Preliminary Study On The Regulation Mechanism Of FvCO4 In The Early Development Of Strawberry Fruit

CO（CONSTANS）encodes the B-box protein belonging to the zinc finger protein family,and regulates the key gene FT to perform functions in the plant photoperiodic flowering.Previous studies have shown that in strawberries,FvCO4,a strawberry CO homologue,is tissue-specifically expressed in the strawberry fruit.With the knockout of FvCO4,development of strawberry receptacle is significantly inhibited,indicating that FvCO4 is involved in the regulation of the strawberry receptacle into fruit.However,the mechanism of it is still not clear.In this study,seedlings of the FvCO4 knock-out T1 generation and the offspring coming from tissue culture propagation were checked for the edition of FvCO4 sequence.Then we performed the hormone treatment and hormone content detection,the transient over-expression transformation of FvCO4 and FvFT-2 in strawberry fruits,q RT-PCR,etc.to initially reveal the function mechanism of FvCO4 in the early development of strawberry fruit.The main findings are as follows:1.Sg RNA detection was performed on the FvCO4 CRISPR T1 seedling lines and the offspring from tissue culture.PCR-sequencing results showed that both of them still showed the gene edition near the FvCO4 site.The gene edition by CRISPR-Cas9 could be inherited sexually or asexually.2.When treated with NPA,the auxin inhibitor,and S-3307,the gibberellin inhibitor,fruit development of the Yellow Wonder was significantly hindered.The q RT-PCR results showed that not only the expression of FvCO4,but also the expression of FvFTs and cell division related genes,such as FvCYC,Fv EDE1 and Fv KNOLLE,were all inhibited significantly,indicating that FvCO4 is regulated by auxin and gibberellin.3.After the exogenous NAA and GA₃treatments to the receptacles of FvCO4 CRISPR line,the aborted receptacles can be further enlarged and the development of the fruit can be partially restored,indicating that lack or reduction of the endogenous hormones may cause the hindered fruit development in the FvCO4 knock-out lines.4.Hormone detection results showed that contents of GA₁and GA₃in the receptacles of the FvCO4 knock-out line decreased significantly,similar results were also found in the IBA content detection.However,content of IAA was found to be more higher than the control.Hormone content variation may be a factor that affecting the early development of strawberry fruit in FvCO4 knock-out lines.5.Artificial pollination on the receptacles of the FvCO4 knock-out line with the pollens of the wild-type Yellow Wonder plants can restore the normal fruit development.6.After over-expressing FvCO4 and FvFT-2 in receptacles of the FvCO4 knock-out line by transient transformation,the receptacles can be further enlarged and developed into fruit.Such result further confirmed that FvCO4 plays a key role in the early development of strawberry fruit,which was potentially mediated by FvFT-2.