杨树PagRAX2_16G及其启动子的克隆与功能分析

doi:10.13560/j.cnki.biotech.bull.1985.2025-1259

摘要/Abstract

摘要：

目的 R2R3-MYB转录因子在植物的生长发育过程中具有广泛的调节作用，探究‘84K’杨（Populus alba × P. glandulosa 84K）R2R3-MYB成员PagRAX2_16G的生理功能，为揭示‘84K’杨树次生木质部形成的分子机制提供科学理论依据。方法以‘84K’杨为试材，采用同源克隆方法获得PagRAX2_16G及其启动子，并进行序列分析；使用RT-qPCR和基因特异启动子驱动GUS报告基因的检测方法分析PagRAX2_16G的表达模式；构建pBI121-35S::PagRAX2_16G过表达载体、转化‘84K’杨，经PCR和GUS检测获得过表达PagRAX2_16G阳性植株；以‘84K’杨野生型为对照，测量并统计PagRAX2_16G过表达植株的株高、茎粗、根长、根粗等外部形态特征，并使用石蜡切片技术观察茎和根的内部组织结构。结果 PagRAX2_16G编码区全长999 bp，编码332个氨基酸；PagRAX2_16G启动子序列长度为1 648 bp，含转录起始核心元件TATA-box等启动子基本元件。RT-qPCR结果表明，PagRAX2_16G在次生茎、老根、成熟叶中的表达水平显著高于其在初生茎、幼根、幼叶中的表达水平，且在老根中表达量最高。GUS染色结果表明，PagRAX2_16G主要在器官的维管组织中表达；在具有次生生长的茎和根中，PagRAX2_16G在形成层及其邻近衍生的维管细胞中表达。经表型分析，与‘84K’杨野生型相比，PagRAX2_16G过表达株系的株高、茎粗、根长、根粗均显著增加，且茎和根中的次生木质部宽度与层数、形成层层数、导管和纤维细胞面积均显著增加。结论 PagRAX2_16G正向调节‘84K’杨形成层向次生木质部的分化和木质部细胞的扩张，在根、茎次生木质部形成过程中发挥重要的调节作用。

关键词: ‘84K’杨, R2R3-MYB转录因子, PagRAX2_16G, 表达模式, 功能分析

Abstract:

Objective R2R3-MYB transcription factors play extensive regulatory roles in plant growth and development. The physiological function of PagRAX2_16G, an R2R3-MYB member in ‘84K’ poplar (Populus alba × Populus glandulosa ‘84K’), was investigated to provide a scientific theoretical basis for elucidating the molecular mechanisms underlying secondary xylem formation in ‘84K’ poplar. Method Using ‘84K’ poplar as the experimental material, homologous cloning was adopted to obtain PagRAX2_16G and its promoter, and they were subsequently subjected to sequence analysis. Detection methods of RT-qPCR and gene-specific promoter-driven GUS reporter gene expression were used to analyze the expression pattern of PagRAX2_16G. The pBI121-35S::PagRAX2_16G overexpression vector was constructed, transformed into ‘84K’ poplar, and then PagRAX2_16G-overexpressed positive plants were obtained through PCR and GUS detection. Using the wild-type ‘84K’ poplar as a control, external morphological traits such as plant height, stem diameter, root length, and root diameter of PagRAX2_16G overexpression plants were measured and statistically analyzed. Paraffin sectioning techniques were employed to further observe the internal tissue structures of stems and roots. Result PagRAX2_16G coding region is 999 bp, encoding 332 amino acids. The length of PagRAX2_16G promoter is 1 648 bp and contains essential promoter elements such as the TATA-box transcription initiation core element. The RT-qPCR results showed that the expression of PagRAX2_16G was significantly higher in secondary stems, old roots, and mature leaves than in primary stems, young roots and young leaves, and with the highest expression observed in old roots. GUS staining results revealed that PagRAX2_16G was primarily expressed in the vascular tissues of organs; in stems and roots undergoing secondary growth, PagRAX2_16G was expressed in the cambium and adjacent derived vascular cells. Phenotype analysis indicated that compared to the wild-type ‘84K’ poplar, PagRAX2_16G overexpression plants exhibited significant increases in plant height, stem diameter, root length, and root diameter; moreover, the width and number of secondary xylem layers, the number of cambium cell layers, and the area of vessel and fiber cells in both stems and roots were significantly increased. Conclusion PagRAX2_16G positively regulates the differentiation of the cambium into secondary xylem and the expansion of xylem cells in ‘84K’ poplar, playing a crucial role in the formation of secondary xylem in the roots and stems.

Key words: ‘84K’ poplar, R2R3-MYB transcription factor, PagRAX2_16G, expression pattern, functional analysis

郑淑雅, 苏玉婷, 何玉美, 赵盼, 周心怡, 刘冉, 刘平丽, 郭惠红. 杨树PagRAX2_16G及其启动子的克隆与功能分析[J]. 生物技术通报, 2026, 42(9): 186-195.

ZHENG Shu-ya, SU Yu-ting, HE Yu-mei, ZHAO Pan, ZHOU Xin-yi, LIU Ran, LIU Ping-li, GUO Hui-hong. Cloning and Functional Analysis of Gene PagRAX2_16G and Its Promoter from Poplar[J]. Biotechnology Bulletin, 2026, 42(9): 186-195.

图/表 10

图1 PagRAX2_16G蛋白序列的多重比对A和B代表保守结构域；Pag：‘84K’杨；Pal：银白杨；Ptr：毛果杨；At：拟南芥

Fig. 1 Multiple alignments of PagRAX2_16G protein sequencesA and B indicate conserved domains. Pag: Populus alba × P. glandulosa; Pal: P. alba; Ptr: P. trichocarpa; At: Arabidopsis thaliana

图2 ‘84K’杨PagRAX2_16G与其他物种RAX2_16G蛋白系统发育树分析

Fig. 2 Phylogenetic tree analysis of PagRAX2_16G in '84K' poplar and RAX2_16G proteins from other plant species

图3 PagRAX2_16G的启动子序列及顺式作用元件分析TATA-box：转录起始核心元件；CAAT-box：增强子元件；TGACG-motif和CGTCA-motif：茉莉酸甲酯响应顺式作用元件；GARE-motif：赤霉素响应元件；GC-motif：厌氧特异性诱导相关增强子样元件；ABRE：脱落酸响应顺式作用元件

Fig. 3 Promoter sequence and cis-acting elements analysis of PagRAX2_16GTATA-box: Core components of transcription initiation. CAAT-box: Enhancer elements. TGACG-motif and CGTCA-motif: Cis-acting element of methyl jasmonate response. GARE-motif: Gibberellin response element. GC-motif: Anaerobic-specific inducible enhancer-like element. ABRE: Cis-acting element of abscisic acid response

图4 ‘84K’杨PagRAX2_16G的表达模式分析不同小写字母表示显著性差异（P<0.05）。下同

Fig. 4 Expression pattern analysis of PagRAX2_16G in '84K' poplarDifferent lowercase letters indicate significant differences (P<0.05). The same as below

图5 proPagRAX2_16G的GUS染色结果

Fig. 5 GUS staining result of proPagRAX2_16G

图6 PagRAX2_16G转基因杨树的PCR和GUS检测及RT-qPCR分析A：PagRAX2_16G转基因杨树的PCR检测；B：PagRAX2_16G转基因杨树的GUS检测（Bar=1 cm）；C：PagRAX2_16G转基因杨树的RT-qPCR分析。WT：野生型植株，阴性对照；#2：假阳性植株；#1、#3、#4：转基因阳性植株。下同

Fig. 6 PCR detection, GUS assay and RT-qPCR analysis of PagRAX2_16G transgenic poplarA: PCR detection of PagRAX2_16G transgenic plants. B: GUS assay of PagRAX2_16G transgenic plants (Bar = 1 cm). C: RT-qPCR analysis of PagRAX2_16G transgenic plants. WT: Wild-type plants, negative control. #2: False positive plant. #1, #3, #4: PagRAX2_16G transgenic positive plants. The same below

图7 过表达PagRAX2_16G植株茎与叶的外部形态分析A：‘84K’杨野生型和PagRAX2_16G转基因植株茎与叶的外部形态（Bar=10 cm）；B‒F：茎与叶的外部形态分析

Fig. 7 Analysis of the external morphology of stems and leaves in plants overexpressing PagRAX2_16GA: External morphology of the stems and leaves between wild-type ‘84K’ poplar and PagRAX2_16G-overexpressed ‘84K’ poplars (Bar = 10 cm). B‒F: Analysis of the external morphology of stems and leaves

图8 过表达PagRAX2_16G植株茎的内部结构分析A：‘84K’杨野生型和过表达PagRAX2_16G植株茎第20节间的横切面（Bar=100 µm）；B：‘84K’杨野生型和过表达PagRAX2_16G‘84K’杨植株茎第20节间的形成层区域（Bar=100 µm）；Ph：韧皮部；Xy：木质部；Ca：形成层；C‒G：茎的内部结构分析

Fig. 8 Analysis of internal structure in the stems of plants overexpressing PagRAX2_16GA: Cross-sections of the 20th internodes of stems from wild-type and PagRAX2_16G-overexpressed ‘84K’ poplars (Bar = 100 µm). B: Cambium region of the 20th internodes in stems of wild-type ‘84K’ poplar and PagRAX2_16G-overexpressed ‘84K’ poplars (Bar = 100 µm). Ph: Phloem. Xy: Xylem. Ca: Cambium. C‒G: Analysis of the internal structure of stems

图9 过表达PagRAX2_16G植株根系的外部形态分析A：‘84K’杨野生型和PagRAX2_16G转基因植株根系的外部形态（Bar=10 cm）；B‒F：根系的外部形态分析

Fig. 9 Analysis of the external morphology of roots in plants overexpressing PagRAX2_16GA: External morphology of the roots between wild-type ‘84K’ poplar and PagRAX2_16G-transgenic poplar (Bar = 10 cm). B‒F: Analysis of the external morphology of roots

图10 过表达PagRAX2_16G植株根系的内部结构分析A：‘84K’杨野生型和过表达PagRAX2_16G植株根基部的横切面（Bar=100 µm）；B：‘84K’杨野生型和过表达PagRAX2_16G ‘84K’杨植株根基部的形成层区域（Bar=100 µm）；C‒G：根系的内部结构分析

Fig. 10 Analysis of internal structure in the roots of plants overexpressing PagRAX2_16GA: Cross-sections of the root crown from wild-type ‘84K’ poplar and PagRAX2_16G-overexpressed ‘84K’ poplar (Bar = 100 µm). B: Cambium region of the root crown of wild-type ‘84K’ poplar and ‘84K’ poplar overexpressing PagRAX2_16G (Bar = 100 µm). C‒G: Analysis of the internal structure of roots

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