Expression Patterns Of NbrgsCaMs Family Genes And Their Roles In Developmental And Environmental Stress Of Nicotiana Benthamiana

Calmodulin-like protein(CML),as a calcium sensor,plays an important role in mediating abiotic and biotic stress.Rgs Ca M,a unique branch of calmodulin-like(CML),has been known as regulator of gene silencing induced by viral infection in Nicotiana.Three members of CML37,CML38 and CML39 were found in the Atrgs Ca Ms family of Arabidopsis thaliana.We believe that there might be several Nbrgs Ca Ms in Nicotiana benthamiana,like Atrgs Ca Ms,which can respond to different environmental and developmental stimuli.In order to further understand the regulatory role of rgs Ca M in plant growth and development,disease response and stress resistance,the following studies were carried out 1 Sequence analysis of rgs Ca M family: We found a total of 5 Nbrgs Ca M coding sequences through screening the N.benthamiana genome among 7 homologs which named rgs Ca M 1-7.The amino acid sequence and phylogenetic tree analysis of EF chiral domain containing conserved rgs Ca M protein showed that five of them were Nbrgs Ca M coding sequences,and the other two rgs Ca M2 and rgs Ca M6 might be pseudogenes or unknown proteins.Thus,we focused on rgs Ca M1,3,4(rgs Ca M4 is the previously reported Nbrgs Ca M),5 and 7 only,to elucidate the characters of rgs Ca M family in N.benthamiana.Amino acid sequence alignment and phylogenetic tree analysis showed that the Nbrgs Ca Ms coding sequence shared 70.4% to 93.3% similarity with each other,and the amino acid sequence homology was 60.2% to 83.2%.2 Analysis of rgs Ca M family promoters' activity: Based on the ?-glucuronidase(GUS)reporting system,we constructed chimeric rgs Ca M promoters(rgs Ca Mp::GUS).Under a series of abiotic and biotic stresses treatment,the activity of rgs Ca M family promoters was measured by GUS staining and RT-q PCR.Rgs Ca Mp::GUS reporting system analyzed GUS transcription levels at different developmental stages and under specific stress treatments.The results showed that each Rgs Ca Mp::GUS drives a similar but distinct GUS expression pattern.Therefore,Nbrgs Ca Ms in plants may have overlapping and functional compensation expression patterns to regulate plant development and response to environmental stimuli.Each Nbrgs Ca Mp drives an overlapping but specific GUS expression pattern.Nbrgs Ca Mps are mainly expressed in the veins and roots of seedlings and mature plants and Nbrgs Ca Mp4 had highest activity among all Nbrgs Ca Mps.At the same time,Nbrgs Ca Mps coding sequences have high similarity and many enhancer regulatory elements with the same functions are predicted in the sequences.Therefore,we believe that Nbrgs Ca Ms in plants may have overlapping expression and functional compensation modes to regulate plant growth and development and respond to environmental stimuli.3 Under biological stress,GUS staining of the leaves of NbrgsCaMp4::GUS inoculated with TYLCCNV-A,TYLCCNV-A+?,PVX,PVX+ ? and TMV showed that Nbrgs Ca Mp4::GUS had the strongest response to TMV virus which was more destructive to plants,followed by the combine infection group(TYLCCNV-A+?,PVX+?)at the later stage of virus infection.GUS staining and RT-q PCR analysis showed that there was no expression difference of Nbrgs Ca Mp4::GUS between TYLCCNV A treatment along and TYLCCNV A+?C1 combination infection at the early stage.In thelate stage of TYLCCNV infection,TYLCCNV A+?C1 treatment showed higher promoter activity.TYLCCNV A inoclution can not cause visible phenotype.As a pathogenic factor,?C1 is the key factor to cause curly leaves and vein convexity in the plants.Therefore,we believe that ?C1 may induce the expression of Nbrgs Ca M4 through toxic damage effects.4 Analysis of expression level of NbrgsCaM4: There was no notable difference between wild type and transgenic TYLCCNV A+? plants and TYLCCNV-A treatment in the expression level of Nbrgs Ca M4 at early stage of virus infection.The expression level of Nbrgs Ca M4 in wild type plants were higher than that in wild type plants at the late stage of TYLCCNV A+? infection compared with TYLCCNV-A inoculation alone.The expression level of Nbrgs Ca M4 in ?C1 transgenic N.benthamiana was 2-3 times higher than that in wild type,whether in seedling or mature plants.The expression level of Nbrgs Ca Mp4 increased by tens of times after inoculation with virus-free buffer and responsed strongly to mechanical damnification.In addition,leaf age also impacts the expression of Nbrgs Ca M4,and the expression transcripts in old leaves was tens of times higher than that in young leaves.the expression transcripts in mature plants was also tens of times higher than that in younger plants.Therefore,in addition to the diversity of Nbrgs Ca Mps activities can be induced at the general stage of plant development,Nbrgs Ca M4 is a member of the CML subfamily,which can be caused by different degrees of plant damage stress(salt,drought,mechanical damage,virus infection types).