大麦是全球第四大谷类作物，是食品和饲料生产的基本原料。在表型上，不分枝的大麦穗以两种主要的穗结构出现: 二棱或六棱。在六棱的品种中，三棱分生组织的三个小花都能发育成种子，而在两棱的品种中，只有中心小花能形成种子。
VRS5(HvTB1)作为横向种子生长的抑制剂，VRS5(HvTB1)突变体呈现六棱状穗状结构。VRS5(HvTB1)是TCP转录因子(TF)家族成员之一，常与其他转录调控因子形成蛋白-蛋白相互作用，调控其靶基因的表达。尽管VRS5(HvTB1)在调控大麦植株结构中起着关键作用，但目前对其分子作用模式知之甚少。
我们对TCP转录因子家族进行了广泛的系统发育分析，然后使用酵母-双杂交进行了体外蛋白-蛋白相互作用研究。
我们的分析表明，VRS5(HvTB1)具有多种互作能力，可以与II类TCP, NF-Y TF互作，也可以与染色质建模器互作。
进一步分析VRS5(HvTB1)与其他TCP TFs的互作能力表明，VRS5(HvTB1)较好地与TB1分支内的其他II类TCP TFs相互作用。其中一个互作蛋白由HvTB2编码，与VRS5(HvTB1)相比表现出类似的表达模式。
单倍型分析表明HvTB2基因高度保守，在品种和野生大麦中几乎没有变异。CRISPR-CAS9在cv中诱导HvTB2突变。“黄金承诺”导致大麦植株失去了其特有的无分枝穗状结构。hvtb2突变体在主穗处出现分枝，表明与VRS5(HvTB1)类似，hvtb2具有抑制分支的作用。
综上所述，我们通过对VRS5(HvTB1)蛋白间相互作用的研究，鉴定出了另一个调控大麦穗状结构的关键调控因子HvTB2。
了解植物结构调控因子VRS5(HvTB1)的分子网络，包括蛋白-蛋白相互作用，为鉴定大麦其他植物结构调控因子提供了新的途径。
图 大麦TCP636转录因子在TB1和CIN分支中的蛋白质相互作用酵母验证
图3 hvtb2突变体的表型
Barley is the fourth largest cereal crop grown worldwide, and essential for food and feed production. Phenotypically, the barley spike, which is unbranched, occurs in two main architectural shapes: two-rowed or six-rowed. In the 6-rowed cultivars, all three florets of the triple floret meristem develop into seeds while in 2-rowed lines only the central floret forms a seed. VRS5(HvTB1) , act as inhibitor of lateral seed outgrowth and vrs5(hvtb1) mutants display a six-rowed spike architecture. VRS5(HvTB1) is a member of the TCP transcription factor (TF) family, which often form protein-protein interactions with other transcriptional regulators to modulate the expression of their target genes. Despite the key role of VRS5(HvTB1) in regulating barley plant architecture, there is hardly any knowledge on its molecular mode-of-action. We performed an extensive phylogenetic analysis of the TCP transcription factor family, followed by an in-vitro protein-protein interaction study using yeast-two-hybrid. Our analysis shows that VRS5(HvTB1) has a diverse interaction capacity, interacting with class II TCP’s, NF-Y TF, but also chromatin modellers. Further analysis of the interaction capacity of VRS5(HvTB1) with other TCP TFs shows that VRS5(HvTB1) preferably interacts with other class II TCP TFs within the TB1 clade. One of these interactors, encoded by HvTB2 , shows a similar expression pattern when compared to VRS5(HvTB1). Haplotype analysis of HvTB2 suggest that this gene is highly conserved and shows hardly any variation in cultivars or wild barley. Induced mutations in HvTB2 trough CRISPR-CAS9 mutagenesis in cv. Golden Promise resulted in barley plants that lost their characteristic unbranched spike architecture. hvtb2 mutants exhibited branches arising at the main spike, suggesting that, similar to VRS5(HvTB1) , HvTB2 act as inhibitor of branching. Taken together, our protein-protein interaction studies of VRS5(HvTB1) resulted in the identification of HvTB2 , another key regulator of spike architecture in barley. Understanding the molecular network, including protein-protein interactions, of key regulators of plant architecture such as VRS5(HvTB1) provide new routes towards the identification of other key regulators of plant architecture in barley.
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