光皮桦开花发育过程的miR156调控机制

The study on regulation mechanism of flowering-development by the miR156 in perennial forestry tree, has great scientific meanings for elucidating the forming mechanism of juvenile period. Many progresses on regulation of flowering-development have been obtained in model herbaceous species. However, it is unclear that the upstream regulation network for controlling the flowering-development in perennial forestry tree. Recent studies showed that, miR156 function at the upper level of gene regulation, play important regulatory roles during flowering process. In order to elucidate whether the similar mechanism exists in perennial forestry tree as in herbaceous species, several related experiments would be conducted. Firstly, the identification of miR156 and its' target genes based on data sets of small RNA library and transcriptome by Solexa technique in Betula luminifera, and validate by corresponding experiments. Secondly, the comparison of expression for miR156 and target gene SPLs between two different materials is conducted, flowering in 1.5-year-old and non-flowering in 8-year-old respectively, which is used for mutual grafting, and the expression level of miR156 and SPL is detected, with the aim to elucidating the connection between miR156 expression level and flowering-development in B. luminifera. Thirdly, the expression levels of miR156 are monitored among a series of different flowering-development material, and the threshold of miR156 expression level is ascertained by the statistics technology. Fourthly and lastly, the study on biological function of miR156 was based on the overexpression of miR156, which elucidating the regulatory role and mechanism of flowering-development by miR156 in B. luminifera. It lays foundation for the study on regulation mechanism of flowering-development by miR156 in perennial forestry tree.

多年生林木开花发育调控机制的研究对认识童期形成原因具有重要意义。草本模式植物开花发育调控机制的研究已取得很大进展，但多年生林木开花发育控制有关基因的上层调控机制还不清楚。研究表明miR156在开花过程中起重要调控功能，可成为研究林木开花发育调控机制的新切入点。为了揭示miR156对多年生林木开花发育的调控是否与草本模式植物相似，以光皮桦小RNA文库和转录组的高通量数据挖掘miR156及其靶基因SPL，并实验验证；以1.5年生开花和8年生仍未开花的两类种质为实验材料，比较miR156与SPL表达差异，同时开展两类种质的相互嫁接，检测miR156与SPL表达水平，揭示光皮桦开花发育与miR156表达水平的相关性；检测一系列不同开花发育种质的miR156表达水平，统计学确定光皮桦开花发育的miR156表达阀值；miR156超表达转化光皮桦，阐明miR156对光皮桦开花发育的调控功能和机制。

多年生林木开花发育调控机制的研究对认识童期形成的原因具有重要意义。研究表明miR156在开花过程中起重要调控功能，可成为研究林木开花发育调控机制的新切入点。因此，我们开展了：（1）我们开展光皮桦小RNA和降解组测序，结合生物信息学分析，鉴定了包括10个miR156在内的114个保守miRNA（miRNA*）、24个新miRNA和15个潜在miRNA；降解组测序鉴定了19个保守miRNA家族和7个miRNA*的49个靶基因，7个miRNA的7个靶基因，为光皮桦miRNA及其靶基因的功能研究奠定了基础。（2）通过对10个候选基因筛选得知：不同时期花中RPL39和TUB是表达最稳定的基因，可用作内参基因，而S24和TUA最不稳定。（3）应用RACE扩增得到18个SPL基因全长，在6个亚组均有分布，即SPL家族虽有较高保守性，但光皮桦SPL基因存在功能分化。12个SPL具有miR156的互补位点，其中SPL3、SPL15和SPL17的互补位点位于3’UTR，其余成员的位于编码区。5'RACE验证表明大部分SPL基因的剪切位点位于miR156序列的第10和11位间，而SPL8的剪切位点位于第11和12位间。（4）从不同年龄叶片的定量表达可看出miR56a/b随年龄增长呈下降趋势，而靶基因表达随年龄增加呈上调趋势，尤其G1的SPL1、SPL6、SPL7、SPL8、SPL9、SPL11最为明显，而SPL18和G2、G3中SPL14—SPL17在18月时明显上调，7年龄后下调，预测这几个SPL可能参与光皮桦营养期向生殖生长的转换，而其他非miR156靶基因的SPL基因表达与年龄叶片无明显趋势，在各个时期叶片中表达均较高。（5）miR156与SPL在葡萄糖和甘露醇处理时的表达分析表明，miR156受可溶性糖诱导表达，在葡萄糖处理12h时表达量达到最高；miR156受甘露醇快速诱导表达，50mM甘露醇处理48h达到表达高峰。SPL2在不同浓度葡萄糖处理6h时上调表达，SPL7和SPL10分别在两种浓度葡萄糖处理12h和48h时有一定上调表达，而SPL3在低浓度葡萄糖处理48h时表达显著下调，且SPL4和SPL9亦呈一定程度的下调表达。SPL2受低浓度甘露醇诱导表达，而高浓度甘露醇对其表达有一定抑制作用。（6）构建了miR156a超表达载体，使用叶盘法进行遗传转化，获得了5株阳性植株，目