从转录组和表观遗传角度探讨嫁接提高黄瓜耐冷性的分子机制

Cucumber(Cucumis sativus L.) is a typical chilling-sensitive vegetable crop. Grafting can improve the chilling tollerance of cucumber obviously, but the molecular mechanisms still remains to be clarified. In this study, chilling-tolerant cucurbita ficifolia and a chilling-sensitive inbred line of cucumber will be used as test materials. Illumina platform-based RNA-seq and small RNA-seq will be performed to analyze the transcriptome and microRNA expression profile of leaves from grafted seedlings and self-grafted cucumber seedlings under ordinary temperature and low temperature(chilling stress), respectively.Differentially expressed mRNA and microRNA will be screened out respectively, and the function annotation and gene ontology analysis will be carried out to find the most meaningful metabolic pathway. This will lay the foundation of the study of chilling tolerance of grafted cucumber from a single metabolic pathway. At the same time, we aim to determined the genome-wide methylation profile of leaves from grafted seedlings and self-grafted seedlings under ordinary temperature and low temperature using methylated DNA co-immunoprecipitation sequencing technology（MeDIP-seq）, to target the DNA methylation sites that have differences. Combined with the sequence consequence of mRNA and microRNA above, this study is aimed to discuss the molecular mechanism of how does grafting improving chilling tolerance of cucumber from transcriptomics and epigenetics. This project can provide a new way in the research of plant stress resistance, and the research results will have great significance for further discussion on the molecular mechanism of chilling tolerance in plants.

黄瓜为典型的冷敏蔬菜作物，嫁接可有效提高其耐冷性。目前，嫁接提高黄瓜耐冷性的分子机制尚不明了。本项目以耐冷性强的黑籽南瓜和冷敏黄瓜高代自交系为试材，利用Illumina测序技术，对常温和冷胁迫下嫁接苗和自根苗叶片的转录组、microRNA表达谱进行平行检测，分别鉴别出存在明显表达差异的RNA和microRNA，通过对以上基因的功能注释、gene ontology分析，找到统计学上最有意义的代谢途径，为从单一代谢途径研究嫁接黄瓜的耐冷性奠定基础；同时，利用甲基化DNA 免疫共沉淀测序技术（MeDIP-seq）绘制常温和冷胁迫下嫁接苗和自根苗叶片的全基因组甲基化图谱，捕获存在差异的DNA甲基化位点，结合以上的mRNA和microRNA测序结果，从转录组和表观遗传角度协同探讨嫁接提高黄瓜耐冷性的分子机制。本项目可为植物抗逆性研究提供一个新的思路，研究成果对于深入探讨植物耐冷分子机制具有重要意义。

本项目以耐冷性强的黑籽南瓜和冷敏黄瓜自交系为试材，利用第二代测序技术，对常温自根苗（S-N）、冷胁迫自根苗（S-C）、冷胁迫嫁接苗（T-C）和常温嫁接苗（T-N）叶片进行深度测序，共计检测到2821个差异表达基因。在S-C和S-N分析组合中，共筛选得到1748个差异表达基因，冷胁迫处理下849个基因下调表达，899个基因上调表达；在T-C和T-N分析组合中，共筛选得到差异表达基因2035个，冷胁迫处理下861个基因下调表达，1274个基因上调表达；常温下，嫁接苗和自根苗叶片中存在453个差异表达基因，其中嫁接苗中上调表达158个，下调表达295个；低温下，嫁接苗和自根苗叶片中存在585个差异表达基因，其中嫁接苗中上调表达241个，下调表达344个；有35个基因在以上4种分析组合中均有差异；差异表达基因共富集到32个GO条，包括光合作用膜、光系统Ⅰ反应中心、光系统II、类囊体等；差异表达基因共富集到11个KEGG 通路，包括光合生物碳固定、光合反应、卟啉与叶绿素代谢、乙醛酸盐代谢、植物激素信号转导、植物病原物相互作用等。我们对冷胁迫初期，黄瓜幼苗叶片转录组进行了测序，发现随着处理时间的延长，差异表达基因数目越多，冷处理后2小时，有53个基因上调表达，22个基因下调表达；处理6小时后，共有730个差异表达基因，其中上调表达461个，下调表达269个；处理12小时后，共计有1773个差异表达基因，其中上调表达639个，下调表达1134个。最终共检测到13个显著富集GO条目，11个显著富集的KEGG通路。本研究对黄瓜幼瓜和营养器官的RNA文库进行了深度测序，检测到有102个miRNA匹配到已知miRNA上，分属于69个家族的88个miRNA是在黄瓜中新发现的。