山苍子PAT1基因亚家族鉴定及其在嫁接愈合过程中的表达分析

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

摘要/Abstract

摘要：

目的探索山苍子（Litsea cubeba）PAT1亚家族（phytochrome A signal transduction 1 subfamily）在嫁接愈合中的作用，对其成员进行系统鉴定与表达分析，为后续的功能验证与分子改良提供理论基础。方法利用生物信息学方法对山苍子GRAS基因家族进行全基因组鉴定，共鉴定出45个成员，其中PAT1亚家族包含6个基因。通过分析其理化性质、基因结构、保守基序、顺式作用元件和共线性关系，结合转录组和RT-qPCR结果，探讨其在不同组织及嫁接愈合过程中的表达模式。同时利用加权基因共表达网络分析（WGCNA）挖掘核心调控基因及其潜在功能模块。结果 6个LcPAT1基因不均匀分布于4条染色体上，其启动子富含光响应元件及ABA、MeJA、SA等激素响应元件。基于山苍子不同组织转录组数据的WGCNA结果显示，核心基因LcPAT1-3与346个基因显著共表达，这些基因主要富集于转录调控、激素信号转导及碳水化合物和次生代谢通路，提示其在嫁接愈合过程中的枢纽作用。不同组织的转录组和RT-qPCR结果均显示，6个LcPAT1基因在茎中表达较高。不同嫁接愈合时期的RT-qPCR结果显示，LcGRAS04与LcPAT1-2表达量在形成层连接期均升高，LcPAT1-3和LcPAT1-4在维管分化期持续高表达，呈现明显的时空特异性。结论山苍子PAT1亚家族成员在顺式元件和表达模式上存在分化。LcPAT1-3可能作为关键调控因子，协同其他成员在嫁接愈合的不同阶段调控形成层激活、维管再生及防御加固等过程。

关键词: 山苍子, PAT1亚家族, 嫁接, WGCNA, 表达模式, GRAS基因家族, 系统发育分析, 顺式作用元件

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 had 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

梁慧琪, 陈益存, 汪阳东, 赵耘霄, 高暝. 山苍子PAT1基因亚家族鉴定及其在嫁接愈合过程中的表达分析[J]. 生物技术通报, doi: 10.13560/j.cnki.biotech.bull.1985.2025-1133.

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

图/表 10

图1 山苍子及拟南芥GRAS基因系统发育进化树红色字体代表山苍子，黑色字体代表拟南芥

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

表1 LcPAT1蛋白理化性质与亚细胞定位

Table 1 Physicochemical properties and subcellular localization of LcPAT1 protein

基因名称Gene name	蛋白长度Number of amino acids	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	-

图2 LcPAT1基因染色体定位图

Fig. 2 Chromosomal localization of LcPAT1s genes

图3 LcPAT1基因保守基序组成及基因结构预测

Fig. 3 Composition of conserved motifs of the LcPAT1s genes and prediction of gene structure

图4 LcPAT1转录因子启动子顺式作用元件分析

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

图5 LcPAT1基因的共线性分析

Fig. 5 Synteny analysis of LcPAT1s genes

图6 LcPAT1-3共表达网络黄色菱形表示基于COG注释的功能簇，圆形表示基因。基因节点颜色对应其功能大类：紫色代表信息存储与处理，绿色代表代谢，蓝色代表细胞过程与信号转导

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

图7 山苍子不同组织转录组中LcPAT1基因的表达量热图

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

图8 山苍子不同组织中PAT1亚家族基因的相对表达量RT-qPCR结果（柱状图，左Y轴）与转录组log2（FPKM+1）值（折线图，右Y轴）展示于同一图中。样品采集自与转录组分析相同的植株。qPCR数据表示为均值±SD（n=3）。*P<0.05，下同

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

图9 山苍子嫁接愈合过程中PAT1亚家族基因的相对表达量U：结合处上部未嫁接砧木茎段；C：嫁接结合处砧穗茎段；B：结合处下部未嫁接砧木茎段；Stage 1：隔离层形成期；2：愈伤组织形成期；3：形成层连接期；4：维管组织分化期

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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