Cloning Of Gene Fragments Related To Carotenoid Biosynthesis In Sweetpotato, Ipomoea Batatas (L.) Lam.

The Carotenoids are the most diverse and widespread group of pigments in nature and serve an extraordinary variety of functions in plant, animal, algae and cyanobacteria. A sweetpotato variety, Nongdafu 14, is a carotenogenic mutant obtained from Kokei No.14 irradiated with gamma-ray. To elucidate the molecular machinism of the mutagenesis, the variation of carotenoid concentration in storage roots of Nongdafu 14 and its original variety Kokei No.14 was analyzed during the root thickening and the fragments of genes related to sweetpotato carotenoid biosynthesis were cloned. The main results are as follows:1. The carotenoid content of Nongdafu 14 is higher five to ten times than that of Kokei No.14. During storage root thichening, the carotenoid concentration of Nongdafu 14 increased rapidly, while the change of carotenoid concentration in Kokei No.14 was not obvious.2. Degenerate primers were designed from the conserved motifs of carotenogenic genes isolated from other dicotyls and cDNA fragments of the genes encoding geranylgeranyl pyrophosphate synthase (GGPS), phytoene synthase (PSY), phytoene desaturase (PDS), zeta-carotene desaturase (ZDS) and lycoene beta-cylase (LYCB), were cloned and sequenced. The sweetpotato sequences obtained shared high similarity with the carotenogenic genes from other plants.3. A full-length cDNA encoding sweetpotato phytoene desaturase (PDS) was isolated, which length was 2,128 bp and had an open reading frame. Based on the deduced amino acid sequence, the sweetpotato PDS gene was found to have 572 amino acid residues and is significantly conserved as compared with the PDS genes of other plants. RT-PCR analysis indicated that the expression of PDS gene was constitutive in the mutant and its original variety and real time fluorenscent quantitative PCR analysis showed that the expression of the PDS gene was reduced with the thickening of storage roots. The expression level of the PDS gene was higher in the mutant than in the original variety at different developmental stages of storage roots.4. Differential expression of genes in storage roots of the mutant and its original variety was analyzed by cDNA-AFLP. The results indicated that most of genes showed similar expression in both, but 32 differential fragments were obtained. Six of the 32 fragments were sequenced and made Blast search in GenBank. The identity of the sequence of fragment 2 with the putative senescence -associated protein mRNA sequence (AY436773.1) from Pyrns communis is 100%; The identity of the sequence of fragment 3 with the Ttgl-like protein mRNA sequence (AF220203.1) from Malus domestica is 89%; The identity of the sequence of fragment 4 with the putative adenylate kinase mRNA sequence (BT004398.1) from Arabidopsis thaliana is 81%.