Optimization Of Protoplast Isolation And Subcellular Localization Of PhAAE13a,PhAAE13b And PhERF6 In Petunias

The volatile substances or anthocyanins are the products of secondary metabolites of plants.It is particularly important to study its synthesis pathways.Previous stydy showed that silencing of PhAAE13 a gene resulted in a significant reduction in anthocyanin accumulation,but the silence of PhAAE13b(another gene in the AAE superfamily)did not alter the content of anthocyanin in petunia.The gene is localized in the cytoplasm and mitochondria in Arabidopsis thaliana,but the localization in petunia is not clear.The ethylene-responsive element-binding factor(ERFs)gene is the most downstream of the ethylene signal transduction pathway.The preliminary results of previous study show that the PhERF6 gene of petunia plays a role in regulating flower scent biosynthesis.Howevere,the localization of PhERF6 and the regulate relationship between PhERF6 and ethylene has not been studied.The aim of this study was to investigate the subcellular localization of PhAAE13 a,PhAAE13b and PhERF6.To explore the relationship between the expression of PhERF6 during flower development and the regulation between ethylene and PhERF6 expression.In addition,the extraction method of protoplasts is optimized,which is conducive to subcellular localization.The results are as follows:The protoplast extraction solution and the extraction method were optimized,including the selection of ’rose red’,the plant size was about 30 days,the leaves were treated by vertical shredding,the solution osmotic pressure was 500 Os,the container of enzymatic hydrolysis was 25 ml small beaker and the time o f enzymatic hydrolysis for 1 h.These optimizations can improve the number and quality of protoplasts and facilitate subcellular localization.The PhAAE13 a and PhAAE13 b gene fragments were ligated into pSAT6-eGFP vector,and the recombinant plasmid was introduced into protoplasts of Petunia for observation.The results showed that both PhAAE13 a and PhAAE13 b located in the nucleus and cytoplasm.PhERF6 was located in the nucleus.PhERF6 also showed a certain regularity during the development of flower,whic h was the highest in the early stage of flower buds,and gradually decreased with the elongation of buds.After the full expansion of the flowers,the expression gradually increased,indicating that PhERF6 may play an important role in the early stages of flower development and after opening flowers.After treatment with ethylene,the expression of PhERF6 was significantly increased.