利用新合成异源六倍体挖掘小麦粒长重要候选基因及其功能研究

Grain length, one of the major grain traits, is one of the important factors determining crop yield. In wheat, however, genes involved in grain development, especially grain length development are hardly reported. We previously found significant grain length growth vigor in newly synthesized allohexaploid wheat derived from a combination of tetraploid wheat accession PI94655 (Triticum turgidum, 2n=28) and diploid wheat accession AS2404 (Aegilops tauschii, 2n=14). By comparative analysis of transcriptome profiles between the progeny and the progenitors, we found nine highly expressed genes in pistil and immature seed. Furthermore, these genes exhibited expression patterns potentially correlated with grain length inheritance patterns during the synthesis of allohexaploid wheat. Together with their natures as genes in auxin pathways, these data strongly suggest that these nine genes are candidate genes involved in grain length development. In this proposal we hence plan to conduct the following works: (1) To further confirm their roles in grain length development using virus-induced gene silencing (VIGS) techniques, supported by tissue-specific and spatiotemporal expression patterns and auxin response assays as well as segregation analysis by genetic population; and (2) To study the functions and regulatory modes of one confirmed gene by studying its homoeoallele expression patterns using in situ hybridization, homoeoallele-specific real time PCR, and GUS expression driven by native promoters. Finally, transgenic wheat expressing the chosen candidate gene using tissue-specific promoter will be generated to further study its potential values for wheat yield improvement. This study takes advantage of newly synthesized allohexaploid wheat as bridging materials to screen and identify grain length genes and to characterize their functions, which may provide a new strategy to mine agronomically important genes from wheat donor species.

粒长作为籽粒重要性状之一，是影响作物产量的重要因素。然而，小麦粒长性状相关基因报道却十分有限。前期工作发现一个新合成异源六倍体小麦后代与亲本相比表现出明显的粒长优势，通过表达谱分析获得了9个表达模式可能影响粒长表型的基因，这些基因不仅在雌蕊或未成熟籽粒中特异高表达，且均为生长素路径成员，预示其很有可能与小麦粒长发育相关。本项目拟采用病毒介导的基因沉默方法（VIGS）结合基因时空表达、激素应答以及遗传群体验证等进一步明确其与粒长的关系；对初步明确功能的1个基因，结合BAC文库筛选、in situ、内源启动子驱动GUS表达、基因组特异VIGS以及亚硫酸氢盐测序等方法，深入探讨其三个部分同源基因共同参与粒长性状调控的协同作用模式，揭示其在合成过程中的表达调控机制,并通过转基因小麦最终验证其功能。本研究从新合成异源六倍体出发，以粒长基因为例，以期为挖掘小麦供体种重要农艺性状基因开辟一条新途径。

粒长作为籽粒重要性状之一，是影响作物产量的重要因素。已有研究表明，生长素通过影响细胞分裂，细胞增殖和分化在器官发育中起着至关重要的作用。尽管已有多个生长素信号传导途径成分被发现参与籽粒发育，但还未见AUX/IAA作用的报道。本研究中，我们围绕小麦IAA21基因开展了以下四个方面的工作，并获得了较好的发现：1)实时PCR分析表明小麦TaIAA21在授粉后4天的籽粒中高表达，原位杂交显示TaIAA21主要在授粉后4天的果皮 (Pe)，珠心突起 (NP)和胚乳核中表达。在三个部分同源基因中，TaIAA21-B的表达占主导，表明亚基因组基因间有一定的功能分化和演变；2)通过获得和观察栽培小麦Cadenza突变体表型我们发现，TaIAA21突变导致粒长，粒宽和千粒重与野生型相比均显著增加，其中粒长增加最为显著。组织切片发现TaIAA21突变导致果皮细胞长度和果皮细胞数目增加，表明TaIAA21参与小麦粒长调控；3) 酵母双杂交互作蛋白筛选实验发现TaIAA21-A，TaIAA21-B和TaIAA21-D可以分别与TaARF16，TaARF19和TaARF25互作，形成异源二聚体，并且TaIAA21-A和TaIAA21-B还可以与TaTPL形成异源二聚体，而TaIAA21-D第一个外显子缺少了与TaTPL互作所需的63个关键氨基酸，导致TaIAA21-D无法与TaTPL互作，暗示D拷贝在蛋白互作方面可能存在亚功能化；4)进一步的序列多样性分析发现TaIAA21-A有6种单倍型。通过在262份中国小麦微核心种质中的11种重要农艺性状关联分析发现，Hap2, Hap3与高千粒重显著关联，且栽培种中Hap2和Hap3的出现比例显著高于地方品种，表明该基因在现代育种中受到选择。总之，我们的工作首次在小麦中鉴定了一个参与小麦粒长发育调控的AUX/IAA基因TaIAA21。该基因突变后粒长、粒宽以及千粒重均增加，表明其与小麦的产量关系密切。而在小麦的亚基因组基因中，B拷贝主导表达，D拷贝失去与TaTPL的互作，以及鉴定到的2个在现代育种中受选择的A拷贝的单倍型则揭示了异源六倍体小麦基因调控的复杂性。本工作从新合成异源六倍体籽粒发育特点出发，以粒长基因为目标开展研究，为挖掘小麦供体种重要农艺性状基因开辟了一条新途径。