ZmmiR139调控玉米抗旱的功能及作用机制研究

Maize is an important food crops, however, high temperature and drought is one of the main factors limiting maize production. MicroRNAs are vital regulatory factors that regulate plant growth and development as well as adaptability in response to environmental stress at a post-transcriptional level or translation level. At present, it has been proved that miRNAs have close relationship with drought and other adversities in the model plant. However, there are few reports about maize miRNAs in response to drought stress and their participation in drought resistance mechanism. In our preliminary work, we explored novel maize ZmmiR139 that highly expressed under drought stress by using small RNA deep sequencing technology and bioinformatics analysis for drought-resistant maize inbred lines. Then we found that ZmmiR139 affected maize drought resistance through transgenic technology. Our project plans to further confirm the function of ZmmiR139 with gene chip and detection of drought tolerance related genes. Then we would identify the relationship between ZmmiR139 and its target genes via degradome sequencing technology, and verify the important loci of target genes that mediated by ZmmiR139 and expressive abundance of targets’ mRNA. At last, we could carry out overexpression and RNAi analyses of target genes, obtaining different kinds of transgenic plant and acquiring the compound control plants by hybridizing miRNA transgenic plant and targets transgenic plant. Thus we will compare the drought resistance of different kinds of transgenic plant and interactional mechanism among different genes in response to drought stress. Our study will clear the interaction between drought-resistant related miRNA and its target genes, which lays the foundation for maize drought-resistant molecular mechanism.

玉米是重要的粮食作物，而高温干旱是限制玉米生产最主要的因素之一。在模式植物中已经证明miRNA与干旱等逆境有密切的关系，目前玉米抗旱相关miRNA报道较少，且其参与抗旱机制不清楚。前期对抗旱玉米自交系进行小RNA测序和生物信息学分析，发掘玉米干旱胁迫下高表达新的ZmmiR139基因，转基因发现ZmmiR139具有抗旱效应。本项目拟通过基因芯片与抗旱相关基因检测对ZmmiR139基因功能进一步确证，再通过mRNA降解组测序技术鉴定干旱响应miRNA与其靶基因的对应关系，对其中重要的靶基因受ZmmiR139介导的降解位点与mRNA表达丰度进行验证，运用转基因方法开展靶基因过表达、RNAi分析，获得不同类型转基因株，对转miRNA与靶基因调控株系进行杂交获得复合调控株，比较不同株系玉米抗旱功能及基因间互作机制，该研究将明晰玉米抗旱miRNA与其靶基因间相互作用，为玉米抗旱分子机制解析奠定基础。

本研究干旱处理玉米，进行小RNA测序，获得两个受干旱强烈诱导的2个miRNA ZmmiR139与ZmmiR190，将ZmmiR139的靶基因ZmDST44过表达进玉米的植株，获得了阳性植株苗以后，对其进行干旱处理，在处理之前野生型玉米和过表达ZmDST44的植株其表型相似，在进行干旱处理之后，转基因玉米与野生型玉米相比具有明显的抗旱性，并对其丙二醛的含量，相对电导率以及辅氨酸的含量进行了测定，测定结果显示与野生型相比，转基因玉米的MDA的含量呈现一个下降的趋势，但是其辅氨酸的含量与野生型相比呈现上升的趋势，这些实验结果表明ZmDST44提高了转基因玉米抗旱性，同时PsaL蛋白的积累显著增加，并与ABA反应元件（ABRE）结合激活下游抗旱基因的表达，从而提高植株的耐旱性。将ZmmiR190的前体构建玉米过表达载体，对转基因玉米进行干旱分析发现ZmmiR190转基因玉米抗旱性比野生型低。接着我们对转基因玉米和对照组进行了转录组分析，发现转基因玉米中有许多与干旱相关基因的表达量下降，同时其靶基因的表达量也呈现下降趋势。当植物受到外界刺激的时候，在抗旱植株中，ZmmiR190的表达量下降，而靶基因ZmCRP04的表达量相对升高，使玉米获得一个耐旱的表型。以上实验结果说明ZmmiR139与ZmmiR190可以通过调控靶基因的表达，从而调控植物的抗旱性。这一发现为利用基因工程的方法进行玉米抗旱育种提供了基础。