【方法】利用实时荧光定量聚合酶链式反应（quantitative real-time polymerase chainreaction，qRT-PCR）分析ghr-miR394a和GhFBX6的表达模式。在棉花中瞬时超表达ghr-miR394a，利用病毒诱导的基因沉默（virus-induced gene silencing，VIGS）技术在棉花中分别瞬时沉默ghr-miR394a和GhFBX6，研究二者在抗黄萎病中的功能。利用 5^′ 端RNA连接酶介导的cDNA末端快速扩增（5'-RNAligase-mediated rapid amplificationof cDNA ends，5'RLM-RACE）技术并结合荧光素酶（luciferase，LUC）报告系统，分析 ghr-miR394a对GhFBX6的调控作用。【结果】qRT-PCR表明，ghr-miR394a在棉花叶片中的表达量较高，其次为茎和根。与清水处理相比，大丽轮枝菌侵染可显著降低根中ghr-miR394a的表达量，显著提高GhFBX6的表达量。与对照植株相比，在棉花中瞬时沉默ghr-miR394a导致病情指数、病株率显著降低，茎段恢复培养后菌丝积累量较少，棉花对黄萎病的抗性增强；而瞬时过表达ghr-miR394a或瞬时沉默GhFBX6则会降低棉花对大丽轮枝菌的抗性，具体表型为二者的病情指数和病株率均显著升高，过表达ghr-miR394a的茎段恢复培养后菌丝积累量较多，GhFBX6沉默棉株茎秆的维管束褐变程度加重。5'RLM-RACE和LUC报告系统结果表明，ghr-miR394a在转录后水平抑制GhFBX6表达。【结论】ghr-miR394a可靶向抑制GhFBX6的表达，且ghr-miR394a和GhFBX6在棉花黄萎病抗性响应中分别发挥负调控和正调控功能。

ThemiR394a-FBX6module regulatescotton resistance to Verticilliumwilt

Liao Chanjuan1²，Li Dan1²， Zhao Wenjun³，Wu Qi^1，2 ，Yin Xiujuan²， Zhong Guimail²， Wang Zhi³，Hu Guangl，3* Zhai Junfeng3*

（1．ShaoyangPolytechnc，Shaoyang，Hunan422o4，China;2.TechnicalInovationCenterforDevelopmentandIustrial ApplicationofMelonGermplasmResources，Shaoyang，Hunan4220o4，China;3.ChineseAcademyofQualityand Ispectionamp; Testing，Beijing100176，China）

Abstract：[Objective]Thisresearchaimedtoinvestigatethefunctionoftheghr-miR394-GhFBX6moduleincotonresistance to Verticillum wiltandtoprovidecandidategenes forcottondisease-resistantbreeding.Methods]Theexpressionpattrnsof ghr-miR394a and GhFBX6 were analyzedusing quantitativereal-time polymerasechain reaction （qRT-PCR）.Virus-induced genesilencing（VIGS）was employed to transientlysilenceghr-miR394aand GhFBX6 individualy，andincombination withthe transientoverexpressonofghr-miR394aincoton，toinvestigatetheirrolesinresistanceto Verticillumwilt.Therglatory efect of ghr-miR394a on GhFBX6 wasanalyzed using the 5'-RNA ligase-mediated rapid amplification of cDNA ends （ 5^′ RLM-RACE）technique combined with a luciferase （LUC）reporter system.[Results]qRT-PCR analysis revealed that ghrmiR394awas highlyexpressed incotonleaves，folowedbystemsandroots.Comparedwiththewater treatment，inoculation withVertilliumdahliaesignificantlydownregulatedtheexpressionlevelof ghr-miR394ainrots，whilesignificantly upregulated theexpresionlevelofGhFBX6.Comparedwithcontrolplants，transientsilencingofghr-miR394aincoton resultedinasignificantlyreduceddiseaseindexandrateof diseased plantsandless fungal biomassaccumulation in stemsegments duringrecoveryculture，indicatingenhancedresistanceto Verticiliumwilt.Incontrast，transientoverexpreioof ghr-miR394a or silencing of GhFBX6 led to decreased resistance to V. dahliae，asevidencedbyasignificantly higherdisease index andrateofdiseased plants，increased fungal biomassaccumulationin stemsegmentsof ghr-miR394a-overexpressing plants，and more severe browningofvascularbundlesin GhFBX6-silencedplants.The results from5'RLM-RACEandthe LUC reportersystemdemonstratedthatghr-miR394ainhibitstheexpresionof GhFBX6atthepost-transcriptional level. [Conclusion]ghr-miR394a targetsand inhibitstheexpressionof GhFBX6.ghr-miR394aand GhFBX6 playnegativeand positive regulatory roles， respectively， in the cotton response to Verticillium wilt.

Keywords： miR394; GhFBX6; cotton; Verticillium wilt; defense; virus-induced gene silencing

棉花是纺织工业的核心原料，为人们提供优质的天然纤维。棉花及其制品应用广泛，深受消费者喜爱。然而，病虫害常常对棉花生产造成影响，导致严重的经济损失，特别是由大丽轮枝菌（Verticilliumdahliae）引起的黄萎病（Verticilliumwilt）是影响棉花生产力的主要因素之一。大丽轮枝菌通过入侵棉花根部，寄生于棉株内，导致棉花病变，进而显著降低棉花产量和纤维品质[2]。当前，物理和化学方法对黄萎病的防治效果甚微。……