Homologous Cloning Of CmACO And CmDA1 Genes In Chrysanthemum

Medicinal Chrysanthemum is the dried flower of Chrysanthemum morifolium Ramat..It is one of the traditional Chinese medicine and healthy tea in China,which contains volatile oil,flavonoids,chlorogenic acid,trace elements et al active components,with drinking,medicinal and ornamenta valuel.1-aminocyclopropane-1-carboxylic acid oxidase?ACO?plays a role in the last step of ethylene biosynthesis,controlling the rate of ethylene production.DA1 gene encodes a ubiquitin receptor,which is an important cell proliferation regulator in plants.In Arabidopsis,DA1 gene negatively regulates the size of seeds and organs.In this study,the system of regeneration and genetic transformation of medicinal Chrysanthemum were optimized.The genes of CmACO and CmDA1 were cloned and bioinformatics analysis was carried out on them,which provided convenience for further research on the function of CmACO and CmDA1 genes and laid a theoretical foundation for the molecular breeding of medicinal Chrysanthemum.These are the main results:1.The regeneration system of medicinal Chrysanthemum was optimized,and the optimal conditions for inducing the adventitious buds with the leaves of medicinal Chrysanthemum are as follows:MS+6-BA 1 mg/L+NAA 0.5 mg/L;The optimal conditions for inducing the rooting of Chrysanthemum morifolium seedlings are:1/2MS+NAA 0.1 mg/L.2.The genetic transformation conditions of medicinal Chrysanthemum were optimized.Combining with the optimal regeneration conditions,the optimal genetic transformation conditions are as follows:the leaf discs are precultured for 3 days,then are transformed with the agrobacterium solution(OD₆₀₀=0.5-0.6)for 12 min,cultured for another 3 days and decarboxylated for 7 days,and then transferred to the screening medium?MS+6-BA 1 mg/L+NAA 0.5 mg/L+Kan 8 mg/L+carb 300 mg/L?.After two weeks of subculture,the adventitious buds are screened.When the resistant adventitious buds are 2 to 3 centimeters'long,they could be transferred into the rooting screening medium?1/2MS+NAA 0.1 mg/L+Kan 8 mg/L+carb 300 mg/L?for rooting screening.3.A 1196 bp ACO gene sequence and a 1950 bp DA1 gene sequence were cloned from chrysanthemum by homologous cloning and RACE technology,named CmACO and CmDA1,respectively.The full-length open reading frame?ORF?of CmACO and CmDA1were predicted to be 942 bp and 1542 bp,encoding 313 amino acids and 513 amino acids,respectively,using online website ORF finder.Smart BLAST analysis of the ORF sequence showed that the amino acids sequence of CmACO and CmDA1 gene cloned from chrysanthemum are highly homologous with that of ACO and DA1 gene from other species,indicating that both CmACO and CmDA1 genes contain important and typical domains related to their activities.The genome sequences of CmACO and CmDA1 were analyzed and it was found that CmACO contained 4 exons and 3 introns,while CmDA1 contained 12exons and 11 introns.4.The results of phylogenetic tree analysis showed that the amino acid sequence of CmACO gene cloned from medicinal Chrysanthemum had the highest similarity with that of ACO gene in Artemisia annua and the closest evolutionary relationship;the amino acid sequence of CmDA1 gene had the highest similarity with that of DA1 gene in Tanacetum cinerariifolium and Artemisia annua and the closest evolutionary relationship.Therefore,it can be preliminarily confirmed that the CmACO and CmDA1 genes cloned in this study are the genes encoding ACO protein and DA1 protein respectively.