基于高通量测序技术的转录因子、小分子RNA在胡杨异形叶发生中作用分析

Populous euphratica oliv (P. euphratica) shows heterophylly, and its leaf shape is formed during the bud phase. However, so far its exact formation period and molecular mechanism have not been explained clearly. In this project, we aim to solve the above mentioned problems by investigating the lanceolate young leaves in bud and broad-ovate ones in bud of P. euphratica in the Tarim River Basin. The formation period of shaped leaves of P. euphratica is to be determined by observing the morphological changes of the young leaves in bud using microscopy. The abundance of the transcription factor, small RNA, and their downstream genes in the above two kinds of leaves during the leaf formation phase are to be detected by high-throughput sequencing technology. The genes transcription differences of the two shaped leaves are to be contrasted in the course of the development. The bioinformatics method could be used to construct gene networks, and the key genes are to be further validated by using the RNAi method. The aim of this project is to find out transcription factor,small RNA and their downstrem genes which relating to leaf length and leaf width in leaf development of P. euphratica, and further to elucidate their role and regulatory relationships, which would provide a theoretical basis for revealing the genetic regulatory mechanism of leaf shape development and study on the resistance of P. euphratica.

胡杨具有异形叶性，其叶形在芽期决定，但形成的确切时期和分子调控机制尚不清楚。本项目以塔里木河流域的胡杨披针形叶和宽卵形叶芽期幼叶为研究对象，研究胡杨叶形形成的确切时期和分子调控机制。通过显微观察叶芽内幼叶形态变化，确定其叶形形成时期。采用高通量测序技术检测上述两种叶在叶形形成阶段转录因子、小分子RNA及下游基因的转录丰度。通过对比两种胡杨叶发育过程中基因转录差异，用生物信息学方法构建基因调控网络图，对其中起关键作用的基因进一步用RNAi等方法进行验证。旨在找出胡杨叶形发育中与叶长和叶宽相关的转录因子、小分子RNA及下游基因，阐明它们的作用和相互之间的调控关系，为揭示叶形发育的遗传调控机制和研究胡杨的抗逆性提供理论依据。

本项目研究中以第二代高通量测序为基础，结合适时定量PCR、徒手切片显微测量、体外转录实验和包括WGCNA等方法在内的多种生物信息学方法和工具研究分析了miRNA、lncRNA、流产性转录本等非编码RNA和转录因子及下游基因在胡杨异形叶发生过程中的作用，构建了胡杨异形发生的分子调控网络图。发现胡杨异形的发生可分为早、中、后、末四个时期；在胡杨叶形发育过程中总计有374个miRNA被转录（包括165个新miRNA），其中9个miRNA参与了胡杨异形叶的发生；在胡杨叶形发育过程中总共3991个基因发生了差异表达，这些基因中583个基因与胡杨异形叶的发生高度相关，其中50个基因（包括16个转录因子）在这个过程中发挥着更重要的作用；发现有2448个疑似lncRNA在两种叶形中差异表达，其中有86个可以通过拮抗小分子RNA来保护下游基因，从而参与胡杨异形叶的发生；发现4nt和6nt流产性转录本对转录有抑制作用，且6nt的流产性转录本因序列不同而具有基因选择性，这表明流产性起始可能是一种新的转录调控机制。本项目研究表明包括miRNA在内的非编码RNA和转录因子在胡杨异形叶的发生中有重要作用。