Cloning And Functional Analysis Of FaARF4 Gene In Strawberry

ARF4(auxin response factor 4)is the transcription factor of auxin regulation,which plays an important role in auxin signaling pathway.There is only a clear study of the role of ARF4 in pattern plants.But the role of ARF4 in the phosphate signal transduction pathways in the strawberry is still obscure.In this study,the sequence of ARF4 gene coding sequence was cloned from 'Yanli' strawberry,and it was named FaARF4.We constructed an overexpression vector of FaARF4 gene coding sequence mutant(Referred to as FamARF4).And then by useing the method of Agrobacterium-mediated transformation,we obtained ARF4 overexpression plants of Fragaria vesca accession 'Ruegen' and Arabidopsis thaliana.This study lays the foundation for revealing the function and mechanism of ARF4.The main results are as follows:1.The full length of FaARF4 gene's DNA sequence is 5628 bp,and the full length of FaARF4 gene's mRNA sequence is 3236 bp,and the full length of FaARF4 gene coding sequence is 2403 bp.ARF4 gene coding sequence is isolated from strawberry cultivars'Allstar','Sweet charlie','Tochiotome','Toyonoka' and 'Benihoppe' using primers designed according to the full-length gene coding sequence from 'Yanli'.These fragments shared 99.47%amino acid identity.2.With qRT-PCR based on Taqman probe,expressions of FaARF4 were compared among different organs of strawberry,and the expression of FaARF4 in tender leaf,sepal and pistil was higher.The expression of FaARF4 among different periods of fruit growth was detected,the results indicated that with the growth of fruit age a decrease in transcript levels of FaARF4 was found.3.Point mutation of FaARF4 gene coding sequence mutant that tasiRNA3 target sites eliminated without any amino acid change is obtained by Rapid PCR site-directed mutagenesis methodologies.4.Using plant expression vector pRI101 AN as a basis vector and then using Nde I/Sal I restriction enzyme,FamARF4 gene was integrated into the basis vetor,and the overexpression vector named as pRI101-FamARF4 were constructed.5.Using the method of Agrobacterium-mediated transformation with the in vitro leaves as explants,the 'Yanli' strawberry FamARF4 genes was introduced into F.Vesca 'Ruegen'.And transgenic plants were obtained,4 plants were transformed by FamARF4.The 'Yanli'strawberry FamARF4 genes were introduced into A.thaliana by Agrobacterium-mediated transformation.And transgenic plants were obtained,4 plants were transformed by FamARF4.6.It was observed that Arabidopsis thaliana and strawberry transgenic plants had the phenomenon of early flowering.In addition,in the Arabidopsis thaliana transgenic plants,the abortion of the early 1-4 flowers was due to the development of the stamens.In the strawberry transgenic plants,the early fruit produce small amount of seeds.The reasons for this phenomenon need to be further studied.