棉花盐胁迫相关miRNA的表达调控分析

Cotton is one of the most important volunteer crops for the reclamation of salt-alkali land. At present, most of genes which used for transgenic breeding of salt-tolerant crops could produce proteins. They took effect at the transcriptional level. Whiles, miRNAs are a new class of no-coding, single-stranded small RNAs found in eukaryotic cells. They could finely modulate gene expression at the post-transcriptional level and have recently become the subject of intense study. Therefore, the research for the roles of miRNAs in salt-regulatory networks would provide a new class of gene regulators for transgenic breeding of salt-tolerant crops. In pervious study, we found that miRNAs had significantly different expressions between a salt-tolerant cotton cultivar and a salt-sensitive cultivar under salt stress treatment. Their target genes also showed a genotype-specific expression model in response to salt stress, suggesting that miRNAs play a significant role in salt adaptation in cotton at the post-transcriptional level. To make a further study, we will construct small RNA libraries of cotton roots under salt stress, and use cotton genome sequences to identify salt-regulated miRNAs on a genome-wide scale. Furthermore, degradome sequencing and bioinformatics approach will be used to identify their target genes, thereby building up the regulated pathway mobilized by miRNAs in the adaptation responses of cotton to salt. The research result will help us understanding the transcriptome homeostasis in cotton adaptation to salt stress and provide a new class of gene regulators for transgenic breeding of salt-tolerant cotton.

棉花是改良利用盐碱地的先锋作物。目前在耐盐碱转基因分子育种中所应用的基因大多是能够编码蛋白质的结构基因，在基因的转录水平起调控作用。miRNA是近年来发现的一类不能编码蛋白质的、单链小分子RNA，能够在基因的转录后水平调控下游靶基因的表达。因此，研究miRNA应答盐胁迫的调控机制有望为耐盐碱转基因分子育种提供新型基因。本项目在前期研究中发现盐胁迫下miRNA在棉花耐盐品系和盐敏感品系中存在显著差异，并通过调控靶基因的表达应答盐胁迫，由此说明miRNA参与了棉花的耐盐调控过程。本项目在前期研究的基础上，拟构建盐胁迫下棉花根系的小RNA文库，利用棉花全基因组序列，在全基因组水平筛选与盐胁迫相关的棉花miRNA；利用生物信息学结合降解组测序鉴定miRNA所调控的下游靶基因，从而建立棉花通过miRNA表达参与调控盐胁迫的代谢途径，为棉花耐盐碱转基因分子育种提供功能明确的新型基因。

microRNA (miRNA)是近年来发现的一类不能编码蛋白质的、单链小分子RNA，能够在基因的转录后水平调控下游靶基因的表达。棉花是我国重要的经济作物，也是盐碱地改良的先锋作物。目前，在棉花耐盐调控机制方面，已经克隆到许多耐盐相关基因。然而对于miRNA如何调控耐盐相关基因的表达以及彼此间的网络调控路径鲜有报道。本项目在前期研究的基础上，分析了棉花幼苗在盐胁迫下的生理生化特性。构建了3个棉花小RNA文库，通过比对棉花基因组数据，分析得到棉花保守miRNA 201个，novel miRNA 24个。在短期盐胁迫下，88个miRNAs表达量差异显著；在长期盐胁迫下，75个miRNAs表达量差异显著。利用降解组和转录组分析，得到31个miRNAs的靶基因。对部分筛选的miRNA及其靶基因进行荧光定量分析，miRNA及其调控的靶基因呈现负相关的线性关系。此外，本项目分析得到棉花内源siRNA和4个tasiRNA。对雷蒙德氏棉、亚洲棉和陆地棉中的ARF2、ARF3和ARF4基因进行了系统分析。通过检测基因间的表达调控关系，阐明了miR390/tasiRNA-ARFs/ARF4的表达调控路径。生物信息学分析得到部分MAPK类基因可以被内源siRNA调控；系统分析了棉花中的MAPKKK家族，获得MAPKKK基因78个。在本项目资助下，发表SCI论文1篇；获得国家发明专利1项；培养研究生3名；参加中国棉花学会青年论坛并作大会报告，获得优秀青年报告一等奖。