| Propagation via cuttings is the most commonly used asexual propagation method of tea plant.The rooting rate and survival rate are important factors affecting the efficiency of tea asexual reproduction.The formation and development of adventitious root(AR)is the key to improving the survival rate of cutting-based propagation.Therefore,searching for genes related to the regulation of adventitious root development,and studying their regulatory mechanisms are of great significance for asexual propagation of tea plants.In this experiment,a differentially expressed gene of tea plant ARF(CsARF9)was selected from the transcriptome database established by the research team in the early stage.On the basis of previous research,functional identification and regulatory mechanism of CsARF9 in tea plants were studied.The main research results are as follows:1.Combining the tea plant genome database and using bioinformatics analysis,22 tea plant ARF genes were identified and named CsARF1 to CsARF22.Gene structure analysis revealed that CsARFs genes from the same subgroup had similar exon numbers and gene structures.Analysis of protein conserved domains revealed that most CsARFs proteins contain DBD,MR,and CTD regions,while a few CsARFs proteins only contain DBD and MR regions.The expression pattern analysis showed that the expression of CsARF9 at 14 and 28 days of AR development was 4 times and 2.6 times higher than that at 1 day,respectively.The expression level of CsARF9 in the stem is 20 times higher than that in the third leaf.2.Treatment of tea plants with auxin like substance NAA showed that CsARF9 was highly expressed in tea plants treated with NAA,and the relative expression level was 2.5times higher than that of untreated tea plants 6 hours after treatment.The subcellular localization results showed that CsARF9 is located in the nucleus.Overexpression of CsARF9 in Arabidopsis was found to significantly promote the elongation of the main root and an increase in the number of lateral roots.3.Yeast one-hybrid assay showed that CsARF9 could bind to the auxin responsive element Aux RE(TGTCTC)on the CsLBD39 promoter.Dual-luciferase assay found that CsARF9 could promote the expression of CsLBD39.These two experiments demonstrate that CsARF9 positively regulates the expression of CsLBD39 by binding to its promoter.4.After cutting tea nodal cuttings,the expression level of CsARF9 significantly increased at 1 hour compared to 0 min,and its expression level was 1.5 times higher than that at 0 min.CsLBD39 also responds to wound signals,with relative expression levels1.9 and 2.7 times higher at 3 and 6 hours of excision compared to 0 minutes,respectively.The detection results of auxin(IAA)content showed that the auxin content of cuttings cut for 1h and 3h was 1.2 times and 1.4 times of that of cuttings cut for 0 min,respectively.All the above results indicate that CsARF9 is involed in regulating the formation of AR in response to wound signals. |
