MiRNA调控马铃薯侧根发育响应干旱胁迫的研究

Lateral roots are important component of plant root system, which play a critical role in plant facilitate the extraction of water and nutrions from the soil. Although, a large number of genes underlying lateral root development under drought stress have been identified at the transcriptional level. However, Post-transcriptional regulation mechanisms of those genes are incompletely understood. MicroRNAs (miRNAs) are classes of endogenic small RNAs, which can play important roles in the regulation of gene expression at the post-transcriptional level and have been considered to mediate gene silencing in plant development and response to abiotic stress. However little is known concerning the molecular mechnism of miRNA regulate the generation and development of lateral root in response to drought stress. In this reasch, microRNAs sequencing and degradome sequencing were used to global survey miRNAs and their targets underlying Lateral root development in potato root system. Futher sereening the candidate miRNA by functional annotation of miRNA target genes, and obtained over-expression and suppress expression of miRNA transgenic plants by artificial miRNA (amiRNA) and short tandem targets mimic (STTM) respectively, The architecture、length and the number of lateral root of transgenic plants will be analyzed to elucidate role of miRNAs in regulation of lateral root development in potato.

侧根是植物根系的主要组成部分，其在植物吸收养分和水分中发挥着至关重要的作用。然而侧根的发育具有高度的可塑性，干旱胁迫是影响侧根发育的重要环境因素。尽管大量调控侧根发生相关基因在转录水平已被鉴定，但对这些基因的转录后调控机理仍不清楚。MicroRNA是一类重要的内源性小分子基因表达调控因子，其在植物生长发育和胁迫响应中起着重要的调控作用，但关于miRNA调控侧根发育的研究报道极少。本研究将联合小分子RNA-seq和降解组测序技术鉴定干旱胁迫下调控马铃薯侧根发育相关的miRNAs及其靶基因，并通过对靶基因的功能注释筛选获得候选miRNA，利用人工miRNA技术(amiRNAs)和短串联靶标模拟技术(STTM)分别获得过量表达和有效阻碍其内源表达的马铃薯转基因植株，通过对转基因植株进行侧根构型、数量和长度的统计分析，解析miRNA调控马铃薯侧根形态发生来响应干旱胁迫的分子机理。

侧根是植物根系的重要组成部分，其在植物吸收养分和水分中发挥着至关重要的作用。尽管大量调控侧根发生相关基因在转录水平已被鉴定，但对这些基因的转录后调控机理仍不清楚。MicroRNA是一类重要的内源性小分子基因表达调控因子，其在植物生长发育和胁迫响应中起着重要的调控作用。但关于miRNA调控侧根发育的研究报道极少。本研究采用small RNA高通量测序的方法，在马铃薯根系共鉴定出203个已知的miRNA和135个新的miRNA，建立了马铃薯根系发育过程中miRNA的表达谱，通过比较分析马铃薯根系发育前期和成熟期miRNA的表达量，共鉴定出64个显著差异表达的miRNA。通过构建混合降解组文库并测序，共检测到出131个miRNA的2400个候选靶基因。通过对靶基因的功能注释，共筛选出6个miRNA的9个靶基因与马铃薯根系发育有关，其中8个靶基因获得了5’-RLM-RACE验证。qRT-PCR分析也显示这些靶基因与其miRNAs表达模式相反，它们主要参与调控植物的根系发育、根的成熟、根的胚后发育、根毛伸长、细胞壁修复、细胞增殖和细胞生长等生物学过程。另外，本研究利用人工miRNA技术构建了stu-miR156和stu-miR319a的过表达载体，利用短串联靶标模拟技术(STTM)构建了stu-miR160a/b和stu-miR827的抑制表达载体，分别转化马铃薯获得了相应的转基因植株。通过对转基因植株进行侧根构型、数量和长度的统计分析，解析了马铃薯miR156靶向作用SPL基因调控侧根发育，stu-miR160a/b靶向ARF转录因子调控马铃薯根系形态建成，stu-miR319a-3p靶向StNIK蛋白激酶调控马铃薯根系发育，stu-miR827靶向StWRKY48转录因子通过调控气孔密度响应干旱胁迫的分子机理。