Characteristics of Class II Peroxidase Gene Expression During Fruiting Body Development and Stress Response in Flammulina filiformis

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

Abstract

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

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.

Figures/Tables 8

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

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

Fig. 2 Tertiary structures of F. filiformis FfCIIp proteins

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

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

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

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）

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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