苎麻茎皮厚度性状QTL qBT4a候选基因的功能验证及序列进化分析

Bark thickness (BT) is one of the most important components of the fiber yield in ramie. Improvement of the BT trait is helpful for breeding varieties of ramie with high yield. However, the genetic and molecular mechanism for the BT trait is poorly understood in this crop, which seriously hindered its genetic improvement. In our previous study, we have developed a high-density genetic map with 4,338 SNP markers, and mapped the QTLs of bark thickness. A total of 5 BT QTLs were identified, and one of QTLs, qBT4a, has been ascertained its candidate gene that encodes a MYB transcription factor. In the Zhongzhu 1, a parent of population, we have found that the candidate gene of qBT4a included a 760-bp insertion that caused premature termination, thereby producing a protein that lacked part of the MYB domain. In the present study, we will construct an expression vector of qBT4a candidate gene and perform the experiment of genetic transformation, which will validate the function of this candidate gene. We will characterize the function of this MYB gene, including the analysis of the temporal and spatial expression patterns of gene, analysis of the features of gene encoding protein, and identification of the genes interacted with this MYB gene. In addition, we will analyze the haplotypes of qBT4a in the core germplasms of ramie, and ascertain their evolutionary relationship. Based on the DNA sequence of haplotypes, the functional markers of qBT4a will be developed. This study not only will present a base for understanding the molecular mechanism of the formation of the ramie BT trait, but also provide excellent gene resource for improving the BT trait and fiber yield in ramie breeding.

茎皮厚度是苎麻纤维产量性状的重要影响因子，对该性状遗传改良有助于实现苎麻的高产育种。然而，有关苎麻茎皮厚度性状形成的分子遗传机制，目前尚不清楚。为此，本团队前期构建了苎麻高密度SNP标记遗传图谱，完成了茎皮厚度性状遗传分析，共定位到5个性状QTL；鉴定了其中一个QTL qBT4a的候选基因，功能注释发现其编码一个MYB蛋白；在亲本中苎1号中该基因存在760 bp的片段插入，预测导致了蛋白翻译提前终止，形成MYB结构域缺失的蛋白。在此基础上，本项目拟将开展qBT4a的功能互补试验，以验证其候选基因；分析基因的时空表达模式、编码蛋白特征和互作基因，从而阐明qBT4a功能特点；此外，我们还将在苎麻核心种质资源中分析qBT4a的单倍型，进而确定基因的序列演化特征，并基于优异等位基因序列开发功能标记。本项目将不仅为阐明茎皮厚度性状形成机理提供基础，也为分子改良苎麻茎皮厚度和纤维产量性状提供基因资源。

苎麻是我国特色的纤维作物，提高纤维产量是苎麻育种的重要目标。当前有关苎麻纤维产量性状的遗传及分子基础知之甚少，阻碍了性状的遗传改良。为突破苎麻纤维产量性状遗传改良的瓶颈，本人遗传解析了纤维产量及其组成因子，并鉴定到一个茎皮厚度性状QTL qBT4a。在此基础上，本项目重点开展了该茎皮厚度性状qBT4a的克隆、功能及序列进化分析，取得如下研究结果：1）通过对80个薄皮性状F2单株的GBS SNP分析，将QTL定位到约200 kb的基因组区域；基于301份苎麻种质的基因组重测序和全基因组关联分析，在目的QTL区域检测到一关联信号，并发现关联位点下游5.4 kb区域存在一个MYB基因whole_GLEAN_10015497；2）发现whole_GLEAN_10015497过量表达可导致转基因拟南芥茎部木质纤维束增多，次生壁加厚，证实whole_GLEAN_10015497即为qBT4a基因；3）亚细胞定位发现基因编码蛋白在细胞核中表达；转录组和蛋白组分析发现，在纤维发育期的茎皮中whole_GLEAN_10015497 转录本水平并无显著变化，但蛋白水平显著增高；泛素化分析则发现在纤维发育期的茎皮中，whole_GLEAN_10015497蛋白泛素化水平显著降低。据此，我们提出whole_GLEAN_10015497泛素化介导的作用机制模型；4）该基因共有8个单倍型，其中Hap_2仅存在于农家种中，而其它7个单倍型或多或少受到驯化选择，尤其时Hap_1单倍型，约40%含该单倍型的种质为栽培种。在本项目的资助下，我们在Molecule Plant、Plant Biotechnology Journal、Plant Journal等主流SCI刊物发表论文10篇（其中7篇为第一标注），研究成果不仅极大的推动了苎麻纤维性状分子机制研究，也为苎麻纤维产量性状遗传改良提供基因资源。