杉木MYB基因的功能研究及与材性性状的关联分析

Currently, improvement of wood quality is an important objective for Cunninghamia lanceolata breeding. Through dissecting the molecular mechanism of wood formation, the plant breeders can develop the functional makers, and conduct the marker-assisted selection of wood properties. This strategy can shorten the breeding cycle of wood quality improvement, and promote the sustainable development of related industries. In previous studies, a highly differentially expressed protein, identified as the MYB transcription factor, was isolated from the developing xylem of Cunninghamia lanceolata,by the fluorescent difference gel electrophoresis and mass spectrometry on the basis of anatomical observations of a natural variation with specific wood properties. Four MYB genes were cloned in further experiments, namely ClMYB1, 2, 3 and 4. In this study, expression patterns of these genes will be systemically analyzed by quantitative PCR and in situ hybridization. The binding activities of these ClMYBs will be tested using AC-element, the MYB-binding site commonly existing in lignin biosynthetic genes. Furthermore, target genes of transcriptional regulation will be determined through the overexpression of ClMYB genes in transgenic tobacco. On the other hand, single-nucleotide polymorphism of four ClMYB genes in 600 pooled Cunninghamia lanceolata clones will be detected via pooled multiplexed next-generation sequencing and a customized analysis workflow respectively. According to distribution characteristics of SNPs from these genes, the SNP genotyping will be conducted with HRM (high resolution melting analysis) and Taqman probe technology for core clones. After the associations between SNPs and wood quality traits are calculated with a mixed linear model approach, tag SNPs will be further screened. At the same time, the functions of four ClMYB genes will be validated through the association analysis. So the implementation of this project will provide the theoretical basis for revealing the molecular mechanism of wood formation, and molecular marker deriving from these MYB transcription factors will be developed for marker-assisted selection of wood properties in the Cunninghamia lanceolata breeding.

材性性状改良是现阶段杉木育种的重要内容之一。研究杉木木材形成的分子机制，继而开发功能性标记，实现分子标记辅助选择材性，对于缩短其育种周期，促进相关产业可持续发展具重要意义。在前期研究中，在对一杉木材性特异的自然变异材料进行解剖观测的基础上，采用DIGE和质谱技术获得了1个注释为MYB类转录因子的差异蛋白质，并进一步分离到4个相关的MYB基因。本研究围绕这些MYB基因的功能解析及其关联分析，拟运用定量PCR、酵母杂交等技术系统分析其表达模式、基因产物与AC元件的结合活性，以及通过转化烟草分析其功能。在此基础上以保存多年的不同杉木种质为材料，结合样品池策略采用第二代高通量测序发掘存在SNP，进行SNP分型及其关联分析，筛选标签SNP，初步构建基于MYB基因的分子辅助育种体系。期望不仅在理论上为揭示杉木木材形成的分子调控机制提供依据，并且在实践中运用这些转录因子开展杉木材性的分子改良。

在转录组测序的基础上，15个ClMYBs基因的cDNA序列被成功分离。它们皆具有完整的 ORF，且编码MYB蛋白都包含R2R3MYB结构域，与松科植物白云杉R2R3-MYB蛋白的 DBD结构域序列一致性高达96%。进化树分析发现，ClMYB1和ClMYB2与拟南芥、杨树次生壁形成相关的MYB聚在一起，暗示可能参与杉木木材形成。进一步的定量PCR显示，在6种不同的器官中，ClMYB1主要在针叶和木质化茎中表达，在其他器官的表达量都很低；ClMYB2主要在根和木质化茎中表达，在雌花中的表达量最低。进一步的研究发现：ClMYB1与ClMYB2有着类似的表达模式，都主要在木质化的茎中表达，且随着木质化程度的增高表达量增加。sGFP-ClMYB1定位于细胞核中，而sGFP-ClMYB2不仅定位于细胞核，在细胞膜以及细胞质中的某些细胞器上也存在。通过转化烟草证实，ClMYB2可能通过调控一些木质素合成相关基因的表达而参与木质素的形成过程。ClMYB2的基因组序列为1710bp，含有3个外显子和2个 内含子。检测到99个单核苷酸多态性位点，SNP频率为1/17bp；在这些SNPs中，有97个是二态SNPs，其中个54属于转换，43个属于颠换。连锁不平衡分析结果显示，随着核苷酸序列长度的增加，SNPs的连锁不平衡在基因内部迅速衰退。. EST-SSR分析显示，所选杉木群体具较丰富的遗传多样性，可分为3个亚群。通过多态性SSR分子标记与性状的关联分析，筛选出了13个与主要生长与材质性状显著关联位点，发掘了关联位点的优异等位变异，为实现分子标记辅助杉木材质改良奠定了基础。