两个RWP-RK转录因子在玉米胚和胚乳发育过程中的分子调控机理研究

RWP-RK is a new type of transcription factor gene, which is involved in the development of plant embryo and the process of nitrogen metabolism. However, the biological function of these genes in maize, rice and other crops has not been reported. In our previous work, we found that OS1, encoding a transcription factor containing RWP-RK domains, involved in both the endosperm and embryo development. The nutrition constitution in the kernel was influenced in the os1 mutant. The expression analysis showed that OS1 gene was induced by low concentration of nitrogen and inhibited by high concentration of nitrogen. Genome-wide analysis indicated that the RWP-RK family has 15 members in the B73 reference genome. CRISPR/Cas9 technology was used to knock out the other members of the family. The zmrkd1 mutant and the os1 mutant are partly similar in seed. This project will futher study on the molecular function of OS1 and ZmRKD1, making the expression patern more clear in seeds of different periods and parts using RNA SEQ and in situ hybridization; Unerstand the molecular relations between the two genes by constructing double mutants; Identify the downstream regulated target genes of both OS1 and ZmRKD1 by means of Chip-seq and CRISPR / Cas9; Explain the relationship between the two genes and nitrogen using the technique of in vitro culture of kernel. Regulatory mechanism of the two genes in maize kernel development will be elucidated by comprehensive target gene information, expression data and phenotypic analysis.

RWP-RK是植物中一类新的转录因子，在玉米、水稻等作物中的生物学功能还没有报道。在前期工作中，通过图位克隆的方法克隆一个RWP-RK家族的转录因子基因OS1，该基因突变以后影响玉米籽粒胚和胚乳的发育。表达分析发现，OS1基因受到低浓度氮的诱导表达和高浓度氮的抑制表达。该家族在玉米B73基因组中有15个成员，利用CRISPR/Cas9技术对该家族的另一个成员ZmRKD1敲除发现，zmrkd1突变体与os1突变体在籽粒上有部分表型相似。本项目将对OS1和ZmRKD1两个基因的功能开展深入研究，利用RNA-seq和原位杂交技术明确两个基因在籽粒不同时期和部位的表达变化规律；通过构建双突变体，研究两个基因之间的遗传调控关系；利用Chip-seq和CRISPR/Cas9技术确定两个基因所调控的下游靶基因，阐明两个基因和氮之间的关系以及在玉米籽粒发育过程中的分子调控机理。

玉米产量的三大构成因子为亩穗数、穗粒数和粒重，开展玉米籽粒发育遗传调控过程中的重要基因克隆和分子机理研究将对产量性状的遗传改良具有重要意义。本项目主要的研究内容是OS1和ZmRKD1两个基因在胚和胚乳发育过程中的调控机理、两个基因之间的遗传调控关系以及OS1和ZmRKD1与氮的调控关系。在籽粒中，玉米OS1基因控制籽粒中胚和胚乳之间的养分分配。OS1突变以后，籽粒胚变小，但是籽粒大小不变。通过深入研究发现，OS1编码RWP-RK 转录因子，该家族部分成员参与了氮素营养的吸收和代谢。原位杂交和转录组分析结果表明，OS1在早期胚乳中表达最高，尤其在BETL（负责养分吸收和转运）、CZ（负责养分的在胚和胚乳之间的分配）和CSE（养分储藏）细胞类型中的表达量较高。进一步的RNA-seq分析结果显示，胚乳BETL和CSE亚区特异表达基因的转录也表现出下调趋势，如BETL中的ZmMRP-1、Meg1等，CSE中的大多数玉米醇溶蛋白和淀粉调控基因也出现了下调表达。因此，结合遗传和RNA-seq分析发现， OS1在玉米籽粒中胚和胚乳之间的养分分配起着关键作用（JIPB，2019）。氮素（硝态氮）处理分析结果表明，OS1和ZmRKD1基因受到低氮诱导表达，这目前在该领域还没有更多的线索证明硝态氮如何参与到种子中的养分吸收和代谢。OS1除了在种子中表达，还在侧根原基中特异表达，进一步的氮诱导表达分析发现，该基因在低氮培养条件下高表达，推测该基因在根中也参与了硝态氮的信号途径，两个转录因子基因的深入协同调控研究还在进行中。在文章发表方面，已经发表SCI论文4篇，其中第一标注有2篇，第三标注1篇，第四标注1篇。3篇第二标注的为中文核心期刊。培养博士研究生4名，其中2名已经毕业参加工作。