Cloning And Functional Analysis Of Sweetpotato Sucrose Transporter Genes

Sweetpotato(Ipomoea batatas(L.)Lam)is a dicotyledonous crop,whose yield and quality are closely related to the transportation of photosynthetic products.Sucrose is the main form of photosynthate transport,and its transmembrane transport is mediated by sugar transporters.However sucrose transporters(SUTs)play an important role in the distribution and transport of sucrose from source to sink.This study is aimed at the status of the sweetpotato sucrose transporter gene function that has not been systematically reported so far.Firstly,the IbSUT3 and IbSUT4 were cloned from sweetpotato by RACE technology,and their bioinformatics analysis was carried out.Then,in order to preliminarily explore the function of the genes.The four sucrose transporter genes(IbSUT1,IbSUT2,IbSUT3 and IbSUT4)in sweet potato were systematically studied including function of sucrose transport,subcellular localization and gene expression patterns in different tissues and under different stress conditions.Finally,to further explore the function of IbSUT4 gene in sweetpotato.Over-expression of IbSUT4 Arabidopsis and wild-type were carried out in phenotypic observation and abiotic stress analysis.(1)We got two sucrose transporter genes from sweetpoatao including IbSUT3(MN233361)and IbSUT4(MN233360).The open reading frame length,amino acid quantity,protein quality,isoelectric point,transmembrane domain,sequence alignment and phylogenetic tree analysis of IbSUT3 and IbSUT4 genes were carried out by means of bioinformatics.The open reading frame of IbSUT3 is 1518 bp,encoding protein has 505 amino acids.The open reading frame of IbSUT4 is 1512 bp,encoding protein has 503 amino acids.The results of phylogenetic tree showed that the protein encoded by IbSUT3 belonged to SUT1,and the protein encoded by IbSUT4 belonged to SUT4.(2)Identifying whether the sucrose transporter has the function of transporting sucrose provides a basis for subsequent research.Heterogeneous expression of the sweetpotato sucrose transporter IbSUT1,IbSUT2,IbSUT3 and IbSUT4 genes in the yeast-deficient strain SUSY7/ura3.The normal growth on the medium proved that the four sucrose transporters mentioned above have the function of transporting sucrose.The clear protein localization provides the basis for analyzing its function.The subcellular localization analysis of sweet potato sucrose transporter in tobacco protoplasts.These results showed that IbSUT1,IbSUT2 and IbSUT3 were located in plasma membrane,and IbSUT4 was located in plasma membrane and vacuole membrane.Different sucrose transporter genes have different tissue expression patterns.The PCR results suggested that IbSUT1,IbSUT2 and IbSUT3 were highly expressed in source organs and IbSUT4 was highly expressed in sink organs.Low temperature,high salt,drought and exogenous ABA all induced the expression of four sucrose transporter genes,but the response patterns of each gene were different.(3)To explore the IbSUT4 function,we got the transgenic lines(SUT4-1?SUT4-7?SUT4-10).Phenotypic observations indicate that over-expression of IbSUT4 promotes early flowering of Arabidopsis thaliana by increasing AtAF gene expression.In exogenous ABA stress,the expression of AtABI3 gene was significantly increased in transgenic Arabidopsis seeds,results to the transgenic seeds were hypersensitive to ABA.Over-expression of IbSUT4 increases the accumulation of sucrose in transgenic roots,thereby improving the resistance of transgenic plants,and by increasing the expression of the ABA pathways AtABF2,AtABF4,AtSnRK2.2,AtSnRK2.3,and AtPYL8 / RCAR3 genes,reducing AtABI1 express in response to adversity.The results prove that IbSUT4 plays an important role in maintaining plant growth and development and resisting stress.