miRNA参与调控毛竹笋芽初生增粗生长的分子机制研究

The primary thickening growth of shoots seriously affects forest biomass and wood property of bamboo. So far, investigations related to the regulation mechanism of miRNAs in shoot primary thickening remain quite limited. On the basis of our previous studies, we will identify key miRNAs that regulate the meristem differentiation and development of moso bamboo shoots by using conjoint analysis for multi-type characteristics, including miRNA expression profiles, tissue section observation, related hormones levels and enzyme activity determination of shoot apical meristem, vascular and pith meristem in different developmental stages of bamboo shoots underground. Then, we will interpret the effects of candidate miRNAs on the forest biomass and wood property of bamboo, by dissecting the genetic effect of SNPs within differentially expressed miRNAs and their targets on the differences traits related to growth and wood property among individuals of association population by using the association genetics strategy, based on the completed population resequencing and phenotypic data measured previously of 545 individuals of Phyllostachys edulis natural population. We will also verify the biological functions of these key miRNAs by using molecular biology techniques, such as northern blotting, in situ hybridization and genetic transformation. This research will provide new insights for the underlying transcriptional and genetic mechanisms of the primary thickening growth of bamboo underground shoots. Surely, results of this study will lay foundation for further investigation of regulation mechanisms for bamboo specific phenotypes.

笋芽初生增粗生长严重影响竹林生物量和竹材品质。针对前人研究不足，本项目以我国重要材用竹种毛竹为材料，对笋芽不同发育阶段的顶端分生组织、维管和髓分生组织分别进行切片观察、相关激素水平以及酶活力测定和小RNA测序等，通过联合分析多类型表征的时空变化规律，初步发掘调控笋芽各分生组织分化和发育的关键miRNAs；基于已完成的545份毛竹核心种质群体重测序以及17个生长和竹材品质相关性状的群体表型数据，筛选差异表达miRNAs及其靶基因内的SNPs位点，运用联合遗传学策略，全面解析候选基因内SNPs对关联群体个体间生长和竹材品质性状的遗传效应。同时，运用Northern杂交、原位杂交和遗传转化等方法分析并验证关键开关miRNAs及其靶基因的生物功能。通过上述研究，阐释miRNA在毛竹笋芽初生增粗生长过程中的转录调控和遗传调控机制，为进一步揭示竹类植物特异表型的调控机理奠定基础。

竹子笋芽初生增粗生长期间内部各组织持续进行分化和发育，成熟的笋芽已具备地上部分成熟竹秆的“雏形”。该过程严重影响竹林生物量积累、竹材材性和竹笋品质。阐明笋芽发育的分子机制，对于解析竹类植物特异性表型的分子机理与竹类植物的分子育种均具有重要意义。本项目以野生型（Wild Type，WT）毛竹（Phyllostachys edulis）和毛竹壁厚变型厚竹（Thick Wall，TW）（P. edulis ‘Pachyloen’）处于萌动期、发育前期、发育中期、发育晚期和成熟期的笋芽为研究材料，（1）通过细胞学结构观察、激素水平和生理生化指标含量的测定等方法，鉴定了毛竹笋芽5个不同发育阶段顶端分生组织、原形成层、维管分生组织、髓分生组织等的细胞学结构和生理生化动态特征，发现了笋芽各分生组织细胞间存在明显的异质性，并且随着笋芽发育，各分生组织细胞的大小、形态特征等也发生了变化，细胞分裂素或者玉米素/细胞分裂素在促进笋芽发育中发挥关键作用；（2）采用小RNA、转录组和降解组联合分析策略，鉴定了150个miRNAs和58,652个基因，绘制了毛竹及其变型厚竹的不同发育时期笋芽内部miRNA和基因动态表达图谱，鉴定了在两种基因型和5个相邻发育时期样本间存在的差异表达miRNA68个、基因 14,209个，通过比较筛选到在相邻时期间/不同基因型间的差异表达基因，降解组测序验证了387组miRNA-mRNA模块，并通过联合分析筛选到调控竹子笋芽各分生组织发育的关键miRNA-mRNA模块、构建了miRNA-基因-植物激素的调控网络；（3）运用双荧光素酶报告基因系统、eGFP瞬时转化以及荧光原位杂交技术验证了miR319/miR166/miR396/miR160/miR167- PePCF/PeHOX/PeGRF/PeARF/PeARF 5个模块间的靶向调控关系及其在笋芽分生组织中的作用位点；（4）在上述5个miRNAs基因前体序列内鉴定到38个显著SNPs（P < 0.05），它们与竹材弯曲模量和拉伸模量两个力学性状显著关联（q < 0.05）。该成果发掘了调控竹子笋芽发育的关键遗传因子，初步阐释了笋芽组织分化和发育的分子机理，剖析了毛竹与厚竹表型差异的分子基础，提升了竹类植物发育生物学的基础研究水平，为竹林生物量积累、竹材材性和竹笋品质改良提供了重要的理论依据和基因资源。