Identification and Expression Analysis of the RALF Gene Family in Panax notoginseng

doi:10.13560/j.cnki.biotech.bull.1985.2025-0585

Abstract

Abstract:

Objective Rapid alkalinization factors (RALFs) are plant peptide hormones that participate in immune responses. This study is aimed to identify the RALF gene family members in Panax notoginseng and to investigate the roles of PnoRALFs in P. notoginseng immune responses. Method The RALF gene family in P. notoginseng was identified using bioinformatics tools. Chromosomal localization, protein physicochemical properties, phylogenetic relationships, conserved motifs, gene structures, promoter cis-acting elements, and tissue-specific expression patterns were analyzed. Expression profiles were assessed under four hormone treatments-salicylic acid (SA), methyl jasmonate (MeJA), abscisic acid (ABA), and ethylene (ETH) and after infection with C. destructans. The function of a key gene was further validated through transient expression in Nicotiana benthamiana. Result Thirteen PnoRALFs genes were identified and mapped to eight chromosomes. These genes were classified into two subgroups. Conserved motif analysis showed the presence of RRXL and YISY motifs. Promoter regions contained multiple hormone-responsive cis-elements, including those responsive to ABA, MeJA, SA, and gibberellins (GA). Tissue-specific expression analysis revealed considerable variation in gene expressions among different tissues. The expressions of PnoRALF2, PnoRALF5, PnoRALF6, and PnoRALF9 were upregulated by hormone treatments. Transcriptome and RT-qPCR analyses indicated that nine PnoRALFs genes responded to C. destructans infection, with PnoRALF9 showing a marked increase in expression. Transient overexpression of PnoRALF9 in N. benthamiana increased the expressions of immunity-related genes such as NbFPK1 and NbWRKY29 in the pattern-triggered immunity (PTI) pathway and NbMPK6 and NbGAPDH in the reactive oxygen species (ROS) signaling pathway. Conclusion Thirteen PnoRALFs genes are identified in the P. notoginseng genome. Among them, PnoRALF9 responds to multiple hormone treatments and C. destructans infection. Transient overexpression of PnoRALF9 induces plant immunity-related gene expression, suggesting that PnoRALF9 plays a critical role in the immune response in P. notoginseng to disease.

Key words: Panax notoginseng, RALF gene family, hormone treatment, Cylindrocarpon destructans, expression analysis

YANG Zi-han, LI Kui-xiu, LI Jun-liang, LYU Wen-hui, HE Wen-ting, LIU Xu-yan, LIU Guan-ze. Identification and Expression Analysis of the RALF Gene Family in Panax notoginseng[J]. Biotechnology Bulletin, 2026, 42(2): 278-292.

Figures/Tables 15

References 42

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基因 Gene	上游引物Forward primer （5′-3′）	下游引物序列Reverse primer （5′-3′）
PnACT2	GATTGATCTTGGCACCGGGA	TGACCAACCCACGACCTTTC
PnoRALF1	GCTACATGCAACGGTCTGATC	TGCAGTTGTAGTAGGAGGCTC
PnoRALF2	CCACCTGAACTCTATTGGGT	CGCACCGTAGCTGATGTA
PnoRALF3	AGTGATGCTGATGGACTCGG	CGTCTGTAGGGATTGGCTCT
PnoRALF4	CCACCTGAACTCTATTGGGT	GGGAGTACGGATTTGCCTG
PnoRALF5	CGACATGGATTCAGAGAGC	ACGGTTGTAAGGGTTAGCTT
PnoRALF6	CGCCTCCTACTACAACTGTA	ACCAGCATCCATCACAAATC
PnoRALF7	GCAAACAGTCGGTACATCAG	GGGAGTACGGATTTGCCTG
PnoRALF8	GATTCATGGTCGTTTGTCAC	CTGGCCTCGTTTGTTACATG
PnoRALF9	TGATCTTGCTCCCATTTCCA	GGCTGATCTCCGAATCCATA
PnoRALF10	TGTTTCGACGGTGGTGTTTC	CACACACCTGCTCGTTTCTC
PnoRALF11	ATGATGTTGGATTCGGAGGC	CGTCTGTAGGGATTGGCTCT
PnoRALF12	AGTCCTAATCCTGCTGTGTTTC	CGGTTTATCTCCGAATCCATCA
PnoRALF13	CGGAGGTTTCTTGCAGCAAA	CGGAGGTTTCTTGCAGCAAA
pEAQ-HT-PnoRALF9	cccaaattcgcgaccggtATGGGAAAG TCTACTGGTTTTCTCG	atgaaaccagagttaaaggcctcgagACGCC GGCACCGAGTGAT

基因 Gene	上游引物Forward primer （5′-3′）	下游引物序列Reverse primer （5′-3′）
PnACT2	GATTGATCTTGGCACCGGGA	TGACCAACCCACGACCTTTC
PnoRALF1	GCTACATGCAACGGTCTGATC	TGCAGTTGTAGTAGGAGGCTC
PnoRALF2	CCACCTGAACTCTATTGGGT	CGCACCGTAGCTGATGTA
PnoRALF3	AGTGATGCTGATGGACTCGG	CGTCTGTAGGGATTGGCTCT
PnoRALF4	CCACCTGAACTCTATTGGGT	GGGAGTACGGATTTGCCTG
PnoRALF5	CGACATGGATTCAGAGAGC	ACGGTTGTAAGGGTTAGCTT
PnoRALF6	CGCCTCCTACTACAACTGTA	ACCAGCATCCATCACAAATC
PnoRALF7	GCAAACAGTCGGTACATCAG	GGGAGTACGGATTTGCCTG
PnoRALF8	GATTCATGGTCGTTTGTCAC	CTGGCCTCGTTTGTTACATG
PnoRALF9	TGATCTTGCTCCCATTTCCA	GGCTGATCTCCGAATCCATA
PnoRALF10	TGTTTCGACGGTGGTGTTTC	CACACACCTGCTCGTTTCTC
PnoRALF11	ATGATGTTGGATTCGGAGGC	CGTCTGTAGGGATTGGCTCT
PnoRALF12	AGTCCTAATCCTGCTGTGTTTC	CGGTTTATCTCCGAATCCATCA
PnoRALF13	CGGAGGTTTCTTGCAGCAAA	CGGAGGTTTCTTGCAGCAAA
pEAQ-HT-PnoRALF9	cccaaattcgcgaccggtATGGGAAAG TCTACTGGTTTTCTCG	atgaaaccagagttaaaggcctcgagACGCC GGCACCGAGTGAT

基因	基因ID	氨基酸数	分子量	等电点	亲水性	亚细胞定位	信号肽
Gene name	Gene ID	Number of amino acids	Molecular weight （kD）	pl	GRAVY	Subcellular localization	Signal peptide
PnoRALF1	Pno01G005926.t1	128	14.06	9.03	-0.30	细胞外Extracellular	Yes
PnoRALF2	Pno03G001289.t1	126	13.74	8.91	-0.11	细胞外Extracellular	Yes
PnoRALF3	Pno03G005478.t1	131	14.39	0.24	-0.22	细胞外Extracellular	Yes
PnoRALF4	Pno03G005479.t1	125	13.75	9.54	-0.44	细胞外Extracellular	Yes
PnoRALF5	Pno06G000518.t1	114	12.33	8.60	-0.20	细胞外Extracellular	Yes
PnoRALF6	Pno07G004032.t1	158	17.28	9.15	0.24	质膜 Plasma membrane	Yes
PnoRALF7	Pno09G001048.t1	109	12.07	7.50	-0.35	细胞外Extracellular	Yes
PnoRALF8	Pno10G000839.t1	123	13.77	9.83	-0.19	细胞外Extracellular	No
PnoRALF9	Pno11G003888.t1	133	14.33	8.99	-0.08	细胞外Extracellular	Yes
PnoRALF10	Pno12G001525.t1	110	12.31	7.54	-0.07	细胞外Extracellular	Yes
PnoRALF11	Pno284G000001.t1	125	12.75	9.54	-0.44	细胞外Extracellular	Yes
PnoRALF12	Pno606G000002.t1	110	12.30	7.54	-0.07	细胞外Extracellular	Yes
PnoRALF13	Pno606G000003.t1	113	12.46	8.69	-0.29	细胞外Extracellular	Yes

基因	基因ID	氨基酸数	分子量	等电点	亲水性	亚细胞定位	信号肽
Gene name	Gene ID	Number of amino acids	Molecular weight （kD）	pl	GRAVY	Subcellular localization	Signal peptide
PnoRALF1	Pno01G005926.t1	128	14.06	9.03	-0.30	细胞外Extracellular	Yes
PnoRALF2	Pno03G001289.t1	126	13.74	8.91	-0.11	细胞外Extracellular	Yes
PnoRALF3	Pno03G005478.t1	131	14.39	0.24	-0.22	细胞外Extracellular	Yes
PnoRALF4	Pno03G005479.t1	125	13.75	9.54	-0.44	细胞外Extracellular	Yes
PnoRALF5	Pno06G000518.t1	114	12.33	8.60	-0.20	细胞外Extracellular	Yes
PnoRALF6	Pno07G004032.t1	158	17.28	9.15	0.24	质膜 Plasma membrane	Yes
PnoRALF7	Pno09G001048.t1	109	12.07	7.50	-0.35	细胞外Extracellular	Yes
PnoRALF8	Pno10G000839.t1	123	13.77	9.83	-0.19	细胞外Extracellular	No
PnoRALF9	Pno11G003888.t1	133	14.33	8.99	-0.08	细胞外Extracellular	Yes
PnoRALF10	Pno12G001525.t1	110	12.31	7.54	-0.07	细胞外Extracellular	Yes
PnoRALF11	Pno284G000001.t1	125	12.75	9.54	-0.44	细胞外Extracellular	Yes
PnoRALF12	Pno606G000002.t1	110	12.30	7.54	-0.07	细胞外Extracellular	Yes
PnoRALF13	Pno606G000003.t1	113	12.46	8.69	-0.29	细胞外Extracellular	Yes