Towards a better molecular and physiological understanding of spikelet fertility in barley (Hordeum vulgare L.)

这项研究建议我们将重点放在大麦小穗育性的分子阐明上。它主要由两个部分组成：第一个感兴趣的区域涉及一个发育突变体六棱穗2(Vrs2)的分子分析；第二部分关注于vrs4.1下游基因的分子和生理特性。然而，到目前为止，对于vrs2基因座，只发现了一个已知的等位基因(vrs2.e)，这使得对该基因座的分析变得困难。因此，我们建议使用不同的遗传和分子方法来寻找或创造新的vrs2等位基因。新的等位基因将通过(I)中间大麦的Sanger测序来发现。中间)，以及(Ii)两个候选基因的分枝分析。(Iii)应测试推定的新等位基因是否为vrs2.e的等位基因。(4)转基因方法应包括使用两个野生型候选基因对vrs2.e突变体进行补充；但同样通过TALEN技术创造新的等位基因。(V)最后，在我们最近的研究(Koppolu等人)中，vrs2.e和其他假定的等位基因应该经过详细的表型分析以及代谢和激素谱分析，以揭示与两个vrs2候选基因之一的功能联系。我们发现Vrs4调节属于海藻糖生物合成途径的基因的转录。海藻糖-6-磷酸(T6P)被认为是控制分生组织决定的重要信号代谢产物，参与植物生长发育对碳供应的调节。T6P水平可能受一组海藻糖-6-磷酸合成酶(TPS)和海藻糖-6-磷酸磷酸酶(TPP)同源物的控制。本研究拟对这两类酶在大麦花序中的功能进行详细的描述，以阐明它们的系统发育关系、拷贝数、组织特异性、mRNA表达模式和催化活性(即TPS，合成T6P的能力；TPP，T6P的去磷酸化能力)。这项研究旨在更深层次地了解大麦的小穗和小花的育性，以提高未来小粒谷物的产量。

This research proposal we will focus on the molecular elucidation of spikelet fertility in barley. It largely consists of two major parts: The first area of interest deals with the molecular analysis of a developmental mutant, six-rowed-spike2 (vrs2); whereas part two focuses on the molecular and physiological characterization of putative down-stream genes of Vrs4.1) Through genetic mapping we identified a vrs2-candidate chromosomal region containing two predicted gene models in the barley physical map. However, for the vrs2 locus only one known allele has been found so far (vrs2.e), which makes analysis of this locus difficult. Hence, we propose to search for, or create new vrs2 alleles using different genetic and molecular approaches. New alleles shall be found through (I) SANGER-sequencing of intermedium-barleys (H. vulgare convar. intermedium), and (II) TILLING analyses of the two candidate genes. (III) Putatively new alleles shall be tested for being allelic to vrs2.e. (IV) Transgenic approaches shall include a complementation of the vrs2.e mutant using the two wild type candidate genes; but similarly to create new alleles through TALEN technology. (V) Finally, vrs2.e and other putative alleles shall undergo a detailed phenotypic analysis as well as metabolic and hormonal profiling to reveal functional links to one of the two vrs2 candidate genes.2) In our very recent study (Koppolu et al. 2013)* we showed that Vrs4 regulates transcripts of genes belonging to the trehalose biosynthesis pathway. Trehalose-6-phosphate (T6P) has been implicated to control meristem determinacy and generally has been considered as an important signaling metabolite that is involved in the regulation of plant growth and development in response to carbon availability. T6P levels are potentially governed by a group of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) homologs. Here we would like to propose a detailed functional characterization of these two enzyme classes in barley inflorescences to elucidate their phylogenetic relationships, copy number, tissue-specificity, mRNA expression pattern and catalytic activities (i.e. TPS, ability to synthesize T6P; TPP, ability to dephosphorylate T6P).This research proposal aims at a deeper molecular, genetic and physiological insight into spikelet and floret fertility of barley for improving future yields of small grain cereals.