De Novo Transcriptome Sequencing Of The Orange-Fleshed Sweetpotato(Ipomoea Batatas(L.)Lam.)and Cloning And Functional Characterization Of Important Genes Related To Carotenoid Biosynthesis

Sweetpotato,Ipomoea batatas(L.)Lam.,is one important food crop worldwide,and its storage roots can provide both carbohydrates and carotenoids for human consumption.The storage roots of orange-fleshed sweetpotato are an important source of ?-carotene,a precursor of vitamin A.In this study,de novo transcriptome sequencing of the orange-fleshed sweetpotato cv.Weiduoli and its mutant HVB-3 with high carotenoid content was conducted by Illumina pared-end(PE)RNA-sequencing technology and differentially expressed genes related to carotenoid biosynthesis were analyzed.Two important genes,IbNAC29 gene and 1-deoxy-D-xylulose 5-phosphate synthase(IbDXS)gene,were cloned from HVB-3 and their functions were characterized by transforming Arabidopsis.The IbGGPS-overexpressing sweetpotato plants were developed and the IbZDS-overexpressing transgenic sweetpotato plants were evaluated for the carotenoids content and salt tolerance.1.The transcriptome profiles of Weiduoli and HVB-3 were determined by using the high-throughput sequencing technology.A total of 13,767,387 and 9,837,090 high-quality reads were produced from Weiduoli and HVB-3,respectively.These reads were de novo assembled into 58,277 transcripts and 35,909 unigenes with an average length of 596 bp and 533 bp,respectively.In all,874 differentially expressed genes(DEGs)were obtained between Weiduoli and HVB-3,401 of which were upregulated and 473 were downregulated in HVB-3 compared with Weiduoli.2.Using RACE method,IbNAC29 was isolated from HVB-3.The full-length cDNA of IbNAC29 contained an 849-bp ORF encoding a 282-aa polypeptide with a molecular weight of 31.97 kDa and a pI of 8.69.The genome sequence of IbNAC29 was 1054 bp and was deduced to contain 3 exons and 2 introns.The results of qRT-PCR indicated that the expression level of IbNAC29 was significantly higher in the leaves of HVB-3 than in the storage roots and stems.The images from epidermal cells of onion showed that the green fluorescence created by IbNAC29-GFP fusion protein was predominantly localized to nucleus.High level of IbNAC29 expression in transgenic Arabidopsis plants was demonstrated by qRT-PCR.The ?-carotene content was significantly increased in transgenic Arabidopsis plants compared with the wild type(WT).3.bDXS was cloned from HVB-3 with RACE method.The full-length IbDXS cDNA contained a 2160-bp ORF encoding a 719-aa polypeptide with a molecular weight of 77.5 kDa and a pI of 6.69.The genome sequence of IbDXS with 3382 bp was deduced to contain 9 exons and 8 introns.qRT-PCR analysis revealed that there was a higher expression level of IbDXS in the leaves of HVB-3 than in the storage roots and stems.The expression level of IbDXS in transgenic Arabidopsis plants was significantly increased.The ?-carotene content was significantly increased in transgenic Arabidopsis plants compared with WT.4.The IbGGPS-overexpressing sweetpotato plants(cv.Lizixiang)were developed.The ?-carotene,abscisic acid(ABA)and starch contents were significantly increased in transgenic plants compared with WT,while the sugar content was significantly decreased.These results suggest that IbGGPS plays an important role in increasing ?-carotene and starch contents in sweetpotato.5.The three sweetpotato(cv.Kokei No.14)plant lines overexpressing IbZDS were evaluated in a field.The transgenic lines exhibited significantly increased ?-carotene and lutein contents and enhanced salt tolerance.Significant up-regulation of lycopene ?-cyclase(?-LCY)and ?-carotene hydroxylase(?-CHY)genes and significant down-regulation of lycopene ?-cyclase(?-LCY)and ?-carotene hydroxylase(?-CHY)genes were found in the transgenic plants.ABA and proline contents and superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)activities were significantly increased,whereas malonaldehyde(MDA)content was significantly decreased in the transgenic plants under salt stress.The salt stress-responsive genes encoding pyrroline-5-carboxylate reductase(P5CR),SOD,CAT,ascorbate peroxidase(APX)and POD were found to be significantly up-regulated in the transgenic plants under salt stress.These findings indicate that IbZDS has the potential to be applied for improving ?-carotene and lutein contents and salt tolerance in sweetpotato.