Function Analysis Of FvCO4 In The Early Development Of Strawberry Fruit

CONSTANS-like genes encode the zinc finger proteins which belong to the transcription factor.Most of the current research focused on their regulation rules in the phtoperiodic flowering of plants.Our previously study identified that FvCO4,a CO-like gene of strawberry,is specifically expressed in the pith and cortex of strawberry fruit.The treatment of emasculation,which affecting the strawberry fruit development,significantly down-regulated the expression level of FvCO4.Such findings suggested that FvCO4 is involved in the early fruit development of strawberry.In this paper,diploid strawberry(Fragaria vesca)'YW5AF7' was used as the experimental material to illuminate the molecular characteristics of FvCO4.The expressional response of FvCO4 to the treatment of hormones and related inhibitors was also detected.Finally,we knocked out this gene in the forest strawberry genome by CRISPR to confirm its function.The main results are as follows:1.Compared with the fruit development of control,the fruit development of strawberry treated with both auxin inhibitor and gibberellin inhibitor was significantly delayed.2.Subcellular localization experiment indicated that the FvCO4 protein is localized in the nucleus;self-activation identification test found that FvCO4 protein does not have the ability to activate by itself;yeast one-hybrid assays demonstrated that the FvCO4 protein can bind to the promoter sequence of FvFT2.3.With the Agrobacterium-mediated leaf-disc transformation method,FvCO4 in the forest strawberry 'YW5AF7' genome was edited by CRISPR-Cas9 system.PCR and sequencing confirmed that totally 12 lines with the target gene edition were obtained.4.Fruit development of some transformants whose FvCO4 have been knocked out was significantly blocked,incicating the function of this gene invovled in the development of strawberry fruit.Pollen scanning by electron microscopy showed that the pollen morphology of the line in which FvCO4 was knocked out did not change significantly.Tissue section showed that the cell division in the pith and cortex of the fruit belongs to the transgenic lines is inhibited;expression detection by q RT-PCR shouwed that expression levels of FT-like homologues and the genes related to cell division are obviously down-regulated at 10 d after blooming.5.Promoter activity verification vector of Pro FvCO4::GUS was constructed and two transgenic lines were obtained.In addition,overexpression vectors for FvCO4 and FvFT2 were also constructed individually for the subsequent studies.