橡胶树次生乳管分化相关miRNAs的鉴定及功能解析

MiRNAs plays an important regulatory role in cell differentiation in plants and animals. Laticifer is the place of synthesis and storage of natural rubber in rubber tree(Hevea brasiliensis). The number of secondary laticifers, which located in the secondary phloem of bark, is positively correlated with natural rubber yield. It is one of the important indicators of rubber tree breeding program for high yield rubber clone. The secondary laticifers are produced in vascular cambium cells. The process of rubber tree breeding were slow down because there were not secondary laticifers in rubber seedling under natural conditions. Recently, we establish an experimental system about the secondary laticifers inducing by coronatine in young stems of epicormic shoots which only had primary laticifers. We also found that the secondary laticifers were induced by the application of jasmonic acid in young stems. In this study, we identify laticifer differentiation related miRNAs in vascular cambium cells and establish the relationship between miRNAs and laticifer differentiation by using the technologies such as laser microdissection, high-throughput sequencing, bioinformatics analysis and molecular biology. This study is base on the experimental system about the secondary laticifers inducing by coronatine in young stems and the whole-genome sequence data. The results will contribute to establishing a good foundation for further investigating the molecular mechanism for laticifer differentiation and developing molecular markers of the yield traits in rubber tree.

miRNAs在动植物细胞分化中起着重要的调节作用。巴西橡胶树（以下简称橡胶树）的乳管是天然橡胶合成和贮存的场所。树干树皮（次生韧皮部）中次生乳管的数量与天然橡胶产量显著正相关，是橡胶树产量育种的一个重要指标。次生乳管由维管形成层细胞分化而来，但是在自然条件下，橡胶树在苗期并不分化出次生乳管，限制了橡胶树的产量育种进程。最近，我们建立了能诱导苗期的橡胶树分化次生乳管的实验系统，并证明茉莉酸是诱导次生乳管分化的关键信号分子。本项目拟利用冠菌素诱导橡胶树萌条次生乳管分化的实验系统和依托单位拥有的橡胶树全基因组测序数据库信息，采用实验形态学、细胞学和分子生物学的有关技术，结合激光显微切割技术、高通量测序技术及生物信息学分析，鉴定维管形成层中与次生乳管分化相关的miRNAs，通过分析其靶基因，初步解析其对乳管分化的调节功能，为研究橡胶树乳管分化的分子调控机理和研发产量性状相关的分子标记打下良好基础。

橡胶树的乳管是天然橡胶合成和贮存的场所。树干树皮（次生韧皮部）中次生乳管的数量与天然橡胶产量显著正相关，是橡胶树产量育种的一个重要指标。次生乳管由维管形成层细胞分化而来，已有的研究证明miRNAs在动植物细胞分化中起着重要的调节作用。而且茉莉酸是诱导橡胶树次生乳管分化的关键信号分子。本项目利用冠菌素（COR，一种茉莉酸模拟物）诱导橡胶树萌条次生乳管分化的实验系统，采用高通量测序技术测定了橡胶树内层树皮的转录组及小RNA表达谱，并对差异的unigene及miRNA进行验证。同时，对miRNA及比较转录组进行了联合分析，初步鉴定了COR诱导次生乳管分化相关的miRNAs，通过分析其靶基因，初步解析其对乳管分化的调节功能。取得的研究结果如下：.1. 通过比较转录组学分析，初步证实茉莉酸信号途径、CLAVATA-MAPK-WOX信号途径和钙信号可能在乳管分化调节中起着重要的协同作用；.2. 通过对小RNA的数据分析及实验验证，在橡胶树树皮中鉴定了354个已知的miRNA，预测了437个新的miRNA。qRT-PCR分析分析显示多个miRNA的表达受COR诱导。通过对乳管分化能力差异种质中miRNA的表达分析，推测novel-miR47可能与乳管分化具有相关性。另外，对miR156a-5p，miR157a-5p，miR397a和miR408-3p进行了靶基因的5’-RACE验证。.3. 对转录组及小RNA进行了联合分析。miRNA靶基因的GO功能注释及显著性富集分析结果显示，COR早期响应的miRNA靶基因主要富集在14个GO term，而COR晚期响应的miRNA靶基因富集在1个GO term。这些靶基因主要与相关蛋白酶如转氨酶、转移酶、磷酸酶、脂酶、内源性氧化还原酶、ATP酶等活性有关。KEGG显著性富集分析显示miRNA的靶基因主要参与植物-病原体的互作、植物激素转导等信号途径。.本项目的执行，积累了次生乳管分化相关的数据基础，创新性地提出CLAVATA-MAPK-WOX信号途径在次生乳管分化中起着重要的调控作用，并从形成层鉴定数个COR响应的miRNA，研究结果对于进一步研究橡胶树次生乳管分化的分子调控机理以及研发产量性状相关的分子标记打下良好基础。