Molecular Cloning And Expression Analysis Of Two Key Enzyme Genes GGPS And PDS In The Terpenoids Metabolic Pathway From Pyropia Haitanensis

Terpenoids are the most widely distributed natural compounds in nature,playing an important role in improving the excellent quality in Pyropia haitanensis.Terpenoids and their derivatives also play an irreplaceable role in the growing development,hormone synthesis,light absorption,photoprotection and stress resistance of plant.Geranylgeranyl diphosphate synthase(GGPS)and phytoene dehydrogenase(PDS)are two key enzymes in the terpenoids metabolic pathway.In this study we have first cloned the full-length sequence of GGPS gene by methods of RT-PCR and RACE from P.haitanensis(Genbank database accession No.MG431996).The full-length of Ph GGPS is 1416 bp,encoding 264 amino acids while5'UTR(non-coding region)sequence is 493 bp and the 3'UTR sequence be 128 bp.Meanwhile,the deduced protein molecular weight was 27.367 k Da while the theoretical p I was 5.00,the content of glycine and leucine was higher and the average GRAVY was 0.213,which suggesting that protein of Ph GGPS is a hydrophobic stable protein.It was speculated that the secondary structure of the protein is rich in alpha helix,which mainly consisted of an ?-helix of 53.79%,extended chain and random coil accounting for 17.42% and 18.94% of the secondary structure respectively,with the least ?-sheet accounted for 9.85%.On the amino acid level,alignment of Ph GGPS shared 95% sequence identity with GGPS from Porphyra umbilicalis,66% similarity with Emiliania huxleyi,60% similarity with Dunaliella viridis and 60% sequence identity with Chlamydomonas reinhardtii respectively.In this study we have first cloned the full-length sequence of PDS gene from P.haitanensis(Genbank database accession No.MG431995).The full-length of Ph PDS is 2081 bp and the coding length is 1692 bp,which encoding 563 amino acids while5'UTR(non-coding region)sequence is 225 bp and the 3'UTR sequence be 164 bp.Meanwhile,the deduced protein molecular weight was 60.67 k Da while the theoretical p I was 5.89,the content of alanine,glycine and leucine was higher and the average GRAVY was 0.001,which suggesting that protein of Ph PDS is a hydrophobic stable protein with a 51 bp of transit peptide.It was speculated that the secondary structure of the protein is rich in alpha helix and random coil,which mainly consisted of an ?-helix of 36.23% and a random coil of 36.23%,extended chain accounting for 17.23% of the secondary structure,with the least ?-sheet accounted for 10.30%.On the amino acid level,alignment of Ph PDS shared 95% sequence identity with PDS from Porphyra yezoensis,92% similarity with Porphyra umbilicalis,73% similarity with Chondrus crispus and 69% sequence identity with Synechococcus elongatus respectively.In the study,for the first time we used Me JA,BR,ABA,SA to treat Pyropia haitanensis,in order to explore the gene expression difference of the terpenoids metabolic key enzyme.By the gene expression level of GGPS in the foliose thallus and conchocelis,we found there was not significantly different.The expression level of Ph GGPS gene treated with 200 u M Me JA was 7.34 ± 0.348 fold as compared to the control group.The expression level of Ph GGPS gene treated with 100 u M ABA were significantly increased as compared to the control group,up to 4.13± 0.295 fold.Gene expression levels of Ph GGPS with 100 u M SA treatment were significantly higher than the control group,increasing 5.12 ± 0.777 fold.Gene expression of Ph GGPS with 10 u M BR treatment increased 4.12 ± 0.214 fold as compared to the control group.The expression level of Ph PDS gene in two generations also existed significantly different,with the expression of PDS gene in conchocelis was 6.53 ±0.799 fold as compared to that of the thallus.Gene expression of Ph PDS with 200 u M Me JA treatment was 3.14 ± 0.247 fold higher than the control,and the gene expression level of Ph PDS was up-regulated by 3.4 ± 0.066 fold as compared with that of the control group.The gene expression of Ph PDS was significantly increasing 3.02 ±0.487 fold as compared with the control group.The gene expression level of Ph PDS was 2.28 ± 0.352 fold higher than that of the control group under 10 u M BR treatment.Under treatment of the same reagent concentration,carotenoids of Pyropia haitanensis were also extracted.We found that there existed different degrees of increase of the content of carotenoids in experimental groups as compared with the control group.The results showed that one of the final terpenoid metabolism products-carotenoid would increased with the terpenoid metabolism-related genes up-regulated.