Identification and Expression Analysis of the PAT1 Gene Subfamily in Litsea cubeba during Graft Union Healing

doi:10.13560/j.cnki.biotech.bull.1985.2025-1133

Abstract

Abstract:

Objective To explore the role of the Phytochrome A Signal Transduction 1 (PAT1) subfamily in graft union formation of Litsea cubeba, members of this subfamily were systematically identified and analyzed. This provides a theoretical basis for subsequent functional verification and molecular improvement. Method The GRAS gene family of L. cubeba was identified at the genome-wide level using bioinformatics approaches, yielding 45 members, including six genes in the PAT1 subfamily. Their physicochemical properties, gene structures, conserved motifs, cis-acting elements, and synteny relationships were characterized. Expression patterns in different tissues and during graft union formation were examined using transcriptome data and quantitative real-time PCR (RT-qPCR). In addition, weighted gene co-expression network analysis (WGCNA) was employed to identify key regulatory genes and potential functional modules. Result The six LcPAT1 genes were unevenly distributed across four chromosomes, and their promoters were enriched in light-responsive and hormone-responsive elements, including abscisic acid (ABA), methyl jasmonate (MeJA), and salicylic acid (SA). WGCNA results based on transcriptome data from various tissues revealed that the hub gene LcPAT1-3 was strongly co-expressed with 346 genes mainly involved in transcriptional regulation, hormone signaling, and carbohydrate and secondary metabolism pathways, suggesting a central regulatory role in graft healing. Both transcriptome and RT-qPCR analyses showed that the six LcPAT1 genes showed higher expression in stems. During graft union formation, RT-qPCR results indicated that LcGRAS04 and LcPAT1-2 were upregulated during the cambial connection stage, while LcPAT1-3 and LcPAT1-4 maintained high expression during vascular differentiation, displaying distinct spatiotemporal specificity. Conclusion Members of the L. cubeba PAT1 subfamily show functional divergence in their cis-regulatory elements and expression patterns. LcPAT1-3 may act as a key regulatory factor that cooperates with other members to control cambium activation, vascular regeneration, and defense reinforcement during different stages of graft union formation.

Key words: Litsea cubeba, PAT1 subfamily, grafting, WGCNA, expression pattern, GRAS gene family, phylogenetic analysis, cis-acting elements

LIANG Hui-qi, CHEN Yi-cun, WANG Yang-dong, ZHAO Yun-xiao, GAO Ming. Identification and Expression Analysis of the PAT1 Gene Subfamily in Litsea cubeba during Graft Union Healing[J]. Biotechnology Bulletin, 2026, 42(5): 302-311.

Figures/Tables 10

Fig. 1 Phylogenetic tree of GRAS genes from L. cubeba and A. thalianaThe red font indicates L. cubeba, the black font indicates A. thaliana

Table 1 Physicochemical properties and subcellular localization of LcPAT1 protein

基因名称Gene name	蛋白长度Number of amino acids （aa）	分子量Molecular weight （kD）	等电点Theoretical pI	不稳定系数Instability index	脂肪系数Aliphatic index	亲水性平均值Grand average of hydropathicity	亚细胞定位Subcellular localization
LcGRAS04	582	78.07	5.05	50.50	80.43	-0.24	Nucleus
LcGRAS30	591	86.07	6.41	57.11	78.83	-0.32	Nucleus
LcPAT1-1	583	71.07	5.46	53.31	83.60	-0.43	Nucleus
LcPAT1-2	579	110.07	5.84	56.06	79.36	-0.41	Nucleus
LcPAT1-3	589	69.07	5.49	50.44	82.77	-0.27	Nucleus
LcPAT1-4	586	60.07	5.76	48.94	80.89	-0.31	Cytoplasm
平均值 Average	585	79.07	5.67	52.73	80.98	-0.33	-

Fig. 2 Chromosomal localization of LcPAT1s genes

Fig. 3 Conserved motifs composition and gene structure prediction of the LcPAT1s genes

Fig. 4 Cis-acting element analysis of LcPAT1s transcription factor promoters

Fig. 5 Synteny analysis of LcPAT1s genes

Fig. 6 Co-expression network diagram of LcPAT1-3Yellow diamonds indicate functional clusters based on COG annotations, while circles indicate genes. The color of each gene node corresponds to its functional category: Purple indicates information storage and processing, green indicates metabolism, and blue denotes cellular processes and signal transduction

Fig. 7 Expression heatmap of gene LcPAT1s across different tissues based on transcriptome data in L. cubeba

Fig. 8 Relative expressions of PAT1 subfamily genes in different tissues of L. cubebaThe RT-qPCR results (bar chart, left Y-axis) and transcriptome log₂(FPKM+1) values (line chart, right Y-axis) are presented in the same graph. The samples were collected from the same plants used for the transcriptome analysis. qPCR data are presented as mean ± SD (n=3). *P<0.05. The same below

Fig. 9 Relative expressions of PAT1 subfamily genes during graft union healing in L. cubebaU: Upper region of the junction without grafted rootstock stem segment. C: Stem segment of rootstock and scion at the junction of grafting. B: Basal region of the junction without grafted rootstock stem segment. Stage1: Formation period of the isolation layer. 2: Formation period of callus tissue. 3: Cambium connection. 4: Differentiation period of vascular tissue

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