Identification And Functional Analyses Of Calmodulin And Calmodulin-like Genes In Mulberry

As a secondary messenger,calcium(Ca²⁺)is widely involved in the growth and development of plants and participates in plant signal transduction.Calmodulin?CaM?and calmodulin-like proteins?CMLs?are Ca²⁺-binding proteins that play important roles in Ca²⁺signal transduction.Calmodulin and CMLs have been identified and analyzed in diverse species.Those studies have shown that CaMs and CMLs are not only involved in plant growth and development,but also participate in both abiotic and biotic stress responses.However,little research is known about their specific roles in mulberry.In our study,we identified genes encoding CaMs and CMLs in the mulberry genome database,and analyzed their gene structure and conserved motifs.Then,MaCaMs and MaCML1were cloned from Morus multicaulis Perr.‘Hongguo II'.The disease resistance-related genes MaCaM2 and MaCML1 were screened by monitoring changes in the transcript levels of MaCaMs and MaCMLs after treatments with the pathogens Ciboria carunculoides and Botrytis cinerea.Then,MaCaM2 and MaCML1 were overexpressed in tobacco.To analyze the functions of MaCaM2 and MaCML1,we compared the transcript levels of resistance genes and disease susceptibility between wild-type and transgenic plants after treatment with B.cinerea.The main results can be summarized as follows:1.Identification and bioinformatic analysis of CaMs and CMLs in mulberryWe identified four MnCaM genes and 42 MnCML genes in the genome of M.notabilis by bioinformatic methods.Conserved domain analyses demonstrated that the CaMs had a typical CaM structure with four conserved EF-hand motifs,whereas the CMLs normally had one to four EF-hand motifs.Gene structural analyses showed that the MnCaM genes contained one or three introns,and that MnCaM genes were interrupted at the Gly26 codon in the first intron.Most of the MnCML genes consisted of a single exon without intron region.A phylogenetic analysis showed that CaMs and CMLs in mulberry trees are most closely related to CaMs and CMLs in Arabidopsis.2.Expression profiles of MaCaMs and MaCML1 during response to biotic stressTissue-specific expression analyses were conducted for four CaM genes and a CML1gene in five tissues of Hongguo II.The four MaCaM genes and the MaCML1 gene were expressed in all five tissues,but their expression patterns differed.For example,MaCaM1-1 and MaCaM1-2 were highly expressed in all five tissues,MaCaM1-1 was mainly expressed in fruit,and MaCaM1-2 was expressed in young leaves.Subcellular localization analyses indicated that the five gene products were distributed in the cytoplasm,cell membrane,and nucleus.We selected diseased fruits showing symptoms of sclerotial disease at the early stage?Stage 1?,the mid stage?Stage 2?and the middle-late stage?Stage 3?,and fruits from healthy mulberry at corresponding periods for transcriptome sequencing.Statistical analyses of the transcript levels of MaCaMs and MaCMLs confirmed that many of them were up-regulated at stage 2 and 3 of disease development.The results of RT-qPCR analyses of gene expression under B.cinerea stress showed that MaCaM2/3 and MaCML1 were up-regulated at 24 hours post treatment?hpt?.However,the transcript level of MaCaM1-1 did not differ significantly from that of MaCaM1-2.Because MaCaM2 and MaCML1 were up-regulated under several different biotic stress treatments,we selected these genes for further analyses.3.Ca²⁺-binding capacity and functional analysis of MaCaM2 and MaCML1MaCaM2 and MaCML1 proteins were obtained by prokaryotic expression.To detect the Ca²⁺-binding ability of MaCaM2 and MaCML1,we added Ca²⁺or EGTA to the purified target proteins.MaCaM2 was used as the control.Electrophoretic mobility analyses indicated that the target proteins bound to Ca²⁺moved faster than those bound to EGTA,and MaCaM2 had the same mobility as MaCML1.These results showed that the conformation of MaCaM2 and MaCML1 changed after binding Ca²⁺,and they had the same ability to bind Ca²⁺.When mulberry and tobacco leaves were treated with the purified target proteins,MaCML1 induced the production of oxygen species and necrosis,while MaCaM2 showed similar but weaker activity.We constructed MaCaM2 and MaCML1 overexpression vectors and transformed them into tobacco plants.We analyzed the transcript levels of resistance-related genes in transgenic plants and wild-type plants by qRT-PCR,and found that the resistance-related genes PR1,PR2,PR10,and WRKY12were highly expressed in the transgenic plants.In addition,the lesion size was much smaller in transgenic plants than in wild-type plants after treatment with B.cinerea.These results revealed that MaCaM2 and MaCML1 could increase the resistance of plants to B.cinerea.