| Strawberry(Fragaria×ananassa Duch)is a herbaceous perennials belonging to the genus Fragaria of the Rosaceae family and is popular among consumers due to its attractive fruit flavor and abundant nutritional content.Meanwhile,strawberry is also a worldwide cultivated fruit because of its high economic values.For the strawberry production,the flowering time has a great impact on the ripening and marketing time of the fruit,which further influences the yield and economic values.Therefore,it has become the main goal for strawberry growers and breeding researchers to regulate the strawberry flowering time with appropriate cultivation management methods and to select new varieties by elucidating the functions of the genes involved in the flowering time regulation.The strawberry flowering time is affected by a range of external environmental factors,in which including light and temperature.In China,with the expansion of the strawberry cultivation areas,the protected agriculture has been used as a mainstream method in strawberry cultivation.In the strawberry cultivation,light-emitting diodes(LEDs)are often used as a primary or secondary light sources to regulate growth and fruit quality.Photoperiod and light quality are two of the most important light environment settings which affect the flowering time of strawberry.Although the regulation model of photoperiodic control of flowering time in wild strawberries(Fragaria vesca)has been reported,the molecular mechanism of light quality affecting flowering time in strawberries has yet to be investigated in depth.In this study,the octoploid strawberry ‘Benihoppe’(Fragaria×ananassa)as was used as research materials.Using RNA-seq transcriptome sequencing,bioinformatics analysis,phenotypical analysis of transgenic Arabidopsis(Arabidopsis thaliana)lines and other gene function studies as methods,we explored the effects of blue LED irradiation treatment on the flowering time of cultivated strawberries and uncovered the functions of the related key genes.The following findings were obtained :1.The unbloomed strawberry seedlings of ‘Benihoppe’(Fragaria×ananassa)were irradiated with two different light quality treatments(white light for the control group and blue light for the treatment group).The flowering time was observed and recorded.The results showed that strawberry flowering time was significantly advanced by the blue light treatment.A further transcriptome analysis of the strawberry seedling leaves from two treatment groups was performed using RNA-seq technology.As a result,A total of 6875 differentially expressed genes(DEGs)were identified which responded to blue light treatment,including 3138(45.64%)down-regulated genes and 3737(54.36%)up-regulated genes under blue light treatment.According to the gene annotation results,the DEGs could be further enriched into 98 GO items and 71 KEGG pathways.The DEGs involved in light signaling pathways(e.g.Phy B,PIFs and HY5)and plant circadian rhythm pathways(FKF1,CCA1,LHY and CO)were identified.Furthermore,this study identified the BBX transcription factors as a potential key transcription factor family that may responded to blue light and participated in the regulation of strawberry flowering time.2.A total of 51 BBX family members(Fa BBXs)were identified in cultivated strawberry‘Camarosa’(Fragaria×ananassa)and 16 BBX family members(Fv BBXs)were identified in wild strawberry ‘Hawai 4’(Fragaria vesca)by using bioinformatics methods.Based on phylogenetic analysis result,the Fa BBXs and Fv BBXs can be classified into five subfamilies(Group I-Group V).In both strawberry species,whole genome duplication events and segmental duplication events are the main driving forces for the expansion of the BBX family,and gene loss and specific gene duplication may affect agronomic traits such as fruit quality in cultivated strawberry.A large number of light-responsive elements and hormone-responsive elements were identified from the promoters of BBX genes.Expression analysis of Fa BBX genes based on the transcriptome database illustrated that Fa BBX genes have significant differential expression patterns in different tissues and different fruit development stages of cultivated strawberry.Morevoer,16 Fa BBX genes expressed significantly differentially in strawberry leaves under blue light treatment,which indicated that these genes responded to blue light treatment.3.For further functional studies,the protein coding sequence(CDS)of Fa BBX28c1,a BBX family member significantly repressed by blue light treatment,was cloned from‘Benihoppe’strawberry leaves.The results showed that heterologous overexpression of this gene in the Arabidopsis significantly delayed the flowering time as well as significantly more rosette leaves in the transgenic Arabidopsis lines than wild-type Arabidopsis.Using q RT-PCR to detect the expression levels of key genes that participate the flowering time regulation by light signaling.At CO,At FT and At SOC genes were found to be repressed in overexpression transgenic Arabidopsis lines.These results demonstrated that Fa BBX28c1 has a significant ability to modulate the flowering time and the balance between reproductive and vegetative growth in plant.Promoter sequence of Fa BBX28c1 gene promoter(pro Fa BBX28c1)was cloned from cultivated strawberry ‘Benihoppe’.Arabidopsis thaliana transgenic lines harboring a pro Fa BBX28c1::gus report system were constructed to further analysis the activity of pro Fa BBX28c1.The GUS stain was clearly detected in vascular of mature leaves,supporting the role of the Fa BBX28c1 gene in the regulation of flowering in mature leaves.In addition,an inducible GUS staining signal appeared in Arabidopsis roots.Meanwhile,both the q RT-PCR result and transcriptome data analysis showed that Fa BBX28c1 gene expression level was induced by external drought stress.These results suggests that Fa BBX28c1 participates the response to external drought environment in plant.But the details of Fa BBX28c1 function in such response needs to be further investigated. |
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