生长素诱导调节PagHAM4a和PagHAM4b的表达及对杨树次生木质部发育的影响

doi:10.13560/j.cnki.biotech.bull.1985.2025-1092

摘要/Abstract

摘要：

目的生长素在植物生长发育过程中发挥关键作用，探究外源生长素（IAA）对84K杨（Populus alba×Populus glandulosa）PagHAM4a和PagHAM4b表达的调控作用，以及在杨树次生木质部发育过程中的作用，为探索转录因子与IAA互作对树木次生木质部发育提供依据。方法以84K杨为试材，外源施加IAA，使用RT-qPCR检测PagHAM4a和PagHAM4b的表达水平，通过构建2个基因的表达载体：一个载体包含由生长素响应启动子DR5驱动的GFP报告基因和由PagHAM4a特异性启动子驱动的GUS报告基因；另一个载体包含由DR5驱动的GFP报告基因和由PagHAM4b特异性启动子驱动的GUS报告基因，探究PagHAM4a和PagHAM4b的表达区域与生长素分布之间的关系。同时，通过测量、统计，并结合石蜡切片技术观察IAA处理的84K杨和过表达植株（PagHAM4a-OE/PagHAM4b-OE）的外部形态和茎中次生木质部的显微结构。结果 RT-qPCR结果表明，外源IAA的施加显著上调了PagHAM4a和PagHAM4b的表达；双报告基因检测结果显示，PagHAM4a和PagHAM4b在84K杨次生茎中的表达区域与生长素的分布区域一致；表型分析显示，与未施加IAA相比，施加IAA的84K杨和过表达植株的株高、叶面积、茎粗和节间数均显著增加，并且茎中次生木质部的宽度、细胞层数和形成层的宽度、细胞层数均显著增加，这与PagHAM4a和PagHAM4b表达水平的上调趋势一致。结论施用外源IAA，对84K杨PagHAM4a和PagHAM4b的表达以及茎中次生木质部的发育具有促进作用，PagHAM4a和PagHAM4b通过生长素信号途径参与调节次生木质部的发育。

关键词: 84K杨, IAA, PagHAM4a/PagHAM4b, 双报告基因, 次生木质部

Abstract:

Objective Auxin plays a critical role in plant growth and development. This study aims to explore the regulatory role of exogenous auxin (IAA) on the expressions of PagHAM4a and PagHAM4b in 84K poplar (Populus alba × Populus glandulosa), as well as its effect on the secondary xylem development in poplar, providing a basis for exploring the interaction between transcription factors and IAA in the development of secondary xylem in trees. Method Using 84K poplar as experimental material, exogenous IAA was applied. RT-qPCR technology was employed to detect the expressions of PagHAM4a and PagHAM4b. By constructing expression vectors for two genes: one vector containing a GFP reporter driven by the auxin-responsive promoter DR5 and a GUS reporter driven by the PagHAM4a-specific promoter; the other vector containing the DR5-driven GFP reporter and the PagHAM4b-specific promoter-driven GUS reporter, to investigate the relationship between the expression regions of the PagHAM4a/PagHAM4b and auxin distribution. Simultaneously, through measurement and statistical analysis, combined with paraffin sectioning techniques, the external morphology and the microscopic structure of stem secondary xylem in IAA-treated wild-type 84K poplar, PagHAM4a- and PagHAM4b- overexpressing plants (PagHAM4a-OE and PagHAM4b-OE) were observed. Result RT-qPCR result indicated that exogenous IAA application significantly up-regulated the expressions of both PagHAM4a and PagHAM4b. Dual reporter gene assays revealed that expression regions of the PagHAM4a and PagHAM4b in the secondary stem of 84K poplar coincided with the distribution of auxin. Phenotypic analysis showed that, compared with untreated plants, the IAA application significantly increased plant height, leaf area, stem diameter, and internode number in 84K poplar, PagHAM4a-OE and PagHAM4b-OE plants, and also resulted in a significant increase in the width and cell layer number of both the secondary xylem and the cambium in the stems, consistent with the upregulation trend of PagHAM4a and PagHAM4b expression. Conclusion The application of exogenous IAA promotes the expressions of PagHAM4a and PagHAM4b genes and the development of secondary xylem in the stems of 84K poplar. PagHAM4a and PagHAM4b participate in regulating the development of secondary xylem through the auxin signaling pathway.

Key words: 84K poplar, IAA, PagHAM4a/PagHAM4b, dual-reporter gene, secondary xylem

刘冉, 赵盼, 周心怡, 苏玉婷, 郑淑雅, 高宏波, 郭惠红. 生长素诱导调节PagHAM4a和PagHAM4b的表达及对杨树次生木质部发育的影响[J]. 生物技术通报, 2026, 42(9): 178-185.

LIU Ran, ZHAO Pan, ZHOU Xin-yi, SU Yu-ting, ZHENG Shu-ya, GAO Hong-bo, GUO Hui-hong. Auxin Induces and Regulates the Expressions of PagHAM4a/PagHAM4b and Affects the Secondary Xylem Development in Poplar[J]. Biotechnology Bulletin, 2026, 42(9): 178-185.

图/表 8

图1 两个双报告基因植物表达载体的结构示意图A：DR5驱动GFP报告基因和PagHAM4a特异启动子驱动GUS报告基因的双报告基因植物表达载体；B：DR5驱动GFP报告基因和PagHAM4b特异启动子驱动GUS报告基因的双报告基因植物表达载体；DR5pro：生长素响应启动子；GFP：绿色荧光蛋白基因；Tons：胭脂碱合成酶基因的终止子；PagHAM4apro/PagHAM4bpro：PagHAM4a和PagHAM4b的特异启动子；GUS：β-葡萄糖苷酸酶基因

Fig. 1 Schematic diagram of the dual-reporter gene plant expression vector structureA: A dual-reporter gene plant expression vector in which DR5 promoter drives the GFP reporter gene and the PagHAM4a-specific promoter drives the GUS reporter gene. B: A dual-reporter gene plant expression vector in which DR5 promoter drives the GFP reporter gene and the PagHAM4b-specific promoter drives the GUS reporter gene; DR5pro: Auxin-response promoter. GFP: Green fluorescent protein gene. Tons: Terminator of the nopaline synthase gene. PagHAM4apro/PagHAM4bpro: Specific promoter of the PagHAM4a and PagHAM4b gene. GUS: β-glucuronidase gene

图2 外源IAA处理后84K杨茎和叶中PagHAM4a和PagHAM4b的表达量A：PagHAM4a；B：PagHAM4b。3‒5节间的茎和叶：植株次生生长初期的节间及相对应节间的叶；8‒10节间的茎和叶：植株具有显著次生生长的节间及相对应节间的叶。*P<0.05；**P<0.01；***P<0.001。下同

Fig. 2 Expressions of PagHAM4a and PagHAM4b in the stems and leaves of 84K poplar after exogenous IAA treatmentA: PagHAM4a; B: PagHAM4b. Stems and leaves from 3rd‒5th internodes: The internodes during the early stage of secondary growth and the leaves corresponding to those internodes. Stems and leaves from 8th‒10th internodes: The internodes that have showed significant secondary growth and the leaves corresponding to those internodes. *P<0.05; **P<0.01; ***P<0.001. The same below

图3 含有双报告基因植物表达载体转基因植株的PCR鉴定A：PagHAM4a转基因植株的PCR鉴定；B：PagHAM4b转基因植株的PCR鉴定。M：DNA marker；P：含有DR5pro::GFP-PagHAM4apro::GUS、DR5pro::GFP-PagHAM4bpro::GUS载体的质粒；WT：84K杨；#1、#2、#3：转基因植株

Fig. 3 PCR identification of transgenic plants containing a dual reporter gene plant expression vectorA: PCR identification of PagHAM4a transgenic plants. B: PCR identification of PagHAM4b transgenic plants. M: DNA marker. P: Plasmids containing the DR5pro::GFP-PagHAM4apro::GUS and DR5pro::GFP-PagHAM4bpro::GUS vector. WT: 84K poplar. #1, #2, #3: Transgenic plants

图4 转基因植株的GUS鉴定

Fig. 4 GUS assay of transgenic plants

图5 转基因植株次生茎第7‒9节间的GFP荧光和GUS染色

Fig. 5 GFP fluorescence and GUS staining in internodes 7th‒9th of secondary stems from transgenic plants

图6 外源IAA处理后84K杨和过表达PagHAM4a/PagHAM4b植株的表型A：外源IAA处理后植株的外部形态特征（Bar=10 cm）；B：外源IAA处理后植株的叶片（Bar=10 cm）；C：株高；D：叶面积；E：茎粗；F：节间数。WT：84K杨；PagHAM4a-OE/PagHAM4b-OE：过表达转基因植株。不同字母表示具有显著差异（P<0.05）。下同

Fig. 6 Phenotype of wild-type 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b after exogenous IAA treatmentA: External morphological characteristics of plants after exogenous IAA treatment (Bar=10 cm). B: Leaves of plants after exogenous IAA treatment (Bar=10 cm). C: Plant height. D: Leaf area. E: Stem diameter. F: Internode number. WT: 84K plants; PagHAM4a-OE/PagHAM4b-OE: Plants overexpressing PagHAM4a/PagHAM4b in 84K poplar. Different letters indicate significant differences (P<0.05). The same below

图7 外源IAA处理后84K杨和过表达PagHAM4a/PagHAM4b植株的内部组织结构表型A：84K杨和过表达PagHAM4a/PagHAM4b植株茎第20节间的横切面（Bar=100 μm）；B：84K杨和过表达PagHAM4a/PagHAM4b植株茎第20节间的形成层区域（Bar=100 μm）；C：木质部宽度；D：木质部细胞层数；E：形成层宽度；F：形成层细胞层数；G：导管细胞面积；H：纤维细胞面积。Ph：韧皮部；Xy：木质部；Ca：形成层

Fig. 7 Internal tissue struction phenotype of wild-type 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b after exogenous IAA treatmentA: Cross-sections of the 20th internode of stems from 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b (Bar=100 μm). B: Cambium region of the 20th internode in stems of 84K poplar and 84K poplar overexpressing PagHAM4a/PagHAM4b (Bar=100 μm). C: Xylem width. D: Xylem cell layer number. E: Cambium width. F: Cambium cell layer number. G: Vessel cell area. H: Fiber cell area. Ph: Phloem. Xy: Xylem. Ca: Cambium

图8 外源IAA处理后84K杨和转基因植株中PagHAM4a和PagHAM4b的表达水平A：第3‒5节间；B：第8‒10节间

Fig. 8 Expressions of PagHAM4a and PagHAM4b in wild-type and transgenic 84K poplar plants after exogenous IAA treatmentA: 3rd‒5th internodes; B: 8th‒10th internodes

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