| Terpenoids(also called terpenes or isoprenoids)are the largest groups of metabolites in nature,which are most diverse in structure and function.Members of the plant kingdom alone are estimated to produce more than 30,000 terpenoid compounds.Terpenoids of plants mostly have significant biological function.Carotenoids as photosynthesis pigments are the largest groups of pigments in nature,which contain more than 600 known compounds.Because many carotenoids are precursors of VA and have anticancer activity,they are indispensable in food of human and animal.Geranylgeranyl diphosphate synthase catalyzes the condensation of FPP with IPP to give GGPP which is a common precursor of carotenoids,so GGPPS is an important enzyme in the metabolism of carotenoids.To investigate the carotenoid biosynthesis of sweet potato at molecular level and provide a new target gene for metabolic engineering of carotenoid pathway of sweet potato,a new cDNA encoding GGPPS was isolated from a new cultivar Yushu-303 by the RACE method.The full-length cDNA was 1368 bp in length,designated as IbGGPPS(GenBank?Accession Number:EU570195),that contained a 1089 bp open reading frame(ORF)encoding a polypeptide of 363 amino acids.Bioinformatic analysis showed the cDNA sequence of IbGGPPS was homologous with other GGPPS gene and the deduced amino acid sequence of IbGGPPS was highly similar to known plant GGPPSs.IbGGPPS consists notably of two conserved aspartate-rich motifs which are important activity domains of GGPPS. The subcellular location analysis predicted that IbGGPPS was located into plastid. Phylogenic analysis indicated that all GGPPSs could be divided into two groups and IbGGPPS belonged to angiosperm plant GGPPSs' family.The tissue expression profile analysis was carried out to investigate the transcriptional level of IbGGPPS in different tissues including roots,stems,tender leaves,mature leaves and tuberous roots.The results showed that IbGGPPS expression is highest in tuberous roots and tender leaves, the next tissues are mature leaves and roots,however,it could not be detected in stems. Finally,The ORF of IbGGPPS was isolated and substituted the Arabidopsis thaliana ipi gene of plasmid pTrcAtIPI to generate pTrcIbGGPPS.And then pTrcIbGGPPS was introduced into engineered Escherichia coli strain XL1-Blue in which the carotenoid pathway was reconstructed based on plasmid pACCAR25â–³crtE.In engineered XL1-Blue,IbGGPPS could facilitate the metabolic flux to the carotenoids biosynthesis and made the bacteria produce the orange zeaxanthin.These confirmed that IbGGPPS had the typically function of GGPPS gene.In summary,cloning,characterization and functional expression of IbGGPPS will facilitate to understand the function of IbGGPPS at the level of molecular genetics and unveil an important step involved in the biosynthetic pathway of sweet potato carotenoids precursor biosynthesis.Research of Selection method for transformant is an important problem in plant genetic engineering,and using glyphosate as a selection marker is cheap,safe and efficient.Glyphosate is the most widely used herbicide today.It is a non-selectable herbicide,and capable of stopping growth of abroad range of crops and weeds. Glyphosate interferes in the shikimate metabolic pathway by infecting activity of 5-enolpyruvyl-3-phosphoshikimate(EPSPS).This prevents the subsequent synthesis of the three aromatic amino acids tryptophan,phenylalanine and tyrosine.Glyphosate tolerance conferred by the stable introduction of bacterial mutant genes encoding glyphosate-insensitive EPSPS.Moreover,the shikimate metabolic pathway is only existed in plants and microbes,so using glyphosate as a selection marker is safe.In this research,Pinellia ternate was transformed via Agrobacterium tumefaciens strain LBA4404 harboring the plasmid pASM12.Transgenic explants were directly selected on medium containing glyphosate 10mg/L,and transgenic plantlets were confirmed by genomic PCR techniques and glyphosate resistance test.PCR analysis showed transformation frequency of leaves was 23%and petioles was 33%,glyphosate resistance test indicated transgenic plantlets could resist to glyphosate at different levels. We established the transformation method for Pinellia ternate facilitate breeding anti-herbicide varieties of Pinellia ternate and applying the glyphosate-based selection system for Pinellia ternate genetic engineering. |
