Establishment And Validation Of Agrobacterium-mediated Gene Transient Overexpression System In Dianthus Chinensis

For its efficient expressing or silencing the target gene in a short time,the gene transient expression system is often used to study genes function in plants.The flower color of Dianthus chinensis is rich.Establishment of its gene transient overexpression system will be beneficial to quickly screen genes related to the flower color,which facilitates targeted improvement for the flower color.In the previous study,we verified three chalcone synthase（CHS）genes related to flower colors of D.chinensis.Among which,Dch CHS1（KX893854）has a great influence on flower colors.In this study,a transient overexpression system was established by the Agrobacterium-mediated method,and the recombinant vector p BI121-GUS containing ORF of Dch CHS1 was used to verify the system.The main results were as follows:1.The vector p BI121-GUS was transformed into Agrobacterium tumefaciens GV3101 and EHA105,respectively.After detached floral buds of D.chinensis being infiltrated,Agrobacterium tumefaciens strains were screened by blooming percentages,and infiltration concentrations were determined by GUS staining.The blooming percentage in GV3101 was 56.67%,about 15.84%higher than that in EHA105.The color of GUS staining was obvious when the OD₆₀₀of the infiltration solution was 1.4±0.2.Consequently,the suitable transient overexpression system is established in D.chinensis.2.The recombinant vector of p BI121-Dch CHS1-GUS in GV3101 was used to infect detached floral buds with two flower colors,white with red（RW）and double red（RR）.Compared with those in the p BI121-GUS,the flower color decreased and the blue color appeared after GUS stained in the p BI121-Dch CHS1-GUS.DNAs from petals of RW and RR after being infected and uninfected by GV3101 were used to amplify the Dch CHS1fragment by Dch CHS1-F/R.The target fragment was only appeared in the DNAs infected by p BI121-Dch CHS1-GUS.Compared with the counterparts of petals by p BI121-GUS,Dch CHS1 expressions increased over one-fold in the outside part and in the inner part of the petal by p BI121-Dch CHS1-GUS.At the same time,the CHS activities in the outside part and in the inner part of the petal increased 3.87%and 26.04%in RW,and 42.66%and8.64%in RR.The flavonoid contents significantly increased and the anthocyanin contents significantly decreased in both parts.The results suggested that Dch CHS1 was overexpressed in the petals of D.chinensis.The protein encoded by Dch CHS1 possessed its enzyme activity.In summary,the transient overexpression system mediated by Agrobacterium is established and can be used to characterize genes function in D.chinensis.