFB-vs-FB共有158个基因表达量发生了显著变化，其中，162个基因持续发生显著变化。这些差异表达基因主要富集在酪氨酸代谢通路(ko00350)、精氨酸和脯氨酸代谢通路(ko00330)、DNA复制(ko03030)、色氨酸代谢通路(ko00380)、苯丙氨酸代谢通路(ko00360)、苯丙烷生物合成通路(ko00940)上。研究结果表明，酪氨酸代谢通路、苯丙氨酸代谢通路、精氨酸和脯氨酸代谢通路、苯丙烷生物合成通路相关基因可能参与了虫草发育过程中虫体颜色的变化，这与前人报道相一致。例如，酪氨酸酶是黑色、白色、黄色3种色素前体物质合成过程中的关键限速酶，酪氨酸酶活性或其基因表达受影响时，会造成底物大量积累并沿着某一色素合成方向进行反应^[26-27]。鳞翅目昆虫家蚕已有多个与黑色素合成信号通路、苯丙氨酸代谢通路、苯丙烷生物合成通路相关功能基因被报道，如TH(酪氨酸羟化酶)^[28]、DDC(多巴脱羧酶)^[29]、yellow/laccase2(漆酶)^[30]等。此外，精氨酸不仅能保持蓝莓的品质，还能激活其抗氧化系统^[31]。精氨酸能有效抑制柑橘的酶促褐变^[32]，还能抑制刚切开的苹果和生菜的褐变^[33]。本研究中筛选到1个过氧化氢酶基因katG，该基因能显著富集到苯丙氨酸代谢通路、苯丙烷生物合成通路上，这些代谢通路都与褐变、活性氧、黑化等过程密切相关^[22-25]。由此表明，精氨酸处理虫草后，通过调控过氧化氢酶活性或过氧化氢酶基因的表达，进而激活抗氧化系统，调节虫草颜色的变化。

3.2. 结论

精氨酸处理不同发育时期的虫草，活性氧代谢相关酶(PPO、SOD、POD、CAT)活性和H₂O₂含量变化差异有统计学意义。在虫草发育过程中，TMC-vs-MC总共有733个、TST-vs-ST共有815个、TFB-vs-FB共有158个基因表达量发生了显著变化，其中，162个基因持续发生显著变化。这些差异表达基因主要富集在酪氨酸代谢通路、苯丙氨酸代谢通路、精氨酸和脯氨酸代谢通路、色氨酸代谢通路、DNA复制、苯丙烷生物合成通路上。过氧化氢酶基因katG表达变化趋势与CAT酶活性变化相一致。

Figure (6) Table (2) Reference (33)

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