甜高粱茎秆糖积累期特异表达nol-mir271研究

Sweet sorghum is a promising crop for biofuel production; sugars in its stalks are mainly used for ethanol fermentation. However, molecular mechanism of sugar accumulation in sorghum stalks has not been clear. MicroRNAs have been proved to play crucial roles in plant growth, development and stress response. In order to identify which microRNAs involve in sugar accumulation in sorghum stalks, eight libraries of small RNAs were constructed using samples from different sugar accumulation stage for high-throughput sequence. Total RNA of sweet sorghum (Rio) and grain sorghum (BTx623) were extracted from different tissues (stems and leaves) at the heading and dough stage. High-throughput sequencing was used to identify known microRNAs and novel microRNAs of sugar accumulation in the stem of sorghum. Our results showed that a novel microRNA (named novel-mir271), which specifically expressed in stem of sweet sorghum at dough stage. In order to validate the role of novel-mir271 in sweet sorghum sugar accumulation, this project plans to carry out the following research. First, expression pattern of novel-mir271 will be analyzed in 200 sorghum varieties using stem-loop RT-qRCR technology. Second, degradome sequencing technology will be used to identify target genes of novel-mir271. Detection of target genes expression trait and gene functions will help to illuminate the role of target genes in sugar accumulation process, so as to clarify microRNA regulatory mechanism of sweet sorghum stalks in sugar accumulation. This study will make breakthroughs on sugar accumulation of sweet sorghum in RNA regulation level.

甜高粱作为重要的潜在能源作物之一，其应用主要是利用茎秆中的糖，但目前有关高粱茎秆糖积累的分子机制尚不清楚。microRNA是调控植物生长发育和胁迫等生物过程的重要因子之一，为研究microRNA是否参与糖积累的调控，我们比较了甜高粱和粒用高粱在抽穗期和蜡熟期的叶片和茎秆中microRNA的表达差异，筛选到一个在甜高粱茎秆蜡熟期特异表达的新microRNA(暂命名novel-mir271)。为进一步研究该microRNA在甜高粱糖积累中的作用，本项目拟开展如下研究：利用novel-mir271在200份甜高粱和粒用高粱材料中的表达特性分析，确定其与糖积累的相关性；利用降解组测序技术鉴定其靶基因，检测靶基因表达特性，并转化验证基因功能，分析靶基因在糖积累过程中的作用，从而阐明microRNA对甜高粱茎秆糖积累的调控机制。该研究将在甜高粱茎秆糖积累如何在RNA水平进行调控的研究上取得突破进展。

甜高粱作为重要的潜在能源作物之一，其应用主要是利用茎秆中的糖，但目前有关高粱茎秆糖积累的分子机制尚不清楚。microRNA是调控植物生长发育和胁迫等生物过程的重要因子之一。为研究microRNA是否参与糖积累的调控，我们比较了甜高粱Rio和粒用高粱BTx623在抽穗期和蜡熟期的叶片和茎秆中microRNA的表达差异，结果筛选到11个在甜高粱的茎秆或叶片中表达特异的新的microRNA。其中，一个新的microRNA(暂命名为novel-mir271)在甜高粱茎秆蜡熟期特异表达的。为进一步研究该microRNA在甜高粱糖积累中的作用，本项目分析了novel-mir271在90份高粱材料（粒用高粱35份、甜高粱55份）中的表达特性，发现该microRNA基本都在甜高粱蜡熟期的茎秆中特异表达，推测该microRNA参与甜高粱茎秆的糖积累。利用RLM-RACE方法检验microRNA的靶基因，结果证实Sobic.002G208200.1是novel-mir271剪切的靶基因，该候选基因可能编码一个木聚糖糖基转移酶。利用Real-time RT-PCR检测候选靶基因的表达，检测结果显示该候选基因在叶片中表达较高，糖积累前后表达量没有变化；而在茎秆中，粒用高粱和甜高粱的抽穗期表达基本一致，但在蜡熟期甜高粱茎秆中该基因表达明显降低，该表达模式恰恰与novel-mir271的表达模式是相反的，说明miRNA和预测靶基因的表达之间呈现负相关性，也进一步证实了该基因可能受novel-mir271调控。克隆扩增获得靶基因的cDNA序列，构建带有Ubi强启动子的基因超表达载体，并转入高粱Tx430幼胚，已获得转基因再生苗10株，基因功能尚有待验证。特异表达microRNA 及其靶基因的表达调控模式与基因功能研究，为高粱茎秆糖积累的分子机制研究提供了理论依据，将有助于阐明高粱茎秆糖积累的分子调控机制。