Comparative Transcriptome Analysis Of Purple-fleshed Sweetpotato And Cloning And Functional Characterization Of IbMYB4,IbMYB48 And IbMYC2 Genes From Sweetpotato(Ipomoea Batatas(L.)Lam.)

Sweetpotato,Ipomoea batatas(L.)Lam.,is a globally important food crop.The discovery of genes related to anthocyanin synthesis and salt tolerance and drought resistance is of great significance for improving sweetpotato with genetic engineering.In this study,de novo transcriptome sequencing of the purple-fleshed sweetpotato cv.Jingshu 6 and its mutant JS6-5 with high anthocyanin content was conducted by RNA-sequencing technology and differentially expressed genes related to anthocyanin biosynthesis were analyzed.Three genes,IbMYB4,IbMYB48 and IbMYC2,were cloned from JS6-5 and their functions were characterized by transforming Arabidopsis.The main results are as follows:The salt tolerance and drought resistance of Jingshu 6 and its mutant JS6-5 were identified.The results showed that the salt tolerance and drought resistance of JS6-5 were significantly higher than that of Jingshu 6.We characterized the root transcriptomes of the two materials by high-throughput RNA sequencing.A total of 22,873,364 and 27,955,097 high quality reads were obtained from Jingshu 6 and JS6-5,respectively and assembled into 35,592 unigenes.In all,we obtained 1,566 differentially expressed genes(DEGs).Among them,994 were upregulated and 572 were downregulated in JS6-5 compared to the expression in Jingshu 6.A total of 39 DEGs that might be related to anthocyanin synthesis were screened out from 994 up regulated DEGs.Using homologous cloning method,IbMYB4 was isolated from JS6-5.IbMYB4 contained a 720-bp ORF that encoded a 239-amino acid polypeptide with a molecular weight of 27.0 kDa and a pI of 7.65.The genomic sequence of IbMYB4 was 1190 bp and contained 3 exons and 2 introns.qRT-PCR showed that the expression of IbMYB4 in JS6-5 was significantly higher than that in other sweetpotato varieties,and the IbMYB4 gene was induced by NaCl,PEG,ABA,MeJA and H2O2.The subcellular location result indicated that IbMYB4 was located in nuclei.The IbMYB4-overexpressing Arabidopsis plants contained more anthocyanin and exhibited higher salt and drought tolerance compared with the wild-type plants.The ABA,JA content and SOD activities were increased and the MDA and H2O2 content were found decreased in the transgenic Arabidopsis plants.qRT-PCR analyses showed that overexpression of IbMYB4 up-regulated the expression of genes in ABA signalling pathway,JA signalling pathway and ROS-scavenging system,which enhanced the resistance of transgenic Arabidopsis under salt and drought stresses.By Yeast two-hybrid assay and bimolecular fluorescence complementation assay(BiFC),IbMYB4 could interact with AtCYS-3A and AtCSN5B.Using homologous cloning method,IbMYB48 was isolated from JS6-5.IbMYB48 contained a 801-bp ORF that encoded a 266-amino acid polypeptide with a molecular weight of 30.9 kDa and a pI of 8.73.The genomic sequence of IbMYB48 was 2293 bp and contained 3 exons and 2 introns.qRT-PCR analysis revealed that the expression of IbMYB48 was significantly induced by salt,drought,ABA,MeJA,SA and H2O2 treatments.The subcellular location result indicated that IbMYB48 was located in nuclei.The IbMYB48-overexpressing Arabidopsis plants exhibited higher salt and drought tolerance compared with the wild-type plants.The ABA,JA and proline content and SOD activities were increased and the MDA and H2O2 content were found decreased in the transgenic Arabidopsis plants.qRT-PCR analyses showed that overexpression of IbMYB48 up-regulated the expression of genes in ABA,JA signalling pathway,proline synthesis pathway and ROS-scavenging system,which enhanced the resistance of transgenic Arabidopsis under salt and drought stresses.Using homologous cloning method,IbMYC2 was isolated from JS6-5.IbMYC2 contained a 1419-bp ORF that encoded a 472-amino acid polypeptide with a molecular weight of 53.04 kDa and a pI of 6.26.The genomic sequence of IbMYC2 was 1419 bp and contained only 1 exon.qRT-PCR analysis revealed that the expression of IbMYC2 was significantly induced by salt,drought,ABA,MeJA and H2O2 treatments.The subcellular location result indicated that IbMYC2 was located in nuclei.The IbMYC2-overexpressing Arabidopsis plants exhibited higher drought tolerance compared with the wild-type plants.The JA content and SOD activities were increased and the MDA and H2O2 content were found decreased in the transgenic Arabidopsis plants.qRT-PCR analyses showed that overexpression of IbMYC2 up-regulated the expression of genes in JA signalling pathway and ROS-scavenging system,which enhanced the resistance of transgenic Arabidopsis under drought stresses.