Cloning and expression analysis of GGPPS gene from Panax notoginseng / 中国中药杂志

According to the transcriptome dataset of Panax notoginseng, the key geranylgeranyl pyrophosphate synthase gene (GGPPS) in terpenoid backbone biosynthesis was selected to be cloned. Using specific primer pairs combining with RACE (rapid amplification of cDNA ends) technique, the full-length cDNA sequence with 1 203 bp, which containing a 1 035 bp open reading frame, was cloned and named as PnGGPPS. The corresponding full-length DNA sequence contained 2 370 bp, consisted of 1 intron and 2 exons. The deduced protein PnGGPPS contained 344 amino acids and shared more than 73% identity with GGPPS from Ricinus communis and Salvia miltiorrhiza. PnGGPPS also had specific Aspartic acid enrichment regions and other conserved domains, which belonged to the Isoprenoid-Biosyn-C1 superfamily. The quantitative real-time PCR showed that PnGGPPS expressed in different tissues of 1, 2, 3 years old root, stem, leaf and 3 years old flower, and the expression level in 3 years old leaf was significant higher than that in other organs, which suggested that it might not only be involved in the regulation of the growth and development, but also be associated with the biosynthesis of chlorophyll and carotenoids, the development of chloroplast, the shade habit and the quality formation of P. notoginseng.

Cloning of Blakeslea trispora carRA gene by PCR-driven overlap extension and construction of an activity detection system / 生物工程学报

Blakeslea trispora CarRA has both lycopene cyclase and phytoene synthase activity. In order to analyze the double functional activity of CarRA proteins and to detect the active sites of lycopene cyclase, we constructed two detection systems in Escherichia coli by color complementary. Through PCR-driven overlap extension we got carRA gene cDNA, then constructed prokaryotes expression vector pET28a-carRA. pET28a-carRA with plasmid pAC-LYC carrying crtl/crtB/crtE gene clusters were co-transformed to BL21(DE3) to validate lycopene cyclase activity. We constructed the plasmid pAC-LYC delta (crtB) carrying crtl/crtE gene clusters, then co-transtormed them with pET28a-carRA to BL21(DE3) to validate phytoene synthase activity. Based on color complementary, and HPLC analysis of metabolites, we confirmed that the CarRA protein activity detection system was reliable. Our study provides a screening model for specific mutation of lycopene cyclase without affecting phytoene synthase activity.

Path analysis suggests phytoene accumulation is the key step limiting the carotenoid pathway in white carrot roots

Two F2 carrot (Daucus carota L.) populations (orange rooted Brasilia x very dark orange rooted High Carotene Mass - HCM cross and the dark orange rooted cultivated variety B493 x white rooted wild carrot Queen Anne's Lace - QAL cross) with very unrelated genetic backgrounds were used to investigate intrinsic factors limiting carotenoid accumulation in carrots by applying phenotypic correlation and path analysis to study the relationships between major root carotenes, root color and several other morphological traits. Most of the correlations between traits were close and agreed in sign between the two populations. Root weight had a moderate to highly significant positive correlation with leaf length, root length and top and middle root diameter. Although phenotypic correlations failed to identify the order of the substrates and products in the carotenoid pathway the correct order of substrates and products (phytoene -> zeta-carotene -> lycopene) was identified in the causal diagram of beta-carotene for the Brasilia x HCM population. Path analysis of beta-carotene synthesis in the B493 x QAL population suggested that selection for root carotenes had little effect on plant morphological traits. Causal model of beta-carotene and lycopene in the B493 x QAL population suggested that phytoene synthesis is the key step limiting the carotenoid pathway in white carrots. Path analysis, first presented by Sewall Wright to study quantitative traits, appears to be a powerful statistical approach for the identification of key compounds in complex pathways.