Identification And Expression Of Calmodulin/Calmodulin-like Gene In Rosa Roxburghii And Its Relationship With AsA Synthesis

Rosa roxburghii Tratt.is a native fruit tree of Rosa of Rosaceae family from China.Its fruit is rich in nutrients and health care components,especially the accumulation of high content of vitamin C（L-ascorbic acid,AsA）,which has attracted much attention.Calmodulin（CaMs）and Calmodulin-like（CML）are important calcium-binding proteins in plants that specifically bind to and transmit Ca²⁺signals,thereby widely participating in biological processes such as fruit tree growth,stress response and quality formation.Previous studies have also revealed that Ca²⁺and CaM is involved in fruit growth,development and AsA synthesis.In this study,the CaM/CML gene family members of Rosa roxburghii’Guinong 5’were identified systematically,and their expression characteristics and possible relationship with AsA synthesis were analyzed,aiming to lay a foundation for revealing the physiological roles of CaM/CML family members in Rosa roxburghii.The specific research results are as follows:1)Identification and bioinformatics analysis of CaM/CML genes in Rosa roxburghiiIn this work,a genome-wide analysis conducted in Rosa roxburghii and identified 2 RrCaM-encoding genes and 41 RrCML-encoding genes.Both of the RrCaMs contained the typical 4 EF-hands,and the 41RrCMLs contained 1-4 EF-hand.The RrCaM1 and 21 RrCMLs contains 1-6 introns,while the RrCaM2 and20 RrCMLs has no introns.Chromosome mapping analysis indicated that 41 RrCaMs/RrCMLs genes were unevenly distributed in seven chromosomes.The phylogenetic relationship and synteny analyses showed that CaMs/CMLs genetic relationship among Rosa roxburghii,Pyrus bretschneideri,Fragaria vesca and Arabidopsis thaliana is close.RrCaMs and RrCMLs were clustered as 5 different subgroups.Besides,22orthologous CML pairs was found between Rosa roxburghii and Arabidopsis thaliana.Only 2 orthologous CaM pairs were identified between Rosa roxburghii and Fragaria vesca and between Rosa roxburghii and Pyrus bretschneideri,respectively.2)Spatio-temporal expression characteristics of CaM/CML genes in Rosa roxburghii and their expression response to plant growth regulatorsBased on the transcriptome data,the expressions of 43 RrCaMs/CMLs genes in stems,leaves,flowers and different fruit development stages were analyzed.The results showed that 9 RrCaMs/RrCMLs genes expressed low or no in different tissues,and 5 genes expressed low in Rosa roxburghii fruit.Further verification by qRT-PCR showed that 7 genes including RrCML10,RrCML36 were most expressed in fruits at 90 days old,5 genes including RrCaM1 and RrCML24 were most expressed in leaves,6 genes including RrCaM2,RrCML1 and RrCML11 were most expressed in stems,and 8 genes including RrCML13 were most expressed in flowers.The cis-acting element prediction was consistent with the results of exogenous plant growth regulator treatment in vitro and field spraying on Rosa roxburghii Fruit,indicating that the promoter regions of RrCML24 and RrCaM2 genes both contain 3 Abscisic Acid（ABA）responsive elements,which can be induced by ABA to down-regulate and up-regulate expression,respectively.The promoter region of RrCaM2 contains 1 Methy Jasmonate（Me JA）responsive elements,which can be down-regulated by Me JA.The promoter region of RrCML11 contains 3 Gibberellin（GA₃）responsive elements,which are down-regulated by GA₃.3)Possible relationship between calcium and calmodulin and AsA synthesis in Rosa roxburghii fruitThe expression of CaM/CML gene in Rosa roxburghii and its effect on AsA synthesis were studied by in vitro feeding and field spraying of Rosa roxburghii.The results showed that Ca²⁺could up-regulated the expression of RrCaM/RrCML and AsA synthetic genes in Rosa roxburghii fruit,also significantly promote AsA accumulation.The treatment with Ca²⁺chelating agent（Ethylenebis（oxyethylenenitrilo）tetraacetic acid,EGTA）and calmodulin inhibitor（Chlorpromazine,CPZ）significantly inhibited the expression of RrCaMs/RrCMLs gene,and reduced the AsA content in Rosa roxburghii fruit.During the fruit development stage of Rosa roxburghii,after exogenous application of different calcium related preparations,the AsA content was clustered into one class with the same change trend as the expression levels of RrCML13,RrCML10,RrCML36,and AsA synthesis related genes RrGGP2/3,RrDHAR,and RrPMI1,among which the trend of RrCML13 was the most consistent.During the AsA rapid accumulation period of Rosa roxburghii fruit（mid-July）,after in vitro feeding with different calcium related preparations,AsA content and expression levels of RrCaM2,RrCML1,RrCML11,RrCML36,and AsA synthesis related genes RrGMP1,RrGME1/2 and RrGDH1/2 were grouped into one class with the same change trend.These results indicated that the calcium response mechanisms affecting AsA accumulation during fruit development and rapid AsA accumulation in Rosa roxburghii were not the same,but RrCML36 always played a certain role.