作 者：
刘彤;杨文凤;校现周;魏芳;高宏华;罗世巧;吴明;仇键
单 位：
中国热带农业科学院橡胶研究所;海南大学农学院
摘 要：
【目的】MYB转录因子是调控植物木质素合成和次生壁形成的重要转录因子之一。本文分离克隆到一个与拟南芥AtMYB20高度同源的橡胶树MYB转录因子基因HbMYB20,并在拟南芥中对其功能进行研究,以期了解其在橡胶树木质素合成和次生壁发育的分子调控中的作用,为橡胶树木材形成的分子调控机制研究及其遗传改良奠定基础。【方法】采用blast分析从树皮转录组中筛选出与拟南芥AtMYB20序列同一性较高的橡胶树MYB基因HbMYB20;设计ORF区特异性引物,以树皮c DNA为模板进行扩增得到该目的基因cDNA序列。实时荧光定量PCR检测该基因在橡胶树叶片、胶乳、茎干以及木质部与韧皮部的相对表达量。构建HbMYB20过表达植物载体,使用农杆菌蘸花法转化拟南芥,获得该基因过表达转基因株系。采用乙酰溴法和间苯三酚染色法,分析转基因、野生型拟南芥茎的木质素含量以及木质素在拟南芥茎基部横截面中的分布。对转基因、野生型拟南芥茎基部横截面切片进行甲苯胺蓝染色,并测量分析导管、木质纤维和维管束间纤维细胞的细胞壁厚度。最后,采用实时荧光定量PCR分析转基因及野生型拟南芥木质素和纤维素合成相关酶基因的表达。【结果】克隆得到1个橡胶树MYB转录因子基因HbMYB20,该基因开放阅读框(ORF)为927 bp,编码309aa的蛋白,氨基酸序列分析显示,HbMYB20与AtMYB20/43和AtMYB85/42同源性较高,属R2R3MYB转录因子G8亚组成员。表达分析显示HbMYB20在橡胶树茎干和木质部中高表达,胶乳中表达最低。对HbMYB20过表达拟南芥分析显示,该基因在3个转基因株系中均表达;相对野生型拟南芥,转HbMYB20拟南芥植株生长抑制,木质部和维管束间纤维的木质素染色面积较少、染色程度变浅,茎的木质素含量和木质纤维、导管及维管束间纤维的细胞壁厚度均显著低于野生型;同时转基因株系中木质素合成关键酶基因4CL1和CCoAOMT的表达量以及纤维素合成关键酶基因CesA8的表达显著下调。【结论】橡胶树MYB转录因子G8亚组成员HbMYB20,在茎和木质细胞中高表达。拟南芥中过表达HbMYB20导致转基因植株的矮小,细胞壁变薄,阻碍木质部中木质素的合成和积累,同时木质素和纤维素合成相关酶基因的表达显著下降。由此推测HbMYB20对拟南芥的木质素和纤维素合成都具有负调控作用,可能是1个橡胶树次生壁发育的负调控因子。
译 名：
Cloning of HbMYB20 from Hevea brasiliensis and Its Regulation of Secondary Wall Development in Arabidopsis thaliana
作 者：
Liu Tong;Yang Wenfeng;Xiao Xianzhou;Wei Fang;Gao Honghua;Luo Shiqiao;Wu Ming;Qiu Jian;College of Agriculture,Hainan University;Rubber Research Institute,Chinese Academy of Tropical Agricultural Sciences;
关键词：
Hevea brasiliensis;;secondary wall development;;lignin;;MYB
摘 要：
【Objective 】 MYB is one of the major transcription factors( TF) involved in regulation of the lignin biosynthesis and secondary cell wall formation in plants. A MYB TF gene designated HbMYB20,highly homologous to AtMYB20 of Arabidopsis thaliana was cloned from Hevea brasiliensis,and its roles in A. thaliana were also studied. This study was aimed to reveal the important function of HbMYB20 for regulating lignin biosynthesis and secondary cell wall formation in this species,and also to provide theoretical basis for the genetic improvement of the wood formation in H.brasiliensis.【Method】HbMYB20 was selected by BLAST analysis of the bark transcriptome in terms of a high identity withA. thaliana AtMYB20. The c DNA fragments were obtained from bark cDNA by PCR using the specific primers designed according to the open reading frame( ORF). Quantitative real-time PCR was employed in to determine the relative expression of HbMYB20 in different tissues such as leaf,latex,stem,xylem,and phloem in H. brasiliensis. The overexpression vectors with HbMYB20 were constructed and transformed into A. thaliana by agrobacterium dip methods.The lignin contents were measured by acetyl bromide method and presence of lignin was visualized by staining the cross sections with phloroglucinol-HCl. The sections of stem were also stained with toluidine blue,and used for statistical analysis on the cell wall thickness of interfascicular fiber,vessel and xylary fiber between the transgenic and wild-type A.thaliana. Finally,the expression level of the genes involved in lignin and cellulose synthesis were analyzed by quantitative real-time PCR.【Result】A MYB TF gene was cloned from H. brasiliensis and designated HbMYB20,which endowed with ORF of 927 bp,encoding 309 aa protein. The analysis of amino acid sequence indicated that HbMYB20 belonged to G8 subgroup member of R2R3 MYB transcription factor family,which had a high identity to AtMYB20 /43 and AtMYB85 /42.Gene expressions suggested that HbMYB20 had a higher expression in stems and xylem but less in latex. When overexpressing in A. thaliana,HbMYB20 was detected in three transgenic lines but not in wild-type plants. The transgenic A. thaliana showed obvious growth inhibition,smaller areas and less depth of lignin strain in the vessels and fiber compared to wild-type plants. The lignin contents of stem and cell wall thickness of interfascicular fiber,vessel and xylary fiber were significantly lower than those in the control. Furthermore,the genes expression of lignin biosynthetic genes( 4CL1 and CCo AOMT) and cellulose biosynthetic gene Ces A8 were significantly repressed in transgenic plants.【Conclusion】 The experimental data confirmed that HbMYB20,a MYB transcription factor G8 subgroup member from H. brasiliensis,which was highly-expressing in the stem and xylem. Functional analysis showed that the over-expression of HbMYB20 in A. thaliana has led to dwarfism in the transgenic plants,a significant thinning in vessels and fiber cell wall,an obvious decrease in lignin contents. And also,the expression inhibition of the genes related to lignin and cellulose biosynthesis were found. It is reasonable to infer that HbMYB20 plays a negative role in lignin and cellulose biosyntheses,indicating that it is a negative regulation factor for secondary wall development in H. brasiliensis.
