金针菇II类过氧化物酶基因在子实体发育与胁迫应答过程的表达特征

doi:10.13560/j.cnki.biotech.bull.1985.2023-0624

摘要/Abstract

摘要：

II类过氧化物酶（Class II peroxidase，CIIp）是活性氧代谢的重要抗氧化物酶，参与机体氧化应答过程。基于金针菇全基因组数据鉴定到两个II类过氧化物酶基因，并分别命名为FfCIIp1、FfCIIp2，采用在线网站软件对基因进行生物信息学分析，利用实时荧光定量PCR技术分析基因在金针菇不同子实体组织和胁迫应答中的表达模式。结果显示，FfCIIp1全长1 638 bp，包含一个1 131 bp的完整开放阅读框，编码376个氨基酸。FfCIIp2全长1 410 bp，包含一个1 032 bp的完整开放阅读框，编码343个氨基酸。保守结构域及进化树分析表明，FfCIIp1和FfCIIp2分别归属于锰过氧化物酶和通用过氧化物酶家族。RT-qPCR结果表明，两个FfCIIp基因均在伸长期菌柄中高表达，且在快速伸长区段显著上调；损伤和氧化胁迫处理均能诱导FfCIIp1和FfCIIp2上调表达，FfCIIp2的上调幅度明显高于FfCIIp1。以上结果表明，金针菇两个CIIp家族基因参与菌柄伸长与胁迫应答过程，且FfCIIp2对损伤和氧化胁迫更为敏感。

关键词: 金针菇, 锰过氧化物酶, 通用过氧化物酶, 子实体发育, 胁迫应答, 表达分析

Abstract:

Class II peroxidase（CIIp）is an important antioxidant enzyme in active oxygen metabolism, and participates in the oxidative response of organism. Based on the genomic data of Fammulina filiformis, two Class II peroxidase genes were identified and named as FfCIIp1 and FfCIIp2, respectively. Online software was used for bioinformatics analysis of the genes, and real-time fluorescence quantitative PCR was used to analyze the expression patterns of genes in different fruit body tissues and stress responses of F. filiformis. The results showed that the full length of FfCIIp1 was 1 638 bp, including a complete open reading frame of 1 131 bp, encoding 376 amino acids. FfCIIp2 was 1 410 bp in length, containing a complete open reading frame of 1 032 bp and encoding 343 amino acids. Conserved domain and phylogenetic analysis showed that FfCIIp1 and FfCIIp2 belonged to the manganese peroxidase and generic peroxidase families, respectively. RT-qPCR results showed that both FfCIIp genes were highly expressed in the stipe during the elongation stage, and significantly up-regulated in the stipe of fast elongation region. FfCIIp1 and FfCIIp2 were up-regulated by both injury and oxidative stress, and the up-regulated range of FfCIIp2 was significantly higher than FfCIIp1. These results indicate that two CIIp family genes are involved in stipe elongation and stress response, and FfCIIp2 is more sensitive to injury and oxidative stress of F. filiformis.

Key words: Flammulina filiformis, manganese peroxidase, generic peroxidase, fruiting body development, stress response, expression analysis

刘媛媛, 魏传正, 谢永波, 仝宗军, 韩星, 甘炳成, 谢宝贵, 严俊杰. 金针菇II类过氧化物酶基因在子实体发育与胁迫应答过程的表达特征[J]. 生物技术通报, 2023, 39(11): 340-349.

LIU Yuan-yuan, WEI Chuan-zheng, XIE Yong-bo, TONG Zong-jun, HAN Xing, GAN Bing-cheng, XIE Bao-gui, YAN Jun-jie. Characteristics of Class II Peroxidase Gene Expression During Fruiting Body Development and Stress Response in Flammulina filiformis[J]. Biotechnology Bulletin, 2023, 39(11): 340-349.

图/表 8

表1 引物序列

Table 1 Sequences of primers

引物名称Primer name	引物序列Primer sequence（5'-3'）
qFfGAPDH-F qFfGAPDH-R	CCTCTGCTCACTTGAAGGGT
qFfGAPDH-F qFfGAPDH-R	GCGTTGGAGATGACTTTGAA
qFfCIIp1-F qFfCIIp1-R	TGACTTTCGCATTCCCCGAA
qFfCIIp1-F qFfCIIp1-R	ACAGAGAGTCAAGGCAACGG
qFfCIIp2-F	AAGTCGAAAGGCGGGACAAT
qFfCIIp2-R	CCGTCAGGATTGATGGTCGT

图1 金针菇FfCIIp基因结构示意图

Fig. 1 Schematic map of the FfCIIp genes of F. filiformis

图2 金针菇FfCIIp蛋白的三级结构

Fig. 2 Tertiary structures of F. filiformis FfCIIp proteins

图3 金针菇FfCIIp与其他担子菌CIIp的系统进化树标尺：进化距离；分支处数字：自检置信度（Bootstrap=1 000）；分支名称：数据库的ID号、物种拉丁名和序列名称组成；正方形：金针菇FfCIIp序列

Fig. 3 Phylogenetic tree of F. filiformis FfCIIp and CIIp from other basidiomycetes The bar: Evolutionary distance. The values above branches: The degree of bootstrap support from a 1000 replicate analysis. The taxon names of branches: composed of ID in database, Latin name of species and sequence name. Square: F. filiformis FfCIIp sequence

图4 金针菇与其他真菌CIIp蛋白的多序列比对与保守结构分析碱基的背景色：颜色越深位点保守性越高；下划线：过氧化物酶的保守结构域；FfCIIp1、FfCIIp2：金针菇II类过氧化物酶序列；黑色实心倒三角：MnP和VP家族中酸性氨基酸残基对Mn2+氧化起关键作用的位点；空心倒三角：LiP和VP家族中色氨基酸残基对酚类化合物氧化起关键作用的位点；GP家族：无以上4个催化位点

Fig. 4 Multiple sequence alignment and conserved domain analysis of CIIp amino acid sequences from F. filiformis and other fungi Base background color: The darker the color, the more conservative the site. The transverse line: Peroxidase domain. FfCIIp1, FfCIIp2: Class II peroxidase sequences of F. filiformis. Black solid inverted triangle: Three acidic amino acid residues in the MnP and VP families are crucial for Mn2+ oxidation. Hollow inverted triangle: Tryptophan residue in the LiP and VP families is responsible for oxidation of phenolic compounds. GP: No above 4 catalytic sites

图5 金针菇FfCIIp基因在不同子实体组织中的差异表达 A：FfCIIp1在子实体样品中的差异表达（PR：原基；DI：分化期；EL-stipe：伸长期菌柄；EL-pileus：伸长期菌盖；MA-stipe：成熟期菌柄、MA-pileus：成熟期菌盖）；B：FfCIIp2在子实体样品中的差异表达；C：FfCIIp1在菌柄不同区段中的差异表达（elo：为快速伸长区；sta：为稳定区，此区段不再伸长）；D：FfCIIp2在菌柄不同区段中的差异表达。不同字母代表差异显著，P<0.05，n=3

Fig. 5 Expression patterns of FfCIIp during fruiting body tissues of F. filiformis A: Differential expressions of FfCIIp1 in fruiting body samples（PR: Primordium stage. DI: Differentiation stage. EL-stipe: The stipe of elongation stage. EL-pileus: The pileus of elongation stage. MA-stipe: The stipe of maturation stage. MA-pileus: The pileus of maturation stage）. B: Differential expression of FfCIIp2 in fruiting body samples. C: Differential expression of FfCIIp1 in different segment of stipe（elo: Fastest elongating region. sta: Stable region, this region no longer elongated）. D: Differential expression of FfCIIp2 in different segment of stipe. The different letters over the columns within the same graph denote significant differences. P<0.05, n=3

图6 金针菇菌丝损伤胁迫后FfCIIp基因的差异表达 A：金针菇Fv01菌丝损伤胁迫处理；B：FfCIIp1在应答损伤胁迫的差异表达；C：FfCIIp2在应答损伤胁迫的差异表达（*号代表与0 h样品的差异显著，*：P<0.05，**：P<0.01，***：P<0.001，n=3）

Fig. 6 Expression pattern of FfCIIp gene after mechanical injury to F. filiformis hyphae A: Fv01 mycelial injury stress induction. B: The expression pattern of FfCIIp1 in response to mechanical injury of F. filiformis. C: The expression pattern of F. filiformis FfCIIp2 in response to mechanical injury of F. filiformis（The ‘*’ over the columns denotes significant differences with 0 h. *: P<0.05, **: P<0.01, ***: P<0.001, n=3）

图7 金针菇菌丝H2O2胁迫后FfCIIp基因的差异表达 A：FfCIIp1在应答H2O2胁迫的差异表达；B：FfCIIp2在应答H2O2胁迫的差异表达（*号代表与0 mmol/L样品的差异显著，*：P<0.05，**：P<0.01，***：P<0.001，n=3）

Fig. 7 Expression pattern of FfCIIp gene after H2O2 stress of F. filiformis hyphae A: The expression pattern of FfCIIp1 in response to H2O2 stress. B: The expression pattern of FfCIIp2 in response to H2O2 stress.（The ‘*’ over the columns denotes significant differences with 0 mmol/L. *: P<0.05，**: P<0.01，***: P<0.001，n=3）

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