Cloning And Functional Characterization Of IbSWEET10 And IbRAP2-12 Genes From Sweetpotato(Ipomoea Batatas(L.)Lam.)

In this study,a sugar transporter gene IbSWEET10 and an ethylene-responsive transcription factor(AP2/ERF family)gene IbRAP2-12 were cloned from sweetpotato(Ipomoea batatas(L.)Lam.)line ND98 using the RACE(rapid amplification of cDNA ends)method.These two genes were introduced into sweetpotato(cv.Lizixiang)and the model plant Arabidopsis,respectively.Transgenic plants with different stress tolerances were obtained and the underlying molecular mechanisms were deciphered.The major results of this study were as below:1.The cloned 1250-bp full-length cDNA of IbSWEET10 contains a 921-bp ORF that encodes a 306-amino acid polypeptide with a molecular weight of 34.1 kDa and a pI of 9.34.The genomic sequence of IbSWEET10 is 3269 bp long and contains 6 exons and 5 introns.IbSWEET10 showed highest expression level in leaves of sweetpotato and its expression was significantly induced in sweetpotato infected with Fusarium oxysporum Schlecht.f.sp.batatas.Promoter?-Glucuronidase analysis in Arabidopsis showed that the promoter of IbSWEET10 had strongest activity in Arabidopsis leaves.Transient expression in tobacco epidermal cells revealed plasma membrane localization of IbSWEET10,and heterologous expression assays in yeast indicated that IbSWEET10encodes a sucrose transporter.Further characterization revealed that overexpression of IbSWEET10 in sweetpotato(cv.Lizixiang)confered stronger resistance to F.oxysporum;conversely,RNA interference(RNAi)lines showed the opposite results.Additional analyses showed that the overexpression of IbSWEET10 promoted the export of sugars from the leaves of transgenic plants and the sugar content of transgenic sweetpotato was significantly reduced,which diminished the energy supply for the reproduction of pathogens and the F.oxysporum resistance of IbSWEET10-overexpressing sweetpotato was thus enhanced.Meanwhile,overexpression of IbSWEET10 significantly increased the carotenoid content in the storage roots of transgenic sweetpotato.The speed of glycolysis was significantly enhanced and the content of pyruvic acid(PA),the precursor of carotenoid synthesis,was also increased compared with control plants,which led to the increased content of carotenoid in IbSWEET10-overexpressing plants.This is the first report for the cloning of the sugar transporter gene IbSWEET10 from sweetpotato.This study revealed that IbSWEET10 could contribute to the resistance of sweet potato to F.oxysporum and shed new light in the function of IbSWEET10 in carotenoid synthesis in plants.2.The cloned 1767-bp full-length cDNA of IbRAP2-12 contains a 1101-bp ORF that encodes a 366-amino acid polypeptide with a molecular weight of 40.5 kDa and a pI of 5.12.The genomic sequence of IbRAP2-12 is 2443 bp long and contains 2 exons and 1 intron.mRNA expression analysis in sweetpotato showed that IbRAP2-12 is highly expressed in leaves and promoter ?-Glucuronidase analysis in Arabidopsis showed the activity of IbRAP2-12 promoter.qRT-PCR analysis revealed that the expression of IbRAP2-12 was significantly induced by salt,drought,ABA,ethephon and MeJA treatments.The subcellular location result indicated that IbHSP90-6 was located in nuclei.Yeast one-hybrid assay showed that IbRAP2-12 exhibited transcriptional activation and the transcriptional activation domain was located in the C-terminal.TheIbRAP2-12-overexpressing Arabidopsis plants exhibited significantly higher salt and drought tolerance compared with the wild-type(WT)plants.The significant increase of ABA,JA,BRs and proline content and SOD activities and significant reduction of MDA and H2O2 content were found in the transgenic Arabidopsis plants.qRT-PCR analyses showed that overexpression of IbRAP2-12 up-regulated the expression of genes in ABA signalling pathway,JA signalling pathway,BRs signalling pathway,proline synthesis pathway and ROS-scavenging system under salt and drought stresses,which led to the enhanced resistance of transgenic Arabidopsis to salt and drought stresses.In addition,yeast two-hybrid assay(Y2H)and bimolecular fluorescence complementation assay(BiFC)showed that IbRAP2-12 could interact with IbMIEL1(E3 ubiquitin-protein ligase).This is the first report that the IbRAP2-12 gene was cloned from sweetpotato and the overexpression of IbRAP2-12 could increase the resistance of transgenic plants to salt and drought stresses and these results provide a new idea for the molecular breeding of sweet potato with salt tolerance and drought resistance.