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