马铃薯抗寒相关基因表达和MicroRNAs调控模式研究

Taking advantage of fallow fields in southern winter to grow potato production, has an obvious effect on grain yield increasing and food security. At the same time. it is coming up one of the most pressing problems that the seedling suffered frost damage because of off-season cultivation of potato in southern China, leading to unstable yield and late harvest frequently. Depending on insighting into cold response mechanism in potato, how to establish an effective cultivation measures in cold tolerance, and make further efforts on hardiness breeding in potato, is coming up to being a new subject. However, few studies especially at cold resistant gene expression and stress response mediated by the epigenetic regulation of a transposable element small RNA were conducted on potato. Hence, we will perform the two genome potatoes which have different cold sensitivity, transcriptome and miRNA expression profile by Illumina Hi-Seq2000 platform sequencer in order to identify potential genes and miRNA involved in cold responsed during potato cold acclimation. The feature of predicted miRNA promoter and potential target genes will be searched by blasting potential miRNA with potato mRNAs sequences. Interaction between miRNA-gene will be confirmed by dual-lucy assay. Cold stress-responsive miRNA transcriptional regulation status will be discussed. We will Integrate the expression profile and miRNA transcriptional regulation pattern in potato to insight into cold response mechanism during cold acclimation in the end.

冬作马铃薯生产在促进粮食增收、保障国家粮食安全方面的作用日益凸显。同时因反季节栽培由寒冻引起的产量不稳定和收获期延迟问题随之产生。通过抗寒机理研究建立有效防/抗寒栽培措施，促进抗寒品种选育成为马铃薯产业研究的新命题。基于对马铃薯耐寒相关基因表达及miRNA抗寒调控机制缺乏系统了解，本项目拟分别以耐寒和低温敏感马铃薯为研究材料，建立差别冷驯化反应群体，运用Illumina Hi-Seq2000 基因分析系统，研究低温胁迫下马铃薯抗寒相关基因和miRNA 的表达谱；筛选特异低温应答基因和miRNA 调节子；预测并建立相关miRNA 的靶基因群；通过比对特异低温应答基因群和miRNA 的靶基因群，筛选相关目标基因，通过双荧光素酶检测法验证"miRNA-基因"互作；预测miRNA 的启动子序列和转录调控因子，分析马铃薯 "转录因子-miRNA-基因"的低温应答模式,探讨马铃薯低温应答的分子机理。

本研究通过构建差异冷驯化和寒冻群体，利用高通量测序技术对群体进行了转录组和miRNA测序，进行了基因表达、基因结构优化、可变剪接、新转录本发现及编码能力预测、SNP检测等一系列后续分析。转录组测序得到了64002条mRNA的表达量信息；small RNA测序得到了244条已知miRNA的表达量信息以及miRNA与mRNA的靶向关系，预测出33个新的miRNA发夹前体，及其对应的66个miRNA成熟体；完成了对研究群体转录组和miRNA数据在不同处理间的系统比较，并通过基因共表达网络（WGCNA）分析，完成基于差异表达的基因和miRNAs分析，并进行了聚类分析、GO功能显著性富集分析和pathway显著性富集分析；聚焦到与马铃薯冷驯化和寒冻高度相关的基因及其所在生物学途径与主要功能组分；深入关联分析了响应低温胁迫差异显著的miRNA及受其调控的靶基因，获得受miRNA调控的对低温胁迫做出相应的其核心miRNA及其靶基因；运用RLM-RACE法验证stumiR390-5p对靶基因剪切位点后，进一步研究了低温响应stumiR390-5p及其靶基因PGS-09701（抗冻蛋白）、PGS-70158（富含亮氨酸受体蛋白激酶）的功能。项目研究结果为马铃薯基因功能的诠释及miRNA调控靶基因响应低温胁迫机理研究增加了有意义的参考数据。