内源诱捕靶标介导MIR171调控百合体细胞胚发生的功能解析

Somatic embryogenesis is vital to the bulb propagation, germplasm preservation as well as genetic traits improvement in Lilium. miRNA is important in the process of somatic embryogenesis as the gene’s upper control factor. And the function of miRNA on target genes is regulated by endogenous target mimics (eTM). Until now the mechanism of eTM mediating somatic embryogenesis in plants is not yet clear. Our research group has identified miR171 involved in somatic embryogenesis on Lilium, and the target genes SCL6 as well as lpu-eTM171 have also been verified and predicted successfully in our recent work. In this project, we intend to carry out the following work. Cloning the full length cDNA and analyzing the expression profiles of miR171, pre-miR171, eTM, and SCL6 by stem-loop RT-PCR, RACE and qRT-PCR; Explicating the transcription characteristics of miR171 via the functional analysis of the promoter cis-function element; Applying the gain-of-function and loss-of-function study to construct the express profiles of miR171 and SCL6 in over-expression and RNAi transgenic plants of eTM and miR171; Annotating the function and analyzing the specific metabolic regulation pathways of miR171, eTM and SCL6, to clarify the regulating mechanism of eTM-miR171-SCL6 during somatic embryogenesis in Lilium. This study may establish foundation for making clear the mechanism of somatic embryogenesis and its artificial regulation.

体细胞胚发生对于百合种球繁育、种质保存、遗传改良等均具有重要意义。miRNA作为基因上层调控因子在体胚发生中起重要作用，而这种调控受内源诱捕靶标（eTM）的介导，但eTM介导miRNA调控植物体胚发生的机制尚不明确。本研究在确定miR171参与调控百合体胚发生并初步验证其对靶基因SCL6的调控作用、预测获得介导因子lpu-eTM171的基础上，拟通过茎环反转录、RACE及qRT-PCR技术克隆miR171成熟体及前体、eTM和靶基因，并分析其差异表达模式；通过启动子顺式作用元件功能分析明确miR171转录特性；通过功能获得性和缺失性研究，分析过表达、沉默eTM和miR171的转基因百合中miR171、SCL6的差异表达水平；注释三者相关功能并进行特异代谢调控通路分析，阐明eTM-miR171-SCL6参与调控百合体胚发生的机理，为明确百合体细胞胚发生机制、并实现体胚发生的人工调控奠定基础。

体细胞胚具有普遍发生、遗传稳定、适于作为外源基因转化受体等优点，对百合产业中的遗传性状改良和种球优质繁育具有重要意义。miRNA作为一类内源非编码小RNA，在植物体细胞胚发生中发挥调节作用。本课题组前期研究表明，miR171在百合体胚发生中具有重要作用，然而其调控百合体胚发生的内部调控网络尚未明确。本项目为深入探究百合体胚发生机制，采用同源过表达、人工miRNA沉默、基因编辑和点突变等技术，研究了lpu-eTM171，lpu-miR171及靶基因LpSCL6-II/I参与百合体细胞胚发生的调控作用，主要研究结果如下：1.成功克隆lpu-miR171a/b的成熟序列、前体序列，并利用FPNI-PCR法克隆获得MIR171a/b启动子序列。明确了MIR171的启动子可以响应光照、非生物胁迫和激素信号。2.克隆获得靶基因LpSCL6-II/I的cDNA全长。利用RLM-5’RACE技术鉴定了miR171与靶基因SCL6的作用位点；对miR171及其靶基因SCL6在百合体细胞胚发生过程中的表达模式进行了分析；明确了在百合植株体内，lpu-miR171能切割靶基因LpSCL6并抑制其表达。3.lpu-miR171a/b沉默和LpSCL6-II/I超表达株系体胚发生提前、体胚发育进程显著缩短，且子叶形胚形成率较高，并伴随着淀粉含量明显升高，且细胞周期关键基因LpCYCB1和LpCYCD3显著上调，其中lpu-miR171b和LpSCL6-I的作用更加显著。4.克隆得到lpu-eTM171a/b和同义点突变MeTM171a/b的cDNA全长，构建了lpu-eTM171a/b和点突变过表达载体并转化百合。lpu-eTM171b过表达系的体胚发生后期观察到有少量体胚萌发形成子叶形胚。高表达lpu-eTM171a/b 负调控lpu-miR171a/b，对应靶基因LpSCL6-II/I的表达水平显著增加；而点突变过表达系MeTM171a/b中miR171a/b和靶基因LpSCL6-II/I的表达量与对照相比无显著变化。表明lpu-eTM171是miR171的诱饵分子，具有绑定lpu-miR171从而阻止对靶基因LpSCL6的切割作用。本研究明确了eTM-miR171-SCL6模块参与调控细叶百合体胚发生的作用模式，为揭示百合体细胞胚发生以及植物胚胎发生发育机制奠定了基础。