柑橘黄龙病菌与寄主互作过程中4个小RNA的检测及功能分析

[目的]柑橘小RNA不仅是黄龙病抗性的调控因子，也可以开发成为黄龙病早期诊断的分子标记。本研究旨在探明小RNA在柑橘黄龙病与寄主互作中的作用。[方法]分别设计并合成了4个小RNA的反转录茎环引物以及相应的表达检测引物；利用实时荧光定量PCR对引物的特异性和扩增效率进行了测试；进一步检测分析了4个小RNA对靶标基因表达的调控情况。[结果]利用本研究设计的引物在健康样品和黄龙病发病样品中均可特异性检测到4个小RNA。相对定量标准曲线斜率表明，4个小RNA引物的扩增效率相近。表达量的相对定量分析表明：4个小RNA的表达与相应靶标基因的表达均呈负相关性；miR-753E-3p、miR-451b、miR2633和miR838-3p在发病样品中的表达量分别为健康对照样品中的4.69、0.44、0.36和3.08倍，而其靶标Cs6g21280、Cs6g05770、Cs2g08190和Cs8g02530在发病样品中的表达量分别为对照的0.14、8.95、3.06和0.29倍。进一步分析表明：这些小RNA的靶标基因在拟南芥中均存在同源基因：Cs6g21280在拟南芥中的同源基因为ATSMC4，与抑制开花和抑制逆境相关的DNA修复有关；Cs6g05770在拟南芥中的同源基因为BAK1，与病原特征分子诱导的抗病反应有关；Cs2g08190在拟南芥中的同源基因为CIPK23，与促进钾离子吸收、扩大叶片的气孔开度有关；Cs8g02530在拟南芥中的同源基因为PIP2，是一个水通道蛋白，可调节植物耐涝耐旱性。[结论]本研究揭示了黄龙病菌与柑橘在小RNA水平上发生的侵染与防卫的对抗作用，为黄龙病病症形成以及寄主免疫相关分子机制的研究提供了新的线索。
[Objectives] Citrus Huanglongbing (HLB)is the most devastating citrus disease worldwide. Our previous high throughput sequencing has identified some differentially expressed small RNA (sRNA)between healthy and HLB-infected citrus, which are good candidates for early diagnosis markers and immune regulators. In this study, the detection methods and the function of these sRNA in HLB susceptibility or resistance were explored. [Methods] Stem-loop primers and detection primers of the four sRNA were designed according to their sequence. The expression of sRNA and their target genes were explored by using real-time quantitative PCR. [Results] The results showed that the four small RNA could be specifically detected with the designed primers with great efficiency. The relative quantitative analysis of gene expression showed that the four sRNA had reversed expression patterns of their cognate target genes. In particular, miR-753E-3p was up-regulated 4.69 times in HLB-infected citrus, whereas its target gene (Cs6g21280)showed 0.14 times reduction on expression upon HLB infection;miR838-3p was up-regulated 3.08 times in HLB-infected citrus, whereas the target gene (Cs8g02530)showed 0.29 times reduction upon HLB infection;miR-451b was 0.44 times down-regulated in HLB-infected citrus, in contrast, the expression of target gene (Cs6g05770)showed 8.95 times increase;miR2633 was 0.36 times down-regulated in HLB-infected citrus, while the expression of target gene (Cs2g08190)showed 3.06 times increase. Further research showed that Cs6g21280 was the homolog of Arabidopsis ATSMC4, which was related to the early flowering and the activation of disease resistance response;Cs6g05770 was homologous to Arabidopsis BAK1, which was related to PAMP-induced disease resistance response;Cs2g08190 and Cs8g02530 were homologs of Arabidopsis CIPK23 and PIP2,