杨树PdKNAT7基因调控拟南芥次生细胞壁厚度的功能研究

Function of PdKNAT7 gene in poplar regulating the thickness of secondary cell wall in Arabidopsis thaliana

【目的】次生细胞壁的发育对于木材的形成至关重要,揭示林木次生壁形成的分子调控机制将为改良其木材品质提供理论依据。【方法】(1)本研究从速生型杨树NE19中克隆得到PdKNAT7基因,并构建表达载体,采用花序侵染法转化拟南芥,筛选得到过表达植株。(2)利用农杆菌介导的瞬时转化法侵染烟草,对PdKNAT7进行亚细胞定位。(3)通过徒手切片观察不同基因型拟南芥花薹基部的次生细胞壁厚度。(4)通过真空渗透的方法瞬时转化84K杨。(5)qRT-PCR分析不同品系杨树中PdKNAT7基因的表达情况,并揭示拟南芥和杨树中参与次生壁形成的相关基因的表达模式。【结果】(1)PdKNAT7定位于细胞核。(2)PdKNAT7主要在NE19杨茎中表达,且表达量高于107杨。(3)与野生型相比,过表达PdKNAT7的拟南芥花薹基部的束间纤维壁变薄,木质部纤维壁变厚,而突变体正好相反;另外,突变体的导管壁更厚。(4)过表达PdKNAT7的拟南芥植株中,4CL1、C4H1、CCR1、CesA8、IRX9和IRX10基因的表达量均下调,突变体中与之相反。(5)瞬时转化PdKNAT7基因的84K杨中,4CL3、C4H1、CCR1、CesA8、IRX9和IRX10基因的表达量也下调。【结论】PdKNAT7通过调控木质素和纤维素的积累来影响拟南芥次生细胞壁的厚度。

[Objective] The development of secondary cell wall is vital for the wood formation, and revealing the molecular regulation mechanism of secondary wall formation in forest will provide a theoretical basis for improving its wood quality. [Method](1) In this study, PdKNAT7 gene was cloned from fast-growing poplar NE19, and its expression vector was constructed. Arabidopsis thaliana was transformed by inflorescence infection, and overexpression plants were screened.(2) The subcellular localization of PdKNAT7 was implemented by Agrobacterium mediated transient transformation in tobacco.(3) The secondary thickening of flower stalk base in different genotypes of Arabidopsis thaliana was observed by hand sectioning.(4) 84K poplar was transiently transformed by vacuum infiltration.(5) qRTPCR was used to analyze the expression of PdKNAT7 gene in different poplar strains, and reveal the expression patterns of related genes involved in secondary wall formation in Arabidopsis thaliana and poplar. [Result](1) PdKNAT7 was localized in the nucleus.(2) PdKNAT7 was mainly expressed in the stem of NE19, and the expression level was higher than that of poplar 107.(3) Compared with the wild type, the interfascicular fiber wall of the flower stalk base of the overexpression plant became thinner, and the xylary fiber wall became thicker, while the mutant was just opposite to the overexpression plant;in addition, the vessel wall of mutant was thicker.(4) The expression levels of 4CL1, C4H1, CCR1, CesA8, IRX9 and IRX10genes were down regulated in the overexpression plants, and the mutant was on the contrary.(5) The expression levels of 4CL3, C4H1, CCR1, CesA8, IRX9 and IRX10 genes in 84K poplar transiently transformed with PdKNAT7 gene were also down regulated. [Conclusion] PdKNAT7 affects the thickness of secondary cell wall by regulating the accumulation of lignin and cellulose in Arabidopsis thaliana.