MYB58调控蒺藜苜蓿木质素生物合成的研究

The lignin biosynthesis pathway has been well studied in the past decades, and its regulation mechanism and genetic improvement have been earnestly pursued recent years. MYB transcription factors are involved in the regulation of lignin biosynthesis in many species, but their role is not clear in Medicago species. We previously found a stem specific expressing MYB gene (MtMYB58) in Medicago truncatula microarray data and further isolated its knockout mutants. In this study, we plan to carry out the following: (1) To analyze regulation effects of MtMYB58 on lignin content and distribution using MtMYB58 overexpression lines, knockout lines and its complemented lines. (2) To screen potential regulating targets of MtMYB58 by analyzing the expression levels of genes involved in lignin biosynthesis via qRT-PCR. (3) To confirm the interactions between MtMYB58 and promoter elements of target genes by EMSA, ChIP and yeast one hybrid. (4) To create transgenic alfalfa lines with reduced lignin content through RNAi interference of homologous gene, MsMYB58. This study will elucidate the mechanism of MtMYB58 regulation of M. truncatula lignin synthesis and improve alfalfa quality by repression lignin biosynthesis.

木质素生物合成途径已基本清楚，其调控机理及遗传改良成为近年来的研究热点之一。本研究拟从蒺藜苜蓿中挖掘MYB转录因子MtMYB58，深入研究其对木质素生物合成的调控作用，并应用于紫花苜蓿遗传改良。申请人前期在蒺藜苜蓿发现一个茎秆特异高表达MYB基因 (MtMYB58)，并获得其敲除突变体，本研究拟开展如下研究：(1) 创制超表达和回补突变体，明确MtMYB58木质素合成的影响；(2)：qRT-PCR筛选突变体和超表达材料中差异表达的木质素合成关键基因，发现关键调控靶基因；(3)：通过EMSA、ChIP、酵母单杂交实验验证MtMYB58对靶基因的直接调控作用；(4) 遗传改良紫花苜蓿：RNAi干扰紫花苜蓿同源基因MsMYB58，筛选获得木质素含量降低的转基因株系。本研究将阐明MtMYB58调控蒺藜苜蓿木质素合成的机制，并获得低木质素紫花苜蓿新材料，具有重要的理论价值和应用前景。

木质素是影响苜蓿品质的关键因素之一，如何有效获得低木质素、高品质品种始终是研究的重要目标和内容，这有赖于对木质素生物合成途径及调控机制的充分了解。本研究在蒺藜苜蓿基因芯片数据中挖掘到一个MYB转录因子，MtMYB58，发现其表达模式与木质素生物合成关键基因高度一致。实验证实，MtMYB58定位于细胞核中，并具有转录激活活性。Tnt1插入纯合突变体中，MtMYB58的功能缺失导致木质素含量显著降低，而MtMYB58过表达株系的木质素合成和积累显著增加，表明MtMYB58的功能是正向促进木质素的生物合成。qRT-PCR分析发现，MtMYB58影响了HCT、CCoAOMT、COMT、CCR、C4H1等基因的表达水平。酵母单杂交实验证实，MtMYB58可与MtC4H1的启动子互作，驱动下游报告基因。EMSA实验结果表明，MtMYB58直接特异性地结合在MtC4H1启动子区域一段包含两个AC元件的区域。通过RNAi干扰技术获得了MsNST1、MYB58、CCoAOMT、COMT、C4H表达下调的紫花苜蓿转基因株系。测定表明，L10、L22和L38三个株系的木质素含量显著低于WT。过组织切片观察进一步证实，转基因株系的木质化过程显著滞后于WT。木质素含量降低引起了半纤维素含量降低、果胶含量升高，有15种氨基酸含量显著升高，表明转基因株系营养品质有所提升。本研究解析了MtMYB58调控木质素合成的分子机制，获得木质素含量降低的新材料，为紫花苜蓿遗传改良工作奠定了基础。