Isolation And Cloning Of Chalone Synthase Gene In Strawberry And Construction Of Virus-induced Gene Silence Vector

Chalcone synthase (CHS) is important in flavonoids compounds synthesis inplants, and play a crucial role in plant development and diverse resistant reactions. Inthis work, two woodland strawberry CHS homologs FvCHS1and FvCHS2were firstidentified via BLAST analysis againt NCBI Fragaria vesca WGS database usingarabidopsis AtCHS (AT5G13930) and FaCHS (AY997297) EST sequences. Further,these two genes were BLAST analyzied againt all three available databases forstrawberry ESTs (NCBI Fragaria EST,GDR octoploid strawberry ESTs,GDR diploidstrawberry ESTs). It was found that FvCHS1is expressed and has several ESTsequences, while no EST sequence is available for FvCHS2.To isolate CHS genes from octoploid strawberry for VIGS study, specificprimersets were designed using Primer4.0software as following:FaCHS1-U:5'-TGCAAGACAAGACATGGTGGT-3';FaCHS1-L:5'-GGAACATCTTTGAGGAGGTGAA-3'；FaCHS2-U:5'-TACGGATTGCCAAGGACATA-3';FaCHS2-L:5'-CAACAGTCTCCACAGTAAGTC-3'.RT-PCR was then performed to isolate these two genes. The540bp long fragmentspecific to FaCHS1gene was cloned from ripe fruit cDNAs, while the639bp longfragment specific to FaCHS2gene was cloned from the mixed cDNAs from flowersand floral buds. Sequence homology and physical and chemical properties for thededuced strawberry CHS proteins were analyzied using corresponding bioinformaticsmethods.Virus-induced gene silencing (VIGS) is an effective method for gene functionalstudy. The target gene fragment was first introduced into the virus carrier. When thevirus carrier was used to infect plant, target gene will be silenced similar like that theplant's own immune system being activated. In this work, we introduced FaCHS1andFaCHS2fragments into pTRV2virus carrier, respectively.The colony PCR andrestriction enzymes digestion uniformly confirmed that FaCHS1and FaCHS2genefragments were successfully inserted into the virus carrier with reverse orientation. Later, these virus carriers were successfully transformed into agrobacterium strainGV3101. In sum, this work laid a good foundation for functional study of strawberryCHS genes via agrobacterium-mediated infection experiments in the near future.