玉米胚乳成熟期重要转录因子调控网络研究

Endosperm is a terminal organ for storage of proteins and carbohydrates. Since maize has abundant different endosperm mutants and well established genetic analysis systems, it is no doubt the best model for study of endosperm development in grass crops. Maize endosperm enters into the late stage of development from 8 DAP, which is featured as cell expansion rather than mitotic division, endoduplication and fast accumulation of storage products. Opaque 2 (O2) and P-box Binding Factor (PBF) are two functionally conserved storage-protein transcription factors in grass crops belonging to bZIP and Dof transcription factor families, respectively. In maize, the major storage proteins are alcohol-soluble prolamins called zeins. Zeins are divided into four classes, i.e., alpha (19- and 22-kDa), gamma (50-, 27- and 16-kDa), beta (15-kDa) and delta (18- and 10-kDa). O2 and PBF are started to express 10 DAP, concomitant with activation of all zein genes. O2 regulates the expression of alpha- and beta-zeins, while PBF regulates the 22-kDa alpha- and 27-kDa gamma-zeins. In their double mutant, both 19- and 22-kDa alpha-zeins are further reduced, indicating that PBF has additive effect on their expression. In addition, O2 has extensive effect on expression of other genes in endosperm. O2 has two homologous genes, OHP1 and OHP2, which can form the homodimer of themselves or heterodimer with O2, both being able to bind to O2 targets. Due to lack of mutants, their functions are still unknown. Although 19-kDa alpha- and beta-zein promoters contain the conserved P-box, their expression is not affected in the PbfRNAi mutant, indicating that another Dof transcription factor might be involved in their regulation. Bioinformatic analysis has indentified 46 Dof members in maize B73 genome, among which a second Dof transcription factor was found to be exclusively expressed in endosperm from 10 DAP besides PBF by RT-PCR. Since the new Dof member showed the same expression pattern as PBF, it might be the candidate transcription factor for regulating the 19-kDa alpha- and beta-zein genes and therefore was temporarily given a name as Pbf-like. In this project, we have three goals: First, silencing Ohp1, Ohp2 and Pbf-like transcription factors using RNAi and studying their roles in regulation of zein-gene expression; Second, studying the transcriptome changes in all single mutants, i.e. o2, PbfRNAi, OhpRNAi and Pbf-likeRNAi, and their double mutants of o2 and PbfRNAi, o2 and OhpRNAi, and PbfRNAi and Pbf-likeRNAi, and understanding their own and common regulatory networks by using RNA-Seq; Third, investigating the influences of all mutants on the important agronomic traits of seed, like seed hardness, seed contents, amino acid compositions and germination rate.

胚乳正常发育是决定籽粒品质及产量的关键因素。胚乳各种储存物质合成受到严格遗传网络程序控制，使成熟种子蛋白质和淀粉等成分含量相对稳定。O2和PBF分别属于bZIP和Dof转录因子家族，它们除调控玉米醇溶蛋白基因家族转录外，还广泛影响胚乳其它基因表达。O2有两个同源基因OHP1和OHP2，能和O2形成二聚体且都能识别O2靶位点；PBFlike是新筛选到的另一个胚乳特异的Dof转录因子，表达模式和PBF相同。目前，这三个转录因子的功能尚不清楚。本研究在已有的o2及PbfRNAi突变体基础上，1）构建Ohp1、Ohp2及Pbflike RNAi突变体，研究它们在储存蛋白表达中的调控功能；2）用RNA-Seq分析所有单突变及各种组合突变的转录组变化，研究它们各自及共同的调控网络；3）评估各突变体对玉米籽粒农艺形状的影响。通过以上研究，解析玉米胚乳蛋白含量遗传网络调控机理，为改善籽粒营养品质奠定基础。

玉米胚乳主要储存蛋白是醇溶蛋白，即zein，占总蛋白60%以上。Zein根据氨基酸同源性分为α（19和22-kD），γ（50，27和16-kD），β（15-kD）和δ（18和10-kD）四个亚家族。Zein基因家族表达有三个特征：转录水平极高，空间和时间表达非常特异（授粉后10天在胚乳特异表达）。所有zein都几乎不含必需氨基酸赖氨酸和色氨酸，因此玉米胚乳总蛋白中的这两种必需氨基酸含量就极度缺乏。opaque2 (O2)是第一个被鉴定的调控醇溶蛋白表达的转录因子，属于bZIP转录因子家族，通过结合到顺式作用元件O2-box，启动22-kD α-zein等的表达。O2 Heterodimerizing Protein 1 和2 (OHP1和OHP2) 是通过筛cDNA库鉴定到的另外两个bZIP转录因子，它们能和O2形成异源二聚体，但功能一直不清楚；Prolamin-box Binding Factor （PBF）是第二个被鉴定的调控醇溶蛋白表达的转录因子，属于Dof转录因子家族，该转录因子通过结合到顺式作用元件P-box，调控27-kD γ-zein表达。本研究利用生化和遗传手段发现OHP1和OHP2通过结合到顺式作用元件O2-like box，与PBF相互作用共同调控27-kD γ-zein表达。通过构建o2，PbfRNAi和OhpRNAi不同组合的双突变和三突变，揭示这三个转录因子调控了90%的醇溶蛋白表达。本研究结合α-zein和γ-zein在胚乳蛋白体形成过程中的时空表达模式，进一步提出了这三个转录因子调控蛋白体形成的分子模型。这一研究进一步推进了人们对玉米籽粒醇溶蛋白合成及其它作物种子储存蛋白表达调控分子机理的认识。在该项目资助下发表1篇Plant Cell，2篇PNAS和1篇Genetics，其中Plant Cell （Zhang, et al., 2015）和1篇PNAS（Liu, et al., 2016）为第一标注。