Data_Sheet_1_Identification of candidate MYB transcription factors that influence CslF6 expression in barley grain.PDF

(1,3;1,4)-β-Glucan is a non-cellulosic polysaccharide required for correct barley grain fill and plant development, with industrial relevance in the brewing and the functional food sector. Barley grains contain higher levels of (1,3;1,4)-β-glucan compared to other small grain cereals and this influences their end use, having undesirable effects on brewing and distilling and beneficial effects linked to human health. HvCslF6 is the main gene contributing to (1,3;1,4)-β-glucan biosynthesis in the grain. Here, the transcriptional regulation of HvCslF6 was investigated using an in-silico analysis of transcription factor binding sites (TFBS) in its putative promoter, and functional characterization in a barley protoplast transient expression system. Based on TFBS predictions, TF classes AP2/ERF, MYB, and basic helix-loop-helix (bHLH) were over-represented within a 1,000 bp proximal HvCslF6 promoter region. Dual luciferase assays based on multiple HvCslF6 deletion constructs revealed the promoter fragment driving HvCslF6 expression. Highest HvCslF6 promoter activity was narrowed down to a 51 bp region located −331 bp to −382 bp upstream of the start codon. We combined this with TFBS predictions to identify two MYB TFs: HvMYB61 and HvMYB46/83 as putative activators of HvCslF6 expression. Gene network analyses assigned HvMYB61 to the same co-expression module as HvCslF6 and other primary cellulose synthases (HvCesA1, HvCesA2, and HvCesA6), whereas HvMYB46/83 was assigned to a different module. Based on RNA-seq expression during grain development, HvMYB61 was cloned and tested in the protoplast system. The transient over-expression of HvMYB61 in barley protoplasts suggested a positive regulatory effect on HvCslF6 expression.

(1,3;1,4)-β-葡聚糖（(1,3;1,4)-β-Glucan）是一类非纤维素多糖，对大麦籽粒正常灌浆及植株生长发育不可或缺，在酿酒工业与功能食品领域具备重要工业应用价值。相较于其他小粒谷类作物，大麦籽粒中(1,3;1,4)-β-葡聚糖含量更高，这一特性会影响其最终用途：既会对酿酒与蒸馏工艺产生不利影响，同时也与人类健康益处相关。HvCslF6是调控籽粒中(1,3;1,4)-β-葡聚糖生物合成的核心基因。本研究通过对HvCslF6推定启动子区域的转录因子结合位点（transcription factor binding site, TFBS）开展生物信息学（in silico）分析，并结合大麦原生质体瞬时表达系统进行功能验证，探究了HvCslF6的转录调控机制。基于转录因子结合位点预测结果，在HvCslF6起始密码子上游1000 bp的近端启动子区域内，AP2/ERF、MYB以及碱性螺旋-环-螺旋（basic helix-loop-helix, bHLH）这几类转录因子的结合位点显著富集。通过针对多个HvCslF6缺失启动子构建体的双荧光素酶实验，明确了驱动HvCslF6表达的核心启动子片段。进一步将其活性峰值区域缩小至起始密码子上游-331 bp至-382 bp处的51 bp区间。结合转录因子结合位点预测结果，本研究鉴定出两个MYB类转录因子——HvMYB61与HvMYB46/83，作为HvCslF6表达的潜在激活因子。基因共表达网络分析显示，HvMYB61与HvCslF6及其他初级纤维素合酶（HvCesA1、HvCesA2和HvCesA6）同属一个共表达模块，而HvMYB46/83则隶属于另一不同模块。基于籽粒发育过程中的RNA测序（RNA-seq）表达数据，本研究克隆了HvMYB61并在原生质体系统中开展功能验证。在大麦原生质体中瞬时过表达HvMYB61的实验结果表明，其对HvCslF6的表达具有正向调控作用。