毛果杨NAC128基因在次生壁形成中的功能  

Functions of NAC128 Gene from Populus trichocarpa in Secondary Cell Wall Formation

作　　者：李媛 陈金焕[2] 金曌 侯景丫 姜玉松[1] 邢海涛 Li Yuan;Chen Jinhuan;Jin Zhao;Hou Jingya;Jiang Yusong;Xing Haitao(Chongqing Key Laboratory of Economic Plant Biotechnology Chongqing University of Arts and Sciences,Chongqing 402168;Advanced Innovation Center for Tree Breeding by Molecular Design Beijing Forestry University,Beijing 100083)

机构地区：[1]重庆文理学院经济植物生物技术重庆市重点实验室,重庆402168 [2]北京林业大学林木分子育种高精尖创新中心,北京100083

出　　处：《林业科学》2020年第11期62-72,共11页Scientia Silvae Sinicae

基　　金：重庆文理学院引进人才项目(R2018STZ25);重庆市教委基础研究青年项目(KJQN201901303);国家自然科学基金项目(31670610)。

摘　　要：【目的】克隆并鉴定与毛果杨次生壁形成相关的NAC(NAM,ATAF1/2,CUC2)转录因子,在组织表达特异性分析的基础上,通过过量表达分析转基因植株表型及下游调控网路,解析该基因调控杨树次生壁组分生物合成的作用机制,为利用该基因进行林木种质创新奠定基础。【方法】基于前期转录组数据,克隆到1个在毛果杨茎中高表达的NAC转录因子PtrNAC 128。利用qRT-PCR技术分析PtrNAC 128在毛果杨根、茎、叶中的相对表达量。构建35S∷PtrNAC128-GFP植物表达载体,利用农杆菌介导法转化毛白杨;利用基因枪法将PtrNAC128-GFP融合蛋白在烟草叶片中进行瞬时表达,而后对PtrNAC128进行亚细胞定位分析;对毛白杨转基因植株和野生型植株的横切面切片染色观察,并通过qRT-PCR检测木质素、纤维素、半纤维素合成途径关键酶基因及调控次生壁各组分生物合成的NAC、MYB转录因子的表达差异,解析过表达PtrNAC128对杨树次生壁各组分的影响。【结果】毛果杨PtrNAC128与拟南芥ANAC075聚在一支,具有典型NAC结构域和完整的C端激活区。qRT-PCR结果表明PtrNAC128在毛果杨根、茎、叶中均有表达;在老茎中表达最高,是嫩叶中的23.49倍;在嫩茎中也有较高表达,是嫩叶的11.44倍。亚细胞定位分析表明PtrNAC128定位于细胞核内。PtrNAC 128过表达毛白杨转基因株系,次生木质部显著增厚,达到野生型的1.42-1.51倍;木质部细胞层数增加,约为野生型的1.22-1.31倍;转基因株系Klason木质素含量较野生型植株增加12%-22%,纤维素含量较野生型植株增加7.4%-13.1%,但半纤维素含量无显著差异;qRT-PCR检测结果表明,PtrNAC128显著提高木质素和纤维素合成途径关键酶基因以及部分次生壁相关NAC、MYB转录因子的表达。【结论】毛果杨PtrNAC128通过激活木质素和纤维素生物合成的关键酶基因及转录因子调控次生壁组分合成。【Objective】The aim of this study was to clone and identify NAC(NAM,ATAF1/2 and CUC)transcription factors related to secondary cell wall formation in Populus trichocarpa.Based on the analysis of tissue-specific expression pattern,the phenotype and downstream regulatory network of transgenic plants,our results provide some new insights into the mechanism of the gene regulatory network of secondary cell wall biosynthesis in poplar,laying a foundation for the use of this gene in forest germplasm innovation.【Method】Based on the previous transcriptome data,PtrNAC 128,which was preferentially expressed in the stems of P.trichocarpa,was cloned.Relative expression of PtrNAC 128 in roots,stems and leaves were analyzed by qRT-PCR analysis.The subcellular localization of PtrNAC128 was analyzed by gene gun bombardment method;35 S∷PtrNAC 128-GFP plant binary expression vector was constructed and transformed to Populus tomentosa.Anatomical cross-sections of transgenic and wild-type plants were observed by paraffin analysis method,and the effects of PtrNAC128 overexpressing on the components of poplar secondary cell wall were examined.The expression fluctuation of key enzyme genes and NAC,MYB transcription factors which regulating the biosynthesis of lignin,cellulose and hemicellulose synthesis were detected by qRT-PCR analysis.【Result】PtrNAC128 clustered in one branch with Arabidopsis ANAC075,with typical domains and intact C terminal activation regions.PtrNAC128 was expressed in all tissues tested,with the highest expression level in old stems and the lowest expression level in leaves.The expression of PtrNAC128 in old and young stems was 23.49 folds and 11.44 folds higher than that in young leaves,respectively.Subcellular localization analysis indicated that PtrNAC128 was localized into the nucleus.The secondary xylem width of PtrNAC128-overexpression(PtrNAC128-OE)transgenic plants had significantly increased to 1.42-1.51 folds,while the number of secondary xylem cells increased to 1.22-1.31 folds compared to th

关 键 词：毛果杨 PtrNAC128 转基因 次生壁组分 

分 类 号：S718.46[农业科学—林学]