银腺杨生长素受体基因PagFBL3对茎生长发育的影响

doi:10.11707/j.1001-7488.LYKX20230198

摘要/Abstract

摘要：

目的: 本研究分析了银腺杨生长素受体基因 PagFBL3 的序列结构、组织表达特异性和亚细胞定位，并创制了过表达 PagFBL3 转基因杨树，探讨其对茎次生生长的影响，为进一步阐明生长素受体在茎次生生长中的作用机制提供帮助。方法: 利用生物信息学方法及相关软件分析系统进化关系、序列相似性及生化特征；克隆并鉴定了银腺杨PagFBL3基因，利用实时荧光定量PCR技术，分析该基因在根、幼叶、成熟叶、幼茎、维管形成层、木质部和韧皮部中的表达；利用Gateway技术，构建融合表达载体35S::PagFBL3-GFP，通过瞬时转化烟草叶片表皮分析PagFBL3的亚细胞定位；同时，将PagFBL3编码区序列重组进入pMDC32载体，构建植物过表达载体35S::PagFBL3，利用农杆菌介导法创制转基因银腺杨，通过表型观察和组织切片分析过表达PagFBL3基因对转基因杨树茎部生长发育的影响。结果: PagFBL3基因可编码571个氨基酸残基，其编码蛋白定位于细胞核。PagFBL3基因在根、茎和叶中均有表达，并在成熟叶和次生茎中表达量较高。基于抗性筛选和分子水平鉴定，创制了11个过表达PagFBL3转基因杨树株系，选择两个表达量较高的转基因株系进行功能分析。与非转基因植株相比，过量表达PagFBL3基因促进转基因杨树木质部发育，显著增加了转基因杨树的木质部宽度，提高了转基因杨树的径向生长，地径分别增加6.7%和8.5%，第15节间木质部宽度分别增加17.3%和19.9%。此外，过量表达PagFBL3基因也增强了植株的高生长，株高分别增加6.3%和6.9%。结论: PagFBL3基因主要在成熟叶和次生茎中表达并发挥作用，通过影响木质部发育，提高木质部宽度，进而参与调控杨树的径向生长，该研究为进一步揭示PagFBL3基因参与杨树茎部生长发育的分子机制提供了参考。

关键词: 银腺杨, 生长素受体, PagFBL3, 过量表达, 木质部发育

Abstract:

Objective: Auxin plays an important regulatory role in plant growth and development via its signal transduction. Auxin receptor is one of the most critical components in auxin signaling pathway. Therefore, it is of great significance to study the roles of auxin receptor family gene (FBL) in the growth and development of poplar to reveal the mechanism of auxin in the secondary growth of woody plants. In this study, the sequence structure, tissue specific expression and subcellular localization of PagFBL3 in poplar ‘84K’ (Populus alba × P. glandulosa) were investigated to exlore the effects of PagFBL3 on stem secondary growth by creating transgenic poplar overexpressing PagFBL3, so as to provide support in further elucidating the mechanism of action of auxin receptors in secondary stem growth. Method: The bioinformatics methods and related software were used to analyze the phylogenetic relationship, sequence similarity and biochemical characteristics. PagFBL3 gene was cloned and identified from 84K poplar, and its expression levels in roots, young leaves, mature leaves, young stems, vascular cambium, xylem and phloem were detected by real-time quantitative PCR. The fusion expression vector 35S::PagFBL3-GFP was constructed using the Gateway technique, and the subcellular localization of PagFBL3 was analyzed by transient transformation of tobacco leaf epidermal cells. At the same time, the coding region of PagFBL3 was recombined into the PMDC32 vector to construct the plant overexpression vector 35S::PagFBL3. The transgenic poplar was created by Agrobacterium-mediated transformation method. The effects of overexpressing PagFBL3 gene on growth and development of transgenic poplar stem were analyzed by phenotype and tissue sections assays. Result: PagFBL3 gene was able to encode 571 amino acid residues and its encoded protein was localized in the nucleus. PagFBL3 gene was expressed in roots, stems and leaves, and was highly expressed in mature leaves and secondary stems. Based on resistance screening and molecular identification, 11 transgenic poplar lines overexpressing PagFBL3 were generated, and two transgenic lines with high expression levels were selected for further functional analysis. Compared with non-transgenic 84K poplar plants, overexpression of PagFBL3 gene promoted xylem development, significantly increased xylem width of transgenic poplars, and improved stem radial growth. The two transgenic lines showed 6.7% and 8.5% increase in ground diameter, and 17.3% and 19.9% increase in the xylem width of the fifteen internodes, respectively. In addition, overexpression of PagFBL3 gene promoted plant height of transgenic poplar. The two transgenic lines showed 6.3% and 6.9% increase in stem height, respectively. Conclusion: PagFBL3 gene is mainly expressed in mature leaves and secondary stems and plays a role in regulating the radial growth of poplar by affecting xylem development and improving xylem width. This study provides a reference for further revealing the molecular mechanism of PagFBL3 gene involved in the stem growth and development of poplars.

Key words: Populus alba × P. glandulosa, auxin receptor, PagFBL3, overexpression, xylem development.

中图分类号:

S718.46

左雯腾,孟佳慧,卢孟柱,王留强. 银腺杨生长素受体基因PagFBL3对茎生长发育的影响[J]. 林业科学, 2023, 59(11): 59-67.

Wenteng Zuo,Jiahui Meng,Mengzhu Lu,Liuqiang Wang. Effects of the Growth Hormone Receptor Gene PagFBL3 on Stem Growth and Development of Populus alba × P. glandulosa[J]. Scientia Silvae Sinicae, 2023, 59(11): 59-67.

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用于RT-qPCR的基因特异性引物序列 Specific primers used for RT-qPCR
基因/载体结构 Genes/vector structures	正向和反向引物（5’–3’） Forward and reverse primers (5’–3’)
PagFBL3	TGGTTGCAAGAAACTTCGC	CCGATACAAGAACATCTTCT
PagActin1	ACCCTCCAATCCAGACACTG	TTGCTGACCGTATGAGCAAG
PagActin2	AAACTGTAATGGTCCTCCCTCCG	GCATCATCACAATCACTCTCCGA

用于基因克隆和载体构建的引物序列 Primers used for gene cloning and vector construction
pMD19-T-PagFBL3	ATGAATTATTTCCCTGATGAAG	CTATAAAGTCCACACGAACTC
pDONR207-PagFBL3	GGGGACAACTTTGTACAAAAAAGTTGGAATGAA TTATTTCCCTGATGAAGA	GGCGGCCGCACAACTTTGTACAAGAAAGTTGGG TATAAAGTCCACACGAACTC

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