蒙古冰草抗旱相关microRNA发掘与功能研究

Agropyron mongolicum Keng is perennial diploid grasses species of Triticeae，which is stronger drought resistance. It plays an important role on reseeding of natural pasture in drought and semi-desert area of north china and seeding of artificial grassland, as well as have valuable genetic resources for the genetic improvement of wheat crops. MicroRNAs (miRNAs) play critical regulatory roles in responsing to different environmental stresses, and many researches of miRNA of Arabidopsis thaliana , Oryza sativa and Zea mays have been done. But untill now, the miRNAs of grasses have not been reported. In order to exploring miRNAs related to drought stress of Agropyron mongolicum Keng, a mixed small RNA library will be constructed and sequenced by Solexa technology in this study. With solexa high throughput sequencing technology and bioinformatics analysis unifies, miRNA of Agropyron mongolicum Keng which related to drought stress will be explored, and 3～4 miRNA will be analysed by Stem-loop real-time PCR. The plant overexpression vector of miRNA was constructed and transformed via Agrobacterium infestation, and 5～10 transgenic plants of Agropyron mongolicum Keng will be gained and studied by real-time PCR, and the function of 1～2 miRNA will be known. The results can provide the basis of molecular mechanism study for drought resistance and molecular breeding of grasses.

二倍体蒙古冰草（Agropyron mongolicum Keng）隶属于小麦族的多年生牧草，抗旱性极强，在我国北方干旱草原及半荒漠地区天然草场补播改良和旱作人工草地建植中占有重要位置，蕴含麦类作物遗传改良的宝贵基因资源。microRNA在植物应对各种逆境胁迫方面起着重要调控作用，在拟南芥、水稻、玉米等作物上已有不少研究报道，但有关牧草与抗旱相关miRNA研究还尚未见有报道。为发掘蒙古冰草抗旱相关的miRNA，本项目拟构建干旱胁迫下蒙古冰草small RNA文库，通过Solexa高通量测序及生物信息学分析，选出与抗旱显著相关的miRNA；利用Stem-loop real-time PCR技术，对其中的3～4个miRNA进行功能预测；构建过表达miRNA 载体，利用农杆菌介导获得转基因植株5～10株，明确1～2个miRNA的抗旱功能，为深入研究冰草miRNA的抗旱分子机制及分子育种提供依据。

蒙古冰草属禾本科小麦族二倍体多年生牧草，是荒漠草原上抗旱性很强的优势草种，具有重要的饲用、生态和育种利用价值，为国家二级珍稀濒危保护植物。本项目重点开展了干旱胁迫下蒙古冰草抗旱相关miRNA筛选鉴定及靶基因预测，同时为找出能够map到转录组上的大量抗旱相关的miRNA及其靶基因，进行了转录组测序及相关基因筛选。项目获得的主要研究结果：1）构建了干旱胁迫下蒙古冰草small RNA文库，基于Solexa高通量测序，对照和干旱处理下蒙古冰草分别获得16.89M和30.26M原始reads，8.05M和16.61M的clean reads；2）筛选获得蒙古冰草114个miRNA，共预测到1393个靶基因，其中1272个靶基因得到功能注释；3）基于qRT-PCR技术，定量验证了抗旱性相关的7个新预测miRNA，其靶基因大多数参与干旱胁迫响应过程，有光合系统中的相关蛋白、泛素-蛋白酶体系统中的蛋白、转录因子、锌指蛋白等；4）建立了蒙古冰草组织培养再生体系；5）成功构建了7个蒙古冰草抗旱相关miRNA过表达载体pBI121-amo- miR21、pBI121-amo-miR5、pBI121-amo-miR62、pBI121-amo-miR44、pBI121-amo-miR82、pBI121-amo-miR17、pBI121-amo-miR77；6）农杆菌介导获得amo-miR21和amo-miR17蒙古冰草转基因植株8株，amo-miR21、amo-miR5、amo-miR62转基因拟南芥株系T3代和amo-miR44、amo-miR82、amo-miR77转基因株系T1代，PCR检测表明miRNA成功转入拟南芥中，且PEG干旱胁迫下其抗旱明显增强；7）基于高通量测序技术获得41.56 M条的reads，总碱基数为8.39G，组装得到91032条Transcripts和44733条Unigenes，筛选出抗旱相关差异表达基因2171个，获得32089个Unigenes注释结果；并用qRT-PCR技术定量验证了20个差异基因的表达；8）筛选出了蒙古冰草抗旱相关14个WRKY和9个MYB基因。项目的实施和完成可为下一步蒙古冰草抗旱基因表达调控机制解析奠定理论基础，并为麦类作物抗旱性遗传改良提供新的遗传资源，有利于促进我国牧草分子遗传育种研究水平的提高。