摘要
【目的】在前期的种质资源评价过程中,发掘一株中国野生刺葡萄0943,该株系高抗葡萄白腐病。基于mi-croRNA(miRNA)在植物抗病中的重要作用,拟从miRNA水平探讨刺葡萄在受到病原菌侵染后的表达调控机制。【方法】以抗病的中国野生刺葡萄为试材,对比感病的欧亚种'美人指’,分别以病原菌诱导,在0 hpi(hours post inoculation)和病菌诱导后的12 hpi、36 hpi处理后采样,进行二代测序,并对数据进行KEGG及表达量的差异分析。【结果】对比感病葡萄'美人指’,分析了抗病刺葡萄在基础代谢和抗病途径中的差异,结合miRNA的表达量,获得了150个表达量发生变化的miRNA,其中44个miRNA的表达在刺葡萄和'美人指’之间存在差异。5个miRNA在刺葡萄中特异表达,但是在'美人指’中不表达,实施定量验证了这一结果。靶基因预测显示,其靶基因包括与抗病的紧密相关的WRKY、SPL、EFR等转录因子,还包括与抗病直接相关的LRR类的抗病基因。【结论】筛选出5个在刺葡萄上特异表达候选miRNA(miR172a、miR172b、miR845a、novel_81和miR159a),可作为刺葡萄抗白腐病研究的目标。
【Objective】Grape white-rot [Coniella diplodiwlla(speg.) Petrak & Sydow] is one of themost destructive diseases in grape. The traditional fungicide control not only could increases the produc-tion cost, but also would affect the environment. Therefor, it is necessary to use the resistance genes ofwild grape in China and improve the resistance of new varieties. An wild grape strain 0943(Vitis davi-dii) was found to be resistant to the grape white rot in our previous study. It has been known that mi-croRNAs(MiRNAs) play important role in plant disease resistance. This study intended to explore themiRNA mechanism of expression regulation in V. davidii after infection by pathogenic bacteria.【Meth-ods】For providing plant tissues for sRNA sequencing analysis, 2-year-old plant of Vitis davidii and Vitisvinifera'Manicure Finger'were grown in a greenhouse at 28 ℃ with a 16 h photoperiod. These plantswere inoculated with C. diplodiella by fixing four mycelium gelose discs(diameter of 2 mm) on eachleaf with small pins and covering the leaf. Leaf samples were collected at 0, 12 and 36 hours post inocu-lation(hpi). 3 μg of total RNA for each sample was taken to create a small RNA library. Gene Ontolo-gy's study of the distribution of candidate target genes in Gene Ontology would elucidate how the sam-ple differences in experiments were reflected in gene function. KEGG analysis, and significant enrich-ment through pathway were used to identify the most important biochemical and signal transductionpathways for candidate target genes involved. KEGG enrichment analysis was made using KOBAS.【Results】We sequenced three small RNA libraries from Vitis davidii. In this study, we obtained 3.282 Gdata, each library data was over 0.5 G. 106 miRNA mature bodies were obtained through sequencing, ofwhich 91 were able to match the miRNA precursor, and the types of miRNA that could match were6 567. After statistics, the miRNA number of V. davidii was more thanthat of the'Manicure Finger'at0 hpi, but the induced expression of miRNA was lower than the'Manicure Finger'. The number ofmiRNA species increased at 12 hpi and 36 hpi points in both V. davidii and'Manicure Finger', and themiRNA species of V. davidii increased from 862 to 1 300 and 1 115, while'Manicure Finger'miRNAspecies increased from 828 to 1 384 and 1 078. In the differential expression analysis of miRNAs, themiRNAs with significantly different expressions at 12 hpi and 36 hpi were used as references for thethorny grape at the 0 hpi treatment point, and their target genes were analyzed by KEGG enrichment.The analysis showed that miRNAs played a role in 11 life pathways through 847 target genes, of whichthe target genes involved in the protein processing in endoplasmic reticulum pathway were the most,and the target genes involved in the Pantothenate and CoA biosynthesis pathway were the least, account-ing for 23. Through the prediction of miRNA target genes, the basal metabolic pathways with sugar me-tabolism as the core were analyzed. The results showed that in spiny grapes, all basal metabolic path-ways including the Citrate cycle and the oxidative phosphorylation of the NADH energy synthesis path-way, were in a down regulation mode; CoA-mediated energy release Coumaroyl-CoA pathway was inan up regulation mode. In this study, the differences between V. davidii and'Manicure Finger'were an-alyzed. The different expression of 150 was obtained. And 44 miRNA was different between V. davidiiand'Manicure Finger'. At the same time, 5 miRNA were specifically expressed in V. davidii, but werenot expressed in'Manicure Finger'. Target gene prediction showed that the target gene includedWRKY, SPL, EFR and other transcription factors related to disease resistance, and also included LRRdisease-resistant genes. 【Conclusion】In this study, 5 specific expressions of candidate miRNA,mir172 a, miR172 b, miR845 a, novel_81 and miR159 a in V. davidii were screened, which could be usedfor the study of resistance to grape white rot in V. davidii.
引文
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