Abstract

Barley ranks fourth in global cereal production and is primarily grown for animal feed and malt. Hordeins, the principal barley seed storage proteins, are homologous to wheat gluten and when ingested elicit an immune response in people with Coeliac disease. Risø 1508 is a chemically induced barley mutant with low hordein levels imparted by the lys3.a locus that is reported to be caused by an SNP in the barley prolamin-box binding factor gene (BPBF). Reports suggest the lys3.a locus prevents CG DNA demethylation at the Hor2 (B-hordein) promoter during grain development subsequently causing hypermethylation and inhibiting gene expression. In lys3.a mutants, endosperm-specific β-amylase (Bmy1) and Hor2 are similarly downregulated during grain development and thus we hypothesize that the inability to demethylate the Bmy1 promoter CG islands is also causing Bmy1 downregulation. We use whole-genome bisulfite sequencing and mRNA-seq on developing endosperms from two lys3.a mutants and a lys3.b mutant to determine all downstream genes affected by lys3 mutations. RNAseq analysis identified 306 differentially expressed genes (DEGs) shared between all mutants and their parents and 185 DEGs shared between both lys3.a mutants and their parents. Global DNA methylation levels and promoter CG DNA methylation levels were not significantly different between the mutants and their parents and thus refute the hypothesis that the lys3.a mutant’s phenotype is caused by dysregulation of demethylation during grain development. The majority of DEGs were downregulated (e.g., B- and C-hordeins and Bmy1), but some DEGs were upregulated (e.g., β-glucosidase, D-hordein) suggesting compensatory effects and potentially explaining the low β-glucan phenotype observed in lys3.a germplasm. These findings have implications on human health and provide novel insight to barley breeders regarding the use of BPBF transcription factor mutants to create gluten-free barley varieties.

中文翻译：

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大麦 (Horduem vulgare) lys3 突变体的表征鉴定了谷醇溶蛋白盒结合转录因子调控下的基因，并阐明了其在籽粒发育过程中胚乳启动子甲基化中的作用

摘要

大麦在全球谷物产量中排名第四，主要用于动物饲料和麦芽。大麦醇溶蛋白是主要的大麦种子储存蛋白，与小麦麸质同源，摄入后会引起乳糜泻患者的免疫反应。Risø 1508 是一种化学诱导的大麦突变体，具有由lys3.a基因座赋予的低大麦醇溶蛋白水平，据报道，该突变体是由大麦谷醇溶蛋白盒结合因子基因 ( BPBF )中的 SNP 引起的。报告表明，lys3.a基因座可防止籽粒发育过程中Hor2（B-大麦醇溶蛋白）启动子处的CG DNA 去甲基化，从而导致高甲基化并抑制基因表达。在lys3.a突变体中，胚乳特异性 β-淀粉酶 ( Bmy1 ) 和Hor2在籽粒发育过程中类似地下调，因此我们假设Bmy1启动子 CG 岛无法去甲基化也导致Bmy1下调。我们使用全基因组亚硫酸氢盐测序和 mRNA-seq 来开发两个lys3.a突变体和lys3.b突变体的胚乳，以确定受lys3突变影响的所有下游基因。RNAseq 分析确定了所有突变体及其亲本之间共有的 306 个差异表达基因 (DEG)，以及lys3.a突变体及其亲本之间共有的 185 个差异表达基因 (DEG)。突变体及其亲本之间的整体DNA甲基化水平和启动子CG DNA甲基化水平没有显着差异，从而反驳了lys3.a突变体的表型是由籽粒发育过程中去甲基化失调引起的假设。大多数DEGs下调（例如B-和C-大麦醇溶蛋白和Bmy1），但一些DEGs上调（例如β-葡萄糖苷酶、D-大麦醇溶蛋白），表明补偿作用并可能解释在lys3中观察到的低β-葡聚糖表型.种质。这些发现对人类健康具有重要意义，并为大麦育种者提供了关于使用BPBF转录因子突变体培育无麸质大麦品种的新见解。