microRNAs在茉莉酸甲脂诱导的香蕉果实低温抗性形成中的作用研究

Banana fruits were extremely sensitive to chilling injury as kind of tropical fruit. MicroRNA(miRNAs) are short(20-24nt) ,single strand and non-coding small RNAs that negatively regulate their target genes at post-transcriptional level. Research show that miRNAs may involved in plant resistant to biotic and abiotic stress by regulating transcription factors(TFs). Methyl jasmonate(MeJA) is found to be effective on improving chilling tolerance in banana fruit, but little know about the role that miRNAs play in it. In this study, two miRNAs libraries and transcriptomics were constructed from MeJA treated and untreated banana fruits. MeJA induced differential expressed miRNA were screen and their putative targets were predicted. The target genes of these miRNAs were validated using 5' complementary DNA ends (5'-RLM RACE) experiment and stem-loop qPCR analysis. Further more, transcription factors were selected from verified target genes and were used to predicting its downstream regulating genes base on transcriptomics data and bioinformatics analysis. Finally, we conduct yeast one-hybrid experiment and dual-luciferase reporter assay to validate their interaction. All of these results are help to understand how miRNAs- TFs- downstream genes are working in the chilling tolerance induced by MeJA in cold stored banana fruit. The results also providing a new prospective on the study of the involvement of miRNAs in plant resist to biotic and abiotic stress.

亚热带水果香蕉在田间和采后易发生冷害，研究其冷害的发生和预防机制意义重大。microRNAs是一种不编码蛋白质的短链RNA，研究表明microRNAs可通过调控转录因子增强植物的抗逆性。茉莉酸甲脂能有效减轻香蕉果实冷害，但在microRNAs层面的机制尚不清楚。项目组在前期完成了香蕉果实成熟相关microRNAs研究的基础上，以茉莉酸甲脂处理后的香蕉果实作为材料，通过高通量测序，筛选与茉莉酸甲脂诱导低温抗性形成相关的microRNAs，预测其靶基因，采用5′-RLM RACE实验验证其对靶基因的剪切，辅以荧光定量实验明确关键microRNAs和靶基因在低温下的作用模式。最后从靶基因中选择1-2个转录因子，结合转录组数据库、酵母单杂交和双荧光素酶报告系统，研究转录因子和下游基因的互作情况。从而阐明在茉莉酸甲脂诱导下，microRNAs-转录因子-下游基因系列链调控香蕉果实低温抗性形成的机制。

香蕉属于亚热带水果，对低温敏感，11度下易发生冷害。microRNAs是一种不编码蛋白质的短链RNA，研究表明microRNAs可通过调控转录因子增强植物的抗逆性。茉莉酸甲脂能有效减轻许多果蔬的冷害症状，其生理及分子生物学机制已有许多相关报道，但在miRNA层面的机制尚不清楚。本项目通过高通量测序获得香蕉果实中早期响应茉莉酸甲脂的小RNA和转录组数据库，筛选出14个差异显著的miRNAs，进一步开展两大数据库的关联分析，靶基因预测验证，序列比对，通路分析，荧光定量，靶基因剪切验证等试验，发现茉莉酸甲脂处理后，差异表达基因以下调为主，包括叶绿体相关基因，转录因子相关基因等。转录因子中差异表达基因数最多的家族分别是WRKY（33个），NAC（34个），BHLH（35个），MYB（42个），AP2 （43个）。生长素、赤霉素、细胞分裂素、脱落酸、乙烯、油菜素内酯、水杨酸途径均协同参与了反应。miRNAs没有直接调控茉莉酸已知信号通路基因，而是通过调控油菜素内酯信号通路里BRI基因和生长素信号通路里的ARF基因的表达间接参与了茉莉酸甲脂诱导的低温抗性反应。miRNAs参与的代谢途径还包括萜类，苯丙烷类，精氨酸和脯氨酸，抗坏血酸代谢等。此外研究还发现，茉莉酸甲脂处理后SNP的分布发生改变，可变剪切方式主要涉及A3SS和RI两种。研究结果对于更深入解析热带水果低温抗性形成的机制，扩展植物激素间在小RNA层面的互作网络，丰富茉莉酸信号的调控通路等均具有重要的促进作用。