Mining And Expression Analysis Of Amf-induced Papain Family Genes In Citrus

Citrus has rare root hairs,so its nutrient absorption is highly dependent on forming benificial symbiosis with arbuscular mycorrhizal fungus(AMF).Besides,AMF enhance resistance of citrus plant to biotic and abiotic stresses.Therefore,it is vital to study citrus-mycorrhiza symbiosis.The physiological effect of AMF on citrus growth and development has been well known,while little is reported about the molecular mechanism underlying the AM symbiosis with citrus.In our previous study,five citrus Papain genes were found to be greatly induced by mycorrhiza,suggesting their potential function during symbiosis.So we systematically analyzed the spatiotemporal expression of candidate genes and subcellular localization,function of their proteins.The main results in this study are listed as follows:Through phylogenetic analysis of all Papain genes from four main species,we found that 4 Poncirus candidate genes PtrCP1、PtrCP2、PtrCP4、PtrCP5 were in the same clade with 9 Medicago Papain genes,3 rice Papain genes.But none of Arabidopsis Papain genes were clustered in this clade.What’s more,all the poncirus and medicago genes in the clade are greatly induced by AMF and are all annotated as hydrolases.Results of qRT-PCR showed that PtrCP1、PtrCP2、Ptr CP3、PtrCP4、PtrCP5 were significantly upregulated in arbuscular mycorrhizal roots compared with uninnoculated roots(p<0.001),indicating the specific involvement of the five genes in Poncirous-AMF symbiosis.Promoter analysis showed gene PtrCP1 、 PtrCP3 、 PtrCP4 only expressed in arbuscular-containing cells,while a short PtrCP2 promoter(787bp only for limitation of the orange database)didn’t express in a certain pattern.Probably there is one or more decisive motifs in the upstream of the Ptr CP2 promoter.Protein localization analysis showed the PtrCP4 protein driven by its native promoter PtrCP4 pro was expressed in newly formed arbuscular structure especially the terminal instead of the arbuscular trunk or hypha.In addition,we got 2 homozygous mutants(mtcp1 and mtcp9)of the Medicago Papain genes(MtCP1 and MtCP9)which are in the conserved phylogenetic clade and arehomologous to PtrCP1、PtrCP2、PtrCP4、PtrCP5.We will analyze the phenotype of the two mutants to confirm the gene function.According to the cathepsin-L activity of Papain protein and their role in nodule senescence,our research indicated that PtrCP1、PtrCP2、PtrCP3、PtrCP4、PtrCP5 are potentially involved in arbuscular degradation so as to regulate the citrus-AMF symbiosis.