玉米抗丝黑穗病相关miRNA及靶基因的鉴定与功能分析

Head smut is a systemic disease in maize caused by the soil-borne fungus Sporisorium reilianum that poses a grave loss to maize yields in northern China. Resistance against head smut is a quantitative inherited trait controlled by multiple loci, and a major QTL was detected on chromosome bins 2.09.Our group has been selected the candidate gene induced by pathogenic fungi in this region. However, the miRNAs-regulated networks and function in response to head smut are not clear in maize. Two inbred lines, the resistant line Mo17 and the susceptible line Huangzao4, were used in this study. The miRNAs resistant to head smut at seedling stage would be identified by high-throughput sequencing and validated by artificial infection method. Their target genes would be predicted and verified by qRT-PCR, degradome sequencing and 5’RACE. Moreover, over-expression vector for important candidate miRNAs target genes would be constructed and transformed into maize to investigate miRNAs-regulated networks and function in response to head smut in maize. The results of this study could provide a new theoretical basis for understanding the molecular regulation mechanism of maize head smut and the key resistant genes.

由丝轴黑粉菌引发的玉米丝黑穗病是我国北方春玉米生产中一种绝产性病害，抗性属多基因控制数量性状，其一主效抗性位点被定位在2.09区域，本项目组及相关单位前期在此位点已筛选到部分受病原菌侵染所诱导表达的基因，但关于miRNA参与该病的调控研究尚属空白。本研究拟以高抗玉米自交系Mo17和高感自交系黄早四为材料，采用丝轴黑粉菌混合交配型菌液接种技术，通过小分子RNA高通量测序鉴定苗期抗病过程中miRNA，预测其靶基因；通过Real-time PCR分析miRNA及其靶基因的表达情况；通过降解组测序、5’RACE验证靶基因，明确剪切位点；选取重要的靶基因进行时空特异性分析；在对重要抗病靶基因进行体外抑菌试验分析抗病性基础上，构建植物过表达载体，转化高感玉米杂交种HiII，验证基因功能，进而阐明miRNA介导的抗病调控机制。本研究结果可为玉米丝黑穗病抗性机理及抗病关键基因的研究提供新的理论依据。

由丝轴黑粉菌引发的玉米丝黑穗病是我国北方春玉米生产中一种绝产性病害，抗性属多基因控制数量性状，其主效抗性位点被定位在2.09区域，本项目组及相关单位前期在此位点已筛选到部分受病原菌侵染所诱导表达的基因，但关于miRNA参与该病的调控研究尚属空白。本研究拟以高抗玉米自交系Mo17和高感自交系黄早四为材料，采用丝轴黑粉菌混合交配型菌液分别进行浸泡胚根法和针刺中胚轴法人工接种，明确玉米抵抗丝轴黑粉菌侵染的时间点为浸泡胚根法接种后12h，抵抗其扩展时间点为浸泡胚根法接种后6d和针刺中胚轴法接种后1d；针对以上时间点分别进行小分子RNA和降解组测序，结合时空表达分析明确zma-miR164h-5p_R-3及其靶基因ZmNAC21为玉米响应丝轴黑粉菌扩展的重要miRNA及靶基因，用5’RACE方法证实了zma-miR164h-5p_R-3对ZmNAC21的剪切位点为基因664-678bp的GTGCCCTGCTTCTCCA序列；将靶基因ZmNAC21过表达入玉米丝黑穗病高感自交系C01，对T2代阳性株系进行玉米丝黑穗病接种，其抗病性显著提高了19.76-28.55%，进而验证了靶基因ZmNAC21在玉米抗丝黑穗病中的功能；zma-miR164h-5p_R-3可能主要调控NAC、WRKY等靶基因参与茉莉酸、水杨酸和生长素信号通路等反应，进而调节根发育和激素产生抗病功能。本研究结果可为玉米丝黑穗病抗性机理及抗病关键基因的研究提供新的理论依据。