Prediction Of ARF Gene Family Promoter And Analysis Of Transcriptional Activity Of ARF4 Promoter In Strawberry

ARF4 is the transcription factor of plant response to auxin and plays an important role in plant growth and development.There are few researches on the promoter of ARF gene,and the research on the promoter of ARF gene is still blank in strawberry.The cis-acting elements of the promoter of strawberry ARF gene family were predicted,the function of the promoter was speculated and the promoter sequence of strawberry ARF4 gene was cloned.The promoter of ARF4 gene and GUS reporter gene were constructed as fusion genes.The transcriptional activity of the promoter of ARF4 gene was analyzed by different treatments,to lay a foundation for fully revealing the function of ARF4 gene.The main achievements as followed:1.Nine promoter sequences of ARF gene were obtained from strawberry genome website and NCBI website and their specific position in strawberry genome was determined.Bioinformatics analysis shows that they not only contain the core elements,but also contain a large number of response to light,stress,hormone,regulation of physiological mechanisms element and so on.There are differences in the number and types of action elements among different members of the ARF gene family,which are related to hormone response and regulation of physiological mechanisms.In addition to their own unique elements,there are also some common elements among the different members.2.Cloning promoter of ARF4 Gene from'Yanli'Strawberry.By using bioinformatics software,we found that the promoter not only contains TATA-box?CAAT-box,but also has a variety of cis-acting element,such as the photoresponse element SP1,Box 4 and so on.The anaerobic induction element ARE,the fungal elicitor response element Box-W1,the protein metabolism regulating element O₂-site,the gibberellin response element P-box and so on.The promoter sequence of ARF4 gene was cloned in strawberry cultivar'Allstar','Sweet Charlie','Albion'and'Benihoppe',and they have no difference in action elements were found between sequences.3.Based on pRI 201-AN-GUS,the promoter of ARF4 gene was ligated into the vector,and then the fusion vector of strawberry ARF4 gene promoter was obtained,named proARF4::GUS.Five transgenic plants were obtained by genetic transformation of forest strawberry'Ruegen'by Agrobacterium mediated leaf disc transformation.Five transgenic plants were obtained by genetic transformation of Arabidopsis thaliana by dipping flower method.4.Cloning of segment deletion of ARF4 gene promoter and construction of proARF4A::GUS\proARF4B::GUS fusion vectors,By Agrobacterium mediated fruit injection and and GUS tissue staining,it was found that with the length of promoter sequence becoming shorter,expression of GUS gene driven by proARF4B is the weakest.5.Transgenic strawberry plants were stained by GUS tissue staining.The results showed that the expression level of GUS gene was higher in petiole,tender leaf and new stem,and the highest expression level was found in young leaf.Transgenic strawberry plants were treated under low temperature,drought,NaCl,different hormone types and different concentrations.qRT-PCR was used to detected the expression of GUS gene.The results showed that the expression of GUS gene was not affected in the low temperature,drought,NaCl treatments.Compared with other hormones such as IBA,IAA can significantly increase the expression of GUS gene.Further study showed that the expression of GUS gene was the highest at 10?mol L^-1 IAA for 12 h.