Functional Identification Of MiR160a-CmARF16 In Regulating The Resistance Of Chrysanthemum Morifolium To Aphid

Chrysanthemum(Chrysanthemum morifolium)is one of ten traditional famous flowers in China and one of four cut flowers in the world.It is rich in color,flower shape diversity.Chrysanthemum is of great ornamental value,and edible,medicinal functions,as well as high economic value.However,it is susceptible to aphids at the whole growth and development period,and the most serious damage is Macrosiphoniella sanbourni.M.Sanbourni propagates rapidly,and mainly sucks the sap of chrysanthemum stem,leaf and flower bud to obtain nutrients and necessary water,which leads to plant yellowing and wilting,and seriously affects the ornamental and economic value of chrysanthemum.At present,chemical control is mostly used in production,which not only causes waste of resources and environmental pollution,but also easily makes aphid more resistant to pesticides,which leads to more difficulties in aphid control.At present,how to control aphids scientifically and effectively has become a big problem in production.Therefore,it is urgent to study the mechanisms of chrysanthemum in response to aphid feeding.In this study,we studied the effects of different concentrations of exogenous NAA on aphid population,and preliminarily identified the correlation between auxin and aphid resistance of chrysanthemum.To dissect the trascriptional machenisms underlying auxin on aphid feeding,transcriptome sequencing was conducted,we found that CmARF16,a gene in auxin signalling pathway,is differentially expressed in response to aphid feeding.Our previous study had found that miR160 a was differentially expressed in response to aphid feeding,and CmARF16 was predicted to be the target gene of miR160 a.In order to further analyze the role of miR160a-CmARF16 in regulating aphid resistance in chrysanthemum,we verified the cleavage regulation of miR160 a on its target gene CmARF16,and identified the function of miR160 a and its target gene CmARF16 in regulating aphid resistance by genetic transformation of chrysanthemum.The main results were as follows :1.In order to explore the effect of auxin on aphid resistance of chrysanthemum,we investigated the effects of different concentrations of exogenous NAA on the aphid population on chrysanthemum.The results showed that the 5 μM NAA treatment significantly increased the aphid population compared with the control at 14 days after aphid infestation,while there was no significant influence on the aphid population between the 10 μM NAA treatment and the control.The results showed that low concentration of auxin mediated the aphid susceptibility of chrysanthemum.In order to further study how auxin regulated aphid resistance of chrysanthemum at the transcriptional level,the differentially expressed gene CmARF16 responding to aphid feeding in auxin signal transduction pathway was screened out by transcriptome sequencing.2.The cleavage regulation of miR160 a on CmARF16 was verified by RLM-5′-RACE,RNA Ligase-mediated 5′ Rapid Amplification of c DNA ends,technology in chrysanthemum.CmARF16 gene was cloned.Its ORF was 2,037 bp in length,and encoded679 amino acids.The phylogenetic tree analysis showed that it was closely related to At ARF16.Protein domain analysis showed that CmARF16,like other ARF transcription factors,contained a B3 DNA binding domain,an ARF domain and an AUX/IAA binding domain.The subcellular localization experiment showed that CmARF16 was located in the nucleus.The results of yeast experiment showed that CmARF16 had no transcriptional activation activity.The expression pattern of CmARF16 showed that CmARF16 expressed differentially in response to aphid feeding.3.In order to further study the potential role of miR160 a and its target gene CmARF16 in the defense of aphid in chrysanthemum,the corresponding genetic transformation of chrysanthemum was carried out.The results showed that compared with the wild type,the aphid population on CmARF16 overexpressing transgenic chrysanthemum was significantly reduced,while the aphid population of the miR160 a overexpressing transgenic plants was significantly increased,and the sensitivity to aphids was enhanced,suggesting that CmARF16 contributed to the resistance of chrysanthemum to aphids.In addition,the expression level of miR160 a in chrysanthemum was effectively reduced by STTM technology,and the resistance of miR160a-silenced transgenic plants in chrysanthemum to aphid was enhanced compared with wild type.