Screening And Functional Identification Of ABCC Transporter And Sucrose Transporter Gene During Strawberry Fruit Maturation

Anthocyanin belongs to the secondary metabolites of plants and are the main pigments that make up the fruit color of strawberry,which are critical to the appearance of fruit quality.anthocyanin not only have important biological functions,such as protecting the plant itself against ultraviolet light、low temperature and pests and diseases,but also have important medical and health care functions.Anthocyanins are generally synthesized in the cytoplasm and stored in vacuoles.At the same time,sucrose is a kind of primary metabolite widely found in plants,which plays an important role in plant growth and development.Therefore,this paper carries out anthocyanins and sucrose in the cell transport related research has important theoretical and practical value for explaining the molecular mechanism of anthocyanin and sucrose accumulation in fruit and improving the fruit quality by using relevant cultivation measures.（NO₃^--N、NH₄⁺-N、NO₃^--N+NH₄⁺-N、no nitrogen）were treated by sand culture in the early stage of the project.It was found that different nitrogen forms had different effects on promoting fruit coloring.At the same time,by using metabolomics analysis of strawberry fruits treated with different nitrogen forms,biomarkers related to treatment effects were identified and diffracted into KEGG database related metabolic pathways.The results reveal that there are seven biomarkers annotated into the ABC transporters pathway.The results show that ABC transporters were involved in different nitrogen forms to regulate strawberry fruit development and maturation.For this purpose,an ABCC transporter gene Fa ABCC1 and a sucrose transporter gene Fa SUC3 were cloned and analyzed by bioinformatics analysis according to the results of the integration analysis of the previous transcriptome and metabolites.Real-time quantitative PCR was used to analyze Fa ABCC1 and Fa SUC3 in seven different developmental stages of strawberry and four different developmental organ expression patterns.The changes of anthocyanin content in anthocyanin during the seven different developmental stages of strawberry were determined by spectrophotometer.At the same time,the expression pattern of anthocyanin-related functional gene in different developmental stages of strawberry was compared by fluorescence quantitative PCR.To construct interference expression vectors p Hellsgate2-Fa ABCC1 and p Hellsgate2-Fa SUC3and the over-expression vector p CXSN-Flag-Fa ABCC1 and p CXSN-Flag-Fa SUC3 for gene transformation and functional validation study.The experimental study achieved the following main results:1.The results showed that the ORF of Fa ABCC1 was composed of 4518 bases and encoded 1505 amino acids.The phylogenetic analysis of Fa ABCC1 was carried out.The results showed that the Arabidopsis thaliana At MRP4 had the highest homology,and the subcellular localization of onion epidermal cells showed that Fa ABCC1-GFP fusion protein was localized in the cell membrane,and the four different developmental organs of root、leaf、flower and fruit of turning color.Expression pattern analysis showed that the relative expression level was the highest in the roots,followed by flowers and leaves,but the expression level was the lowest in fruit.However,the results of fruit expression analysis of seven different developmental stages of strawberry showed that the expression of Fa ABCC1 was significantly up-regulated,indicating that this gene may be related to anthocyanin transport.The plant expression vector p Hellsgate2-Fa ABCC1 was constructed and successfully transformed into diploid strawberry seedlings.The resistant shoots were obtained and the super-expression vector p Cxsn-flag-Fa ABCC1 was constructed and transformed into Arabidopsis thaliana.The transgenic plants 5 trees.2.The sucrose transporter gene Fa SUC3 and the ORF of Fa SUC3 were cloned from1815 bases,encoding 604 amino acids by collecting sweet strawberry strawberry fruit for30 days（DPA30）.The phylogenetic tree showed that Fa SUC3-GFP fusion protein was located in the cell membrane,and it was found that the Fa SUC3-GFP fusion protein was located in the cell membrane.The results of fruit expression analysis of seven different developmental stages of strawberry showed that the expression of Fa SUC3 was significantly up-regulated,indicating that this gene may be related to fruit maturation.The plant expression vector p Hellsgate2-Fa ABCC1 was constructed and successfully transformed into diploid strawberry seedlings.The resistant shoots were obtained and the super-expression vector p Cxsn-flag-Fa ABCC1 was constructed and successfully transformed into Arabidopsis thaliana.The transgenic plants 2 trees.3.During the seven different developmental stages of the strawberry,anthocyanins accumulated very little during the small green,green,green and ginkgo,and the anthocyanin content began to increase at the turn stage,and the anthocyanin content accumulated at the highest level Level,after crossing the anthocyanin content began to decrease.4.Real-time Fluorescence Quantitative PCR,the results showed that CHS1/3 gene played a key role in the biosynthetic pathway of anthocyanins.Analysis of Genes Related to Anthocyanin Synthesis,the expression patterns of F3H1 and ANS1 genes in red fruit octopus strawberry sweet Charlie showed a significant positive correlation with anthocyanin accumulation,while in the ginkgo diploid strawberry Hawaii4 turn color fruit expression is very low,indicating that ANS1 and F3H1 play a key role in the accumulation of anthocyanins.UFGT1-8 was identified from the strawberry genome,the quantitative analysis of the UFGT1 gene in the octahedral strawberry sweet seizure showed that the expression of UFGT1 gene was higher in the transitional stage,and the other genes were only expressed in the small green fruit of strawberry.The result of diploid strawberry Hawaii4 is opposite,which is almost not expressed in the color change stage,indicating that UFGT1 is a promoter of UFGT gene to promote anthocyanin glycosylation.