高羊茅耐热相关miRNA及靶基因的发掘与功能解析

Tall fescue (Festuca arundinacea Schreb) is a major cool season forage and turf grass species grown in the temperature region of China. Heat stress is one of the limiting factor in the production and using of fescues as forage and turf grass in the southern regions. Some microRNAs (miRNAs) in model plants have been considered to mediate gene silencing in plant response to abiotic stress. Identification of conserved and novel heat-responsive miRNAs in plant could advance our understanding of their functions in plant heat resistance. Therefore, in this study, we constructed small RNA libraries from a heat tolerant and sensitive tall fescue that had been exposed to high temperature for small RNA deep sequencing by Illumina Hiseq 2000 technique, so as to identify conserved and novel miRNAs that are responsive to heat stress in tall fescue; perform differential expression analysis of miRNAs; predict and clone the full length target genes of novel miRNAs; and finally, by using transgenic and RNAi methods to study the function of the key miRNAs and their targets in tall fescue responsive to heat tolerance. The results will broaden our perspective on the important role of miRNA in plant responses to heat, and promote the understanding of the molecular mechanims of heat tolerance in tall fescue. In the same time, our project will provide useful genes for tall fescue breeding based on the transgenic technology.

高羊茅是一种重要的草坪草和牧草，在我国北部及长江流域广泛栽培。但作为冷季型草，高羊茅存在不耐高温，难以越夏的缺点，高温胁迫已成为其在我国南方地区发展的主要限制因子。miRNA可通过调节靶基因的表达使植物对逆境胁迫（包括高温）产生适应，将功能miRNA进行转基因，还可改良植物的抗逆性。鉴于此，本项目以耐热和热敏感的野生高羊茅为研究材料，通过 Illumina Hiseq2000高通量测序，辅以模式植物miRNA芯片技术，鉴定并挖掘耐热相关miRNA；进一步预测和克隆耐热相关miRNA的靶基因；最后，利用转基因过表达结合RNAi基因沉默技术，研究目标miRNA及其靶基因在高羊茅热胁迫响应中的功能。本项目实施将促进对高羊茅耐高温胁迫分子机制的理解，同时，获得的高羊茅抗高温miRNA及其靶基因，也将为高羊茅转基因研究提供有用基因源，最终为高羊茅品种的改良奠定基础。

高羊茅是一种重要的草坪草和牧草，在我国北部及长江流域广泛栽培。但作为冷季型草，高羊茅存在不耐高温，难以越夏的缺点，高温胁迫已成为其在我国南方地区发展的主要限制因子。miRNA 可通过调节靶基因的表达使植物对逆境胁迫（包括高温）产生适应。鉴于此，本项目以耐热和热敏感的野生高羊茅为研究材料，通过Illumina Hiseq2000 高通量测序，辅以模式植物miRNA 芯片技术，鉴定并挖掘耐热相关miRNA 850个，其中从属于145个家族的170个miRNA被首次识别鉴定；此外在耐热材料中筛选到受高温诱导上（下）调表达的miRNA 13个和15个，在热敏感材料中，为7和11个。基于差异表达miRNA，以高羊茅转录组为目标序列进行靶基因预测，共获得790个候选靶基因序列，GO功能注释和KEGG通路富集分析结果显示相较于敏感型，耐热高羊茅热胁迫后‘基因表达与表观遗传学’及‘细胞核与核糖体’GO条目得到显著富集。克隆到bZIP23、NAC2、 ATPD、FP2及LLIX1 5个靶基因全长，明确了bdi-miR159b-5p与bZIP23、NAC2基因，osa-miR5809与 ATPD基因的对应调控关系。克隆了一批sHSP基因并以染色体步移方法获得了sHSP18.8、sHSP18.8上游2Kb启动子，顺式元件预测结果发现了bZIP、NAC类转录因子结合位点。此外我们还揭示了外源小分子物质如亚精胺、褪黑素及次高温锻炼调节高羊茅光系统2耐热性的光化学机理。本项目实施促进了对高羊茅耐高温胁迫生理及分子机制的理解，同时，获得的高羊茅抗高温miRNA 及其靶基因，为高羊茅转基因研究提供有用基因源，最终为高羊茅品种的改良奠定基础。