玉米loss of spikelet determinacy(lsd)基因的生物学功能与调控网络研究

Inflorescence of maize plant is the reproductive organ, and also is the provider of grain yield. Dissecting molecular mechanism of inflorescence architecture and floral organ development is useful for guiding the genetic improvement in grain yield of maize. Early research in our laboratory found a mutant which shows extremely reduced number of spikelet, termed as loss of spikelet determinacy (sld). Furthermore, we had isolated the gene by map-based cloning. In the project, our objectives are i) to link the Lsd gene into the over-expression and RNAi vectors respectively, and then introduce these constructs into maize A188 line. Furthermore, we'll examine the phenotype carefully to reveal the biological function of the Lsd gene. ii) to assay the domain where Lsd gene is expressed on axillary meristem of inflorescence by RNA in situ hybridization, reveal the interacting proteins by the yeast-two hybrid system and validate the interaction by the bimolecular fluorescence complementation (BiFC) assays, and refine the nucleotide motif bound by LSD or its interacting proteins by ChIP-seq, analyze the differentially-expressed genes by RNA-seq, combine all results from above-mentioned examinations to ascertain the regulatory network of Lsd. iii) to establish the association between natural variations of Lsd gene and agronomically important traits such as kernel number per ear, ear length, leaf width and length, and etc), find the causal sites and develop functional markers with potential value for maize genetic improvement.

花序是玉米生殖器官也是籽粒产量的来源，解析花序建成和花器官发育的分子机理对指导玉米产量遗传改良具有重要意义。项目组前期发现了一个玉米雌、雄花序小穗数目明显减少的突变体，暂命名为loss of spikelet determinacy (sld)，并且图位克隆了该基因。在此基础上，本项目拟通过玉米遗传转化及转基因后代遗传分析解析Lsd的生物学功能；采用RNA原位杂交探明Lsd表达的组织区域；使用酵母双杂和双分子荧光互补技术探明LSD互作蛋白；使用ChIP-seq和凝胶阻滞揭示LSD及其互作蛋白特异性结合的顺式元件；基于以上研究，结合转录组比较分析，明确Lsd的调控途径，以期阐明Lsd基因的功能及其调控玉米花序建成的分子机理。另外，采用候选基因关联分析建立Lsd的自然变异与玉米重要性状（穗粒数、穗长、叶片宽度和长度等）数量变异的关联，发掘功能位点，为该基因的育种应用奠定基础。

花序是玉米生殖器官也是籽粒产量的来源，解析花序建成和花器官发育的分子机理对指导玉米产量遗传改良具有重要意义。在本项目中，我们图位克隆了一个玉米雌、雄花序小穗数目明显减少的突变体loss of spikelet determinacy (lsd)，发现gif1基因中一个51bp 插入lsd突变体植株、叶片和花序表型。LSD/GIF1在根系、叶片、节间、花序等组织中均有表达，且在茎顶端分生组织（SAM）、未成熟的雌雄花序中表达量高。其 mRNA在SAM的叶原基和花序分生组织（IM)中心富聚，也在居间分生组织富聚。同时，LSD/GIF1在IM基部及各类花序腋生分生组织(SPM、SM、FM)和花器官分生组织内外稃原基富聚。LSD/GIF1调节叶片细胞增殖和大小和花序中分生组织的确定性。LSD/GIF1与8个GRF和3 个SWI/SNF 复合体成员互作，直接调控UB3、RAMOSA2和CLE4表达，进而调控玉米的花序中分生组织的确定性。另外，候选基因关联分析鉴定到了3个与叶片宽度、雄穗分支数关联和株高关联的位点，为该基因的育种应用奠定基础。