葡萄microRNA对二氧化硫的响应及其在贮藏保鲜中的作用

Sulphur compounds have been used by the viticultural industries as preservatives since Roman times. However, increasing health concerns have prompted international restrictions, leading to legislation against the use of sulphur compounds on fresh produce. However, no effective alternative to sulphur dioxide (SO2) has been found for the table grape industry, despite much research and development. In order to find some new effective alternatives to replace SO2 for postharvest control of table grape, the mechanism of table grape fresh-keeping by SO2 need to be investigated. Plant have developed some mechanisms for adaptation to the environmental challenge during the long term evolution. Transcriptional changes often occur under environmental stress, helping plants to adapt to their environments. During SO2 fumigation, grape berries slowed down their aging rate, and showed enhanced resistance to pathogen infection. Recently study showed that SO2 fumigation induced some defense-related gene expression, meanwhile a few of stress related miRNA molecules expressed differentially, suggesting the important roles of gene transcriptional and past-transcriptional regulation during postharvest fresh-keeping. However, plant miRNA study just has its beginning,although some microRNAs (miRNA) have been found in plant response to abiotic and biotic stress. In order to further understand the mechanism of artificial preservatives, by using high-throughput sequencing platforms, the whole miRNA transcripts of table grape berries exposed to SO2 together with their non-SO2 fumigated controls will be detected over the postharvest fresh-keeping period. Then miRNA transcripts in SO2 fumigation groups and their control groups during the storage period will be analysed, some differentially expressed miRNA will be selected. From total miRNA transcripts, we can find some conserved miRNA and some new miRNA molecules differentially expressed in SO2-fumigated berries. By using bioinformatics analysis, miRNA target genes related to fresh-keeping will be discovered. We select some differentially expressed miRNA molecules which may have more important functions on grape postharvest storage, together with their target genes for further study. The transcription levels of specific miRNA molecules were examined by Northern blotting, for their target genes identification, 5′-RACE analysis was employed. After that, we will get some microRNA-mRNA pairs response to SO2-fumigation and fresh-keeping related. Then we use RT-PCR to analyse the transcription changes of both miRNA and its target mRNA over the storage period, to discover some important miRNA molecules and their regulation pathways for postharvest fresh-keeping. Our study will provide new insight into artificial preservatives to fruit fresh-keeping.

二氧化硫（SO2）是目前鲜食葡萄贮藏期间最有效的保鲜剂，但受残留困扰。为寻找更好的保鲜剂，需要研究SO2保鲜的作用机制。葡萄采后SO2处理组果粒中硫代谢、防御相关基因转录改变，抗逆相关miRNA转录应答，可能与保鲜有关。为此，本项目拟采用高通量测序技术获得贮藏期间不同时段SO2处理组和对照组葡萄果粒miRNA转录组，比较SO2处理组品质无损伤样品与对照组品质下降过程中miRNA的转录差异，选SO2处理和对照组间差异表达的miRNA分子，预测其靶基因并进行生物信息学分析，筛选保鲜相关miRNA-靶基因；Northern杂交检验miRNA分子，5′RACE鉴别其靶基因；RT-PCR分析贮藏不同时段SO2处理和对照组中miRNA及其靶基因的转录水平，分析miRNA变化规律及对靶基因表达的影响；构建防腐保鲜相关miRNA-靶基因调控网络，为揭示鲜食葡萄SO2保鲜机制、建立无害化贮藏保鲜技术做出贡献

二氧化硫是目前最有效的葡萄保鲜剂。为揭示二氧化硫保鲜机制，我们研究了贮藏期间果实内部发生的生物学过程，比较了二氧化硫保鲜组和对照组间存在的分子构成、生物酶活性和细胞代谢途径的差异。在对照组和二氧化硫组贮藏20天、40天和60天时分别取材，连同贮藏起始点材料，共构建了7个小RNA文库，检测miRNA表达谱，在玫瑰香葡萄果实中鉴定到了葡萄中所有保守的miRNA家族。共鉴定到148个known miRNAs，分属于23个保守的miRNA家族和24个非保守的miRNA家族。有49个novel miRNAs为玫瑰香葡萄特有。miR169是鉴定到的最大家族，包含24个成员。各miRNA家族的表达水平不尽相同，同一家族各成员的表达量也各不相同。26个家族的54个 known miRNAs和7个novel miRNAs在SO2组和对照组间差异表达。61个差异表达的miRNAs中，只有miR3635-3p未预测到靶基因，其余的60个miRNAs共预测到具有功能描述的靶基因543个。选择表达水平显著变化的6个miRNAs，通过RT-qPCR验证了不同贮藏时期葡萄果实中SO2熏气组中miR479，miR399g，miR397a和miR3627-5ps的高表达，及miR3627-3p和miR2950-3p的表达量降低。并通过RT-qPCR检测了miR479，miR399g，miR397a，miR3627-5p，miR3627-3pH和miR2950-3p的靶基因β-galactosidase，ACO3，LOX，Grip22，PAL和CHS在贮藏期间的表达水平，证实miRNAs对它们的靶基因负调控。鉴定的6对miRNA-靶基因参与玫瑰香贮藏期间包括果实软化，衰老，乙烯合成，次生代谢等过程，形成了miRNA-靶基因参与SO2对鲜食葡萄保鲜过程的调控网络。随后，在模式植物拟南芥验证了miRNA在SO2诱导植物抗病性增强过程中的作用。之后，通过对贮藏期间葡萄果实防御相关酶的活性检测，基因表达谱检测、RT-PCR验证等，发现次生代谢途径增强及其产物合成增加对果实贮藏保鲜具重要作用，植物抗病防御应答参与果实保鲜过程，SO2对细胞代谢过程的影响是延缓果实生理衰老、维持果实品质的基础。