Cloning of FfCYP98 Gene and Its Functional Analysis in Folioceros fuciformis

doi:10.13560/j.cnki.biotech.bull.1985.2024-0068

Abstract

Abstract:

【Objective】 Cytochrome P450 monooxygenase 98（CYP98）is a key rate-limiting enzyme in the phenylpropanoid pathway, and it is proposed to investigate whether it is involved in the biosynthesis of the phenylpropanoid pathway and in the anti-disease function in Hornworts, so as to provide a reference for the future study of the evolution of the early terrestrial plants and physiological mechanisms of adaptation to adversity stress. 【Method】 The full-length sequence of FfCYP98 cDNA was cloned from Folioceros fuciformis using RACE, analyzed for bioinformatics and subcellular localization, and functionally verified by constructing an overexpression vector to transform the Arabidopsis thaliana mutant FfCYP98-OE1. 【Result】 The FfCYP98 cDNA sequence, with an open reading frame of 1305 bp, is evolutionarily closest to the Bryophytes Anthoceros angustus and Physcomitrium patens CYP98, and subcellular localization results show that FfCYP98 is localized to both the nucleus and the cytoplasm. The overexpression of the FfCYP98 gene revealed that the expressions of total phenolics, total flavonoids, and C4H,4CL and C3H in the phenylpropanoid pathway was significantly higher in FfCYP98-OE1 plants than in A. thaliana WT plants. In addition, infestation of F. fuciformis and A. thaliana FfCYP98-OE1 plants with Botrytis cinerea revealed that the expression of FfCYP98 was significantly up-regulated, that FfCYP98-OE1 plants died significantly slower than WT plants, and that the expressions of total phenolics, total flavonoids, and four genes, C4H, HCT, C3H, and CAD in the phenylpropanoid pathway were significantly higher in A. thaliana FfCYP98-OE1 plants than in WT plants. 【Conclusion】 FfCYP98 may be involved in the biosynthesis of phenolics and flavonoids in the phenylpropanoid pathway by regulating the expressions of genes related to the synthesis of phenylpropanoid pathway, and may be related to disease resistance of plants.

Key words: Folioceros fuciformis, gene cloning, FfCYP98, Botrytis cinerea, resistance

HUANG Dan, JIANG Shan, PENG Tao. Cloning of FfCYP98 Gene and Its Functional Analysis in Folioceros fuciformis[J]. Biotechnology Bulletin, 2024, 40(7): 273-284.

Figures/Tables 6

Table 1 Primer sequences used in the study

引物名称Primer name	引物序列Primer sequence（5'-3'）
JB-F	ATGAGGAGTTTGCGAAGCA
JB-R	CCTTCCCCTCGTCGTCCAC
3'RACE 特异性引物F1	AGGGGCCGCAGTCTCATTTCGT
3'RACE 特异性引物F2	CTCCTGGGGCTGCAGAAGCAGT
5'RACE 特异性引物R1	AACGCGAGGCGCGTAATGTTGT
5'RACE 特异性引物R2	GCCGAGAGGTACGTCTTGGGGTT
FfCYP98-F	TCTAGAATGGGAGCAACTCTGAACGT
FfCYP98-R	GGATCCGACGGTTTTGGCATACAAGG
HACtin-F	ACGCCTGAACATCTCCTGAA
HACtin-R	CGTGCAGAACAAGAACTCGT
qpcr-F	CGCAGTCTCATTTCGTGGAC
qpcr-R	TCCTTCTGCACCTTCCTCTG
跨载体-F	ccaaccacgtcttcaaagca
跨载体-R	atccagactgaatgcccaca
ACtion2-F	GGAAGGATCTGTACGGTAAC
ACtion2-R	GGACCTGCCTCATCTTACT
Action-F	TTCAATGTCCCTGCCATGTATG
Action-R	AATACCGGTTGTACGACCAC
PAL-F	TCTGCGAAAAGGATTTACTCAAAGTT
PAL-R	CTACAAGGATCATCCGCGTATGT
C4H-F	CCTCCAGGACAGTCTAAAGTGGATACTA
C4H-R	CGATTATGGAGTGGTTAAGGATGTG
HCT-F	TCTTTATGGGACCTGGTGGAATT
HCT-R	GATAAGCTGCCATCATTAGTAGGACTTG
4CL-F	TCAACCCGGTGAGATTTGTA
4CL-R	TCGTCATCGATCAATCCAAT
C3H-F	AAGTCTAGTGGAGCGAAACAGCATT
C3H-R	TCCTCACTAAGATCATACTGATCCTTCA
CAD-F	TGCGATTGGATCCGATGGAA
CAD-R	GCTTCCCTGCCTCAGTCATA

Fig. 1 Cloning of FfCYP98 M：DNA maker DL15000 ; 1：FfCYP98

Fig. 2 Bioinformatics analysis of the FfCYP98 A: Physicochemical characterization and secondary structure prediction of FfCYP98. B: Tertiary structure prediction of FfCYP98. C: Analysis of hydrophilicity and hydrophobicity of FfCYP98 amino acid sequences. D: Analysis of the transmembrane structural domains of FfCYP98. E: Phylogenetic tree of FfCYP98

Fig. 3 Subcellular localization of the protein FfCYP98 The asterisk indicates the nucleus, and the arrow indicates the cytoplasm

Fig. 4 Identification of transgenic A. thaliana and relative expressions of total phenols, total flavonoids and phenylpropane synthesis pathway genes in transgenic A. thaliana A: Resistance screening for Arabidopsis thaliana. B: Identification of transgenic A. thaliana, M: Marker = 15 000 bp, 1: Control, 2: WT. C: Expression of the FfCYP98 gene intransgenic A. thaliana（***P< 0.001,****P< 0.0001）. D: Detection of total phenols and total flavonoids in transgenic A. thaliana. E: Expression analysis of transgenic A. thaliana phenylpropane synthesis pathway genes

Fig. 5 Infected behavior of B. cinerea on F. fuciformis and characterization of FfCYP98 expression in transgenic A. thaliana under B. cinerea infestation A: B. cinerea infests Folioceros fuciformis, bar=100 μm. B: Comparison of the invasion rate of hyphae in F. fuciformis. C: Expression of FfCYP98 at different times of F. fuciformis under the stress of B. cinerea. D: Observation of transgenic A. thaliana infection with B. cinerea, bar= 1 cm. E: Expressions of FfCYP98 at different times of transgenic A. thaliana under the stress of B. cinerea, **P< 0.01, ***P< 0.001, ****P< 0.0001

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