RAV转录因子NtMAB1参与植物分枝发育过程的分子机制研究

Shoot branching, a major component of plant architecture, affects the yield of crops crucially. Branch development can be separated into two stages, axillary meristem formation and axillary buds outgrowth. Axillary meristem formation is determined genetically until now, and bud outgrowth is regulated by genetic, hormonal, developmental, and environmental factors, and the interaction between these factors. Recent studies showed that part of RAV subfamily members from B3 transcription factor families participated into the processes of shoot branching, and directly interacted with the pathways of floral control, Brassinosteroid (BR) and Gibberellic Acid (GA). Moreover, the three pathways also took part in regulating shoot branching, and especially BRs pathway had important influence on axillary meristem formation. Although the functions of RAV subfamily in flower development and floral control were deeply analyzed, the functional analysis on shoot branching was poor. A more-axillary-branches (mab) mutant, named mab1, were screened and identified from activation-tagging mutant library of tobacco. The axillary branches of mab1, which was inherited dominantly and controlled by one single gene, were significantly larger than those of wild type in each leaf position during the entire growing period in field. Two flanking sequences were amplified via TAIL-PCR, which means two insertion sites were obtained at least. The objective insertion site 1(IS1) was confirmed when its genotyping results were co-segregated with those of phenotypes. To determine which gene was altered in its expression level, the expression levels of all the genes within 100kb upstream and downstream of IS1 were examined. The results indicated that the candidate over-expressed gene, NtMAB1-S1, encoding a B3 domain transcription factor, belongs to RAV subfamily in B3 superfamily. A homologous gene NtMAB1-T1 was pseudogene. NtMAB1-S1 was most highly expressed in upper axillary buds before decapitation, and the over-expression plants of NtMAB1-S1 indicated obvious phenotype of more axillary branches. Therefore, based on the mab1 mutant of activation tagging, we will thoroughly analyze the biological functions of NtMAB1 participating into the processes of shoot branching, discuss the molecular mechanism of RAV subfamily taking part in shoot branching. All the results will provide new theoretical references for the research of shoot branching development in plants.

植物分枝是植株形态建成的重要构成因素，是影响作物产量的决定因素之一。本研究在烟草激活标签突变体库中获得一个单基因显性遗传的多分枝突变体mab1，通过侧翼序列扩增、共分离分析、Real Time-PCR分析和基因克隆等前期工作，确定并克隆了候选基因NtMAB1-S1。该基因编码一个B3转录因子，属于B3超家族的RAV亚家族。另一个同源基因NtMAB1-T1经鉴定是假基因。NtMAB1-S1在打顶前上部腋芽中表达量最高，其过表达植株表现明显多分枝表型，与突变体表型一致；但相关分子机制还不清楚。基于以上基础，本研究拟通过多分枝的激活标签突变体材料，利用遗传学和功能基因组学手段深入解析NtMAB1过表达导致分枝增多的生物学现象，明确其调控分枝发育的生物学功能，鉴定关键下游靶标基因并分析相应的调控网络，阐明NtMAB1参与植物分枝发育过程的分子机制，为植物分枝发育机理研究提供新的理论参考。

植物分枝是植株形态建成的重要构成因素，是影响作物产量的决定因素之一。RAV转录因子在花发育和开花控制方面的功能得到了较深入的解析，但其参与植物分枝发育过程的分子机制尚未见报道。.本研究从烟草激活标签插入突变体库中筛选到多分枝突变体mab1。通过插入位点侧翼序列和旁侧基因定量分析确定了编码RAV转录因子的候选基因NtMAB1。该基因定位于细胞核，在旺长期腋芽、新鲜叶片、腋生分生组织和叶脉中大量表达。.转基因过表达、RNAi沉默和基因编辑等转基因验证结果显示，NtMAB1基因具有调控分枝发育的生物学功能，是腋芽生长发育的一个正调控因子。NtMAB1基因过表达促进腋芽持续生长为分枝，降低NtMAB1基因表达量可以抑制腋芽发育/生长；敲除可以起部分抑制作用。NtMAB1含有一个B3功能域和一个转录抑制结构域BRD；酵母单杂交和去除BRD结构域的转基因表型显示，NtMAB1基因通过转录抑制功能域直接调控腋芽发育/生长。.在突变体mab1和NtMAB1超表达植株中，NtBRC1表达量降低50%以上，显著低于野生型。转录组分析和EMSA实验表明NtMAB1可以与几丁质酶A和糖转运子SWEET12启动子中相关互作元件结合。激素测定和外施结果显示，NtMAB1对腋芽的调控也与赤霉素信号通路相关。通过ChIP-Seq技术筛选并鉴定了下游调控基因，并通过糖皮质激素DEX处理实验进一步证明NtMAB1可以下调GA2ox8和BBX32的表达。.研究结果表明，NtMAB1过表达之后，降低NtBRC1和糖转运基因的表达量，部分打破了腋芽的休眠状态，导致腋芽破休眠成长为分枝。降低NtMAB1的表达量或敲除后可以抑制腋芽发育/生长。NtMAB1还可能在腋生分生组织分化阶段通过赤霉素途径调控腋芽发育。本研究初步明确了NtMAB1在烟草腋芽发育方面的调控机制，为进一步阐明植物分枝发育的调控机理奠定重要的理论基础。