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林业科学 ›› 2015, Vol. 51 ›› Issue (4): 52-59.doi: 10.11707/j.1001-7488.20150407

• 论文与研究报告 • 上一篇    下一篇

橡胶树HbMYB20基因的克隆及其对拟南芥次生壁发育的调控

刘彤1,2, 杨文凤2, 校现周2, 魏芳2, 高宏华2, 罗世巧2, 吴明2, 仇键2   

  1. 1. 海南大学农学院 海口 570228;
    2. 中国热带农业科学院橡胶研究所 儋州 571737
  • 收稿日期:2014-10-23 修回日期:2014-11-28 出版日期:2015-04-25 发布日期:2015-05-20
  • 通讯作者: 仇键
  • 基金资助:

    国家自然科学青年基金项目(31400579); 海南省自然科学基金项目(314139)。

Cloning of HbMYB20 from Hevea brasiliensis and Its Regulation of Secondary Wall Development in Arabidopsis thaliana

Liu Tong1,2, Yang Wenfeng2, Xiao Xianzhou2, Wei Fang2, Gao Honghua2, Luo Shiqiao2, Wu Ming2, Qiu Jian2   

  1. 1. College of Agriculture, Hainan University Haikou 570228;
    2. Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences Danzhou 571737
  • Received:2014-10-23 Revised:2014-11-28 Online:2015-04-25 Published:2015-05-20

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cDNA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, HbMYB20AtMYB20/43AtMYB85/42 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 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。

关键词: 橡胶树, 次生壁, 木质素, MYB

Abstract:

【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 with A.thaliana AtMYB20. The cDNA 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 309aa protein. The analysis of amino acid sequence indicated that HbMYB20 belonged to G8 subgroup member of R2R3MYB transcription factor family, which had a high identity to AtMYB 20/43 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 CCoAOMT) and cellulose biosynthetic gene CesA8 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.

Key words: Hevea brasiliensis, secondary wall development, lignin, MYB

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