Cloning And Functional Characterization Of IbLCYB2,IbARF5 And IbbZIP1 In Sweetpotato (Ipomoea Batatas(L.)Lam.)

Sweetpotato line HVB-3 with high carotenoid content was employed for cloning the IbLCYB2,IbARF5 and IbbZIP1 genes.Three expressed sequence tag(EST)clones were selected from the transcriptome sequencing data of HVB-3 constructed at our laboratory.Sweetpotato cv.Shangshu 19,a commercial cultivar with low carotenoid content,was used to characterize the function of IbLCYB2 and Arabidopsis was used to characterize the function of IbARF5 and IbbZIP1.1.IbLCYB1 was isolated from HVB-3 with RACE method.Its full-length eDNA contained a 1506 bp ORF encoding a 501 aa polypeptide with a molecular weight of 56.46 kDa and a pI of 8.02.The genome sequence of IbLCYB2 was 1506 bp and was deduced to contain 1 exon.qRT-PCR analysis indicated that the expression level of IbLCYB2 was significantly higher in the leaves of HVB-3 than in the storage roots and stems.The images from epidermal cells of N.benthamiana showed that IbLCYB2-GFP was localized to chloroplasts.Its overexpression significantly increased the contents of ?-carotene,?-carotene,lutein,?-cryptoxanthin and zeaxanthin and enhanced the tolerance to salt,drought and oxidation in the transgenic sweetpotato plants.The genes involved in carotenoid and abscisic acid(ABA)biosynthesis pathways and abiotic stress responses were up-regulated in the transgenic plants.The ABA and proline contents and superoxide dismutase(SOD)activity were significantly increased,whereas malonaldehyde(MDA)and H2O2 contents were significantly decreased in the transgenic plants under abiotic stresses.The overall results indicated that IbLCYB2 enhanced carotenoid contents and abiotic stress tolerance through positive regulation of carotenoid and ABA biosynthesis pathways in sweetpotato.This gene has the potential to improve carotenoid contents and abiotic stress tolerance in sweetpotato and other plants.2.IbARF5 was isolated from HVB-3 with RACE method.Its full-length cDNA contained a 2841 bp ORF encoding a 946 aa polypeptide with a molecular weight of 104.84 kDa and a pI of 5.17.The genome sequence of IbARF5 was 4794 bp and was deduced to contain 13 exons and 12 introns.The results of qRT-PCR indicated that the expression level of IbARF5 was significantly higher in the storage roots of HVB-3 than in the leaves and stems.The images from epidermal cells of onion showed that IbARF5 was localized to nucleus.Its overexpression in Arabidopsis significantly increased the contents of lutein and zeaxanthin in leaves and lutein,?-carotene and zeaxanthin in seeds and enhanced the tolerance to salt and drought.Transgenic Arabidopsis plants also displayed significant up-regulation of the genes involved in carotenoid and ABA biosynthesis pathways and abiotic stress responses.ABA and proline contents and SOD activity were significantly increased,whereas H2O2 content was significantly decreased in the transgenic Arabidopsis plants under salt and drought stresses.The overall results indicated that IbARF5 improved carotenoid contents and enhanced salt and drought tolerance by up-regulating the genes involved in carotenoid and ABA biosynthesis pathways and abiotic stress responses in transgenic Arabidopsis.This study provides a novel ARF gene for improving carotenoid contents and salt and drought tolerance of sweetpotato and other plants.3.IbbZIP1 was isolated from HVB-3 with RACE method.Its full-length cDNA contained a 1080 bp ORF encoding a 359 aa polypeptide with a molecular weight of 41.26 kDa and a pI of 7.89,The genome sequence of IbbZIP1 was 1946 bp and was deduced to contain 4 exons and 3 introns.The results of qRT-PCR indicated that the expression level of IbbZIP1 was significantly higher in the leaves of HVB-3 than in the storage roots and stems.Its overexpression in Arabidopsis significantly enhanced salt and drought tolerance.Under salt and drought stresses,the genes involved in ABA and proline biosynthesis and reactive oxygen species(ROS)scavenging system were up-regulated,ABA and proline contents and SOD activity were significantly increased,whereas H2O2 content was significantly decreased in the transgenic Arabidopsis plants.These results demonstrated that IbbZIP1 is involved in salt and drought tolerance in transgenic Arabidopsis.This study provides a novel bZIP gene for improving the tolerance of sweetpotato and other plants to abiotic stresses.