Cloning and Functional Analysis of Gene PagRAX2_16G and Its Promoter from Poplar

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

Abstract

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

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.

Figures/Tables 10

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

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

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

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

Fig. 5 GUS staining result of proPagRAX2_16G

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

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

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

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

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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