Liu Yang, El-Kassaby Yousry A
Department of Forest and Conservation Sciences, University of British Columbia, Vancouver, BC, Canada.
Methods Mol Biol. 2020;2093:217-225. doi: 10.1007/978-1-0716-0179-2_15.
In recent years, the scientific community has become aware that epigenetic mechanisms play a more important role in gene regulatory networks (GRNs) than was hitherto thought, as accumulating evidence has shown that changes in epigenetics without genetic variation can affect complex traits over multiple generations. Within the epigenetic machinery, small non-coding RNAs (sRNAs, 18-24 nucleotides in length) are evolutionarily conserved RNA molecules that target mRNAs for deregulation or translational repression. They commonly have high-level regulatory functions in GRNs by mediating DNA and/or histone methylation and gene silencing essential for plant developmental programs and adaptability. Local adaptation enables plants to acquire a high fitness by, for example, properly timing developmental transitions to match plant growth stages with organism's favorable seasons. In particular, the seed represents a key evolutionary adaptation of seed plants that facilitates dispersal and reinitiates the development coupled in time with suitable environmental conditions. With the advent of high-throughput sequencing for sRNAs and computational approaches for sRNA detection and categorization, it is now feasible to unravel how sRNAs contribute to the fitness of tree species that can survive hundreds of years (e.g., conifers). Of particular interest is to disentangle the roles of sRNAs from complex genomic information in tree species with intimidating genomic sizes (commonly 20-30 Gb in conifers) and abundant nongenic components (e.g., >60% transposable elements). In this chapter, we use seeds of the conifer Picea glauca as a study system to describe the methods and protocols we used or have recently updated, from high-quality RNA isolation to sRNA identification, sequence conservation, abundance comparison, and functional analysis.
近年来，科学界已经意识到表观遗传机制在基因调控网络（GRNs）中发挥着比以往认为的更为重要的作用，因为越来越多的证据表明，无遗传变异的表观遗传变化能够在多代中影响复杂性状。在表观遗传机制中，小非编码RNA（sRNAs，长度为18 - 24个核苷酸）是进化上保守的RNA分子，其靶向mRNA以进行失调调节或翻译抑制。它们通常通过介导DNA和/或组蛋白甲基化以及对植物发育程序和适应性至关重要的基因沉默，在基因调控网络中具有高级调控功能。局部适应性使植物能够通过例如适当地安排发育转变的时间，使植物生长阶段与生物体的适宜季节相匹配，从而获得高适应性。特别是，种子代表了种子植物的一种关键进化适应性，它促进了传播，并与适宜的环境条件在时间上耦合重新启动发育。随着用于sRNAs的高通量测序以及sRNA检测和分类的计算方法的出现，现在有可能揭示sRNAs如何有助于那些能够存活数百年的树种（例如针叶树）的适应性。特别令人感兴趣的是，从具有庞大基因组大小（针叶树通常为20 - 30 Gb）和丰富非基因成分（例如>60%的转座元件）的树种的复杂基因组信息中梳理出sRNAs的作用。在本章中，我们以针叶树白云杉的种子作为研究系统，描述我们使用或最近更新的方法和方案，从高质量RNA分离到sRNA鉴定、序列保守性、丰度比较和功能分析。
