利用RNA-Seq技术分离鉴定柳枝稷分蘖发育调控相关基因

Tiller is a specialized grain-bearing branch inmonocotyledonous plants. Tillering is one of the most important agronomic traits that determine grain yields. So researching on molecular regulatory mechanisms of tillering make good sense for agriculture. To this day, the research on tillering was mainly carried out in rice. And there were two ways to research on tillering: separating genes by mutant with unnormal tillering and mapping quantitative trait loci（QTL） associated with tillering. There are some important genes had been separated by studying on mutant but no any gene by QTL analysis. However the molecular mechanism of tillering still remains to be elucidated. In order to kown the generally regulatory mechanisms of tillering in monocotyledonous plants, more researchment on tillering in other species and efficient techniques were necessary. Switchgrass (Panicum virgatum L.), an important biofuel crop, were developed into perfect material for studying on tillering because of its high tillering ability. The mutant of Switchgrass with few tillers had been developed in our laboratory by EMS. In this proposal, we plan to sequence the shoot RNA of mutant and wild type in tillering period separately. Sequencing of RNA has long been recognized as an efficient method for gene discovery and remains the gold standard for annotation of both coding and noncoding genes. We hope to separate the genes with different expression levels in mutant and wild type, which may involved in regulating the formation and its subsequent out growth of tiller buds. Then we hope to get the full length cDNA of candidate genes by RACE technic. In order to conform the function of candidate genes we make expressing level of candidate genes increased or decreased by trangenic methods. We expect to elucidate the molecular regulatory mechanisms of high tillering in Switchgrass and molecular regulatory mechanisms of branching in high plants by studying the biology function in detail and the relationship between them. And we expect that the results from this study renew the regulatory network of branching in high plants and provide guidance in theory and gene resource for molecular breeding in crops.

分蘖是禾本科植物分枝的特化形式，分蘖数是禾本科作物的重要农艺性状之一，有效分蘖数直接影响作物的产量，因此研究分蘖的调控机理对农业生产具有重要理论意义。当前有关分蘖的研究主要在水稻中，一般通过突变体克隆相关基因，对分蘖发育的调控机理的认知还有待完善。生物能源植物柳枝稷具有高效的分蘖能力，是研究禾本科植物分蘖的理想材料。本项目组已经通过EMS 诱变创制了丧失高效分蘖能力的柳枝稷突变材料。拟采用高通量RNA-Seq 技术分别对分蘖盛期的突变型和野生型材料的茎尖生长点进行转录组分析，筛选差异表达基因，利用DNA 末端快速扩增技术（RACE）克隆候选基因的全长cDNA，通过基因表达谱分析、转基因超量表达和抑制表达（RNAi）进行候选基因的功能验证，揭示柳枝稷分蘖大量产生的分子机理，探索高等植物分枝发育的调控机理，为高等植物分枝发育的调控理论体系提供新数据，为禾本科作物的育种提供理论依据和基因资源。

柳枝稷是多年生草本C4植物，常作为重要的能源作物和水土保持植物被广泛应用。柳枝稷作为能源作物，其生物产量是重要的生产指标之一，分蘖是构成生物产量的重要因素之一。柳枝稷可以在极端环境下产生大量分蘖。研究柳枝稷分蘖的分子机理对柳枝稷的改良具有重要的应用价值，对其他作物的改良具有重要的参考意义。本文主要对两个相对的EMS诱变分蘖突变体（多分蘖ht和少分蘖lt突变体）进行生理、生化和细胞学分析，探索柳枝稷分蘖产生的机理；对比分析两突变体的转录组发现在ht和lt中差异表达的基因，探索控制柳枝稷分蘖芽伸长的分子机理；克隆相关基因并初步验证其生物学功能，为研究柳枝稷分蘖调控机理提供基础。得出的主要研究结果如下：.（1）ht和lt分蘖数目不同是由分蘖芽伸长速度不同导致的；去顶之后，lt茎节处分蘖芽伸长速度明显慢于ht突变体，说明茎生分蘖芽的伸长和茎基部分蘖芽伸长有类似的调控机制；植物激素含量测定发现lt突变体中独脚金内酯和ABA含量较高，同时检测独脚金内酯途径中的合成基因和信号途径基因发现lt突变体中合成和信号均强于ht突变体；lt突变体地上部分对重力的响应弱于ht突变体。.（2） ht和lt突变体的转录组对比分析发现表达差异大于2倍的基因有5410个，其中在lt中上调表达的基因有2065个，下调表达的有3345个。这些基因参与多个生物过程，尤其在植物对环境的响应和植物激素信号转导途径中发挥重要作用。.（3）成功克隆独脚金内酯信号途径的关键基因PvMAX2；其在各个组织都有表达，在根、茎基部和茎节处表达较高；其表达量可以受到独脚金内酯类似物GR24的诱导；在拟南芥max2突变体中过量表达PvMAX2可以恢复其分枝增多的表型，说明PvMAX2与拟南芥中MAX2在蛋白功能上的具有保守性。.研究结果丰富了柳枝稷分蘖调控的分子调控网络，可以为改良柳枝稷提高柳枝稷生物产量提供理论基础和实践指导。