| Andrographis paniculata is reported to have heat-removing, detoxification, blood-cooling and detumescence activities. It is used as anti-bacterial and anti-inflammatory drugs and the major bioactive constituents are diterpenoid andrographolide and 14-deoxy-11, 12-didehydroandrographolide. However, the biosynthetic pathway of andrographolides has not been elucidated. Biosynthesis of andrigrapholide is very complex, the cyclization of GGPP to form ent-CPP, the core structure of andrigrapholides, catalyzed by the key enzyme ent-copalyl diphosphate synthase. One CPS gene (ApCPS, GenBank:JN216843.1) has been cloned previously from A.paniculata but not yet characterized biochemically. Here we characterized the function of ApCPS in vitro and in planta, which could help us understand the pathway and mechanism of andrigrapholides biosynthesis, and also lay the foundation to use this gene reglation andrigrapholides biosynthesis and for improvement the efficacy of A.paniculata and standardization of production.In this study, the material A. paniculata were grown in greenhouse. RT-PCR were used to analyze ApCPS gene expression. The content of andrographolide and 14-deoxy-11, 12-didehydroandrographolide in each organization were measured by TLC and HPLC. Escherichia coli metabolic engineering was applied to characterize ApCPS function in vitro and determine ApCPS catalytic avtive site by site-directed mutagenesis and bioinformatics ananlysis. Using VIGS to silence ApCPS in planta, the phytoene desaturase of A.paniculata was cloned by RACE technique as a reporter gene for VIGS. ApCPS was silenced with VIGS and the silencing efficiency was verified by semiquatitative RT-PCR and quatitative RT-PCR. The uptream genes HMGR, DXS and GGPS were also chosen to be analyzed by qRT-PCR. In addition, the accumulation of andrographolides was analyzed by HPLC after ApCPS silencing. For deeply investigate the regulatory mechanism of ApCPS, we tested ApCPS gene and its upstream related gene expression after A.paniculata was treated with MeJA. The main results are listed:1. ApCPS was observed to express in various tissues, with high expression in leaf and stem, but low in root, which is consistent with the andrographolides accumulated in this oagans. qRT-PCR analysis showed that MeJA induced ApCPS gene expression strongly. The upstream gene HMGR, DXS, GGPS of andrographolides biosynthesis also exhibited inducible gene expression with MeJA treatment, and response time is earlier than ApCPS, indicating pleiotropic regultion of MeJA on andrographolides biosynthesis.2. ApCPS was cloned successfully from A.paniculata seedings. Characterization of ApCPS in vitro showed that it catalyzed GGPP to generate ent-CPP. Site-directed mutagenesis was employed to change the first Asp of DXDD motif to Ala, which resulted in loss of CPS function, indicating this motif invovled in catalysis. In addition, ApCPS has one conserved Arg close to DXDD motif, implicating putative involvement in specialized metabolism rather than primary metabolism.3. A full-length cDNA of phytoene desaturase (PDS) gene from A.paniculata was obtained through RACE-PCR. The cDNA sequence consists of 2 224 bp with an intact ORF of 1 752 bp (GeneBank:KP982892), encoding a ploypeptide of 584 amino acids. Homology analysis showed that the deduced protein has extensive sequence similarities to PDS from other plants, and contains a conserved NAD(H)-binding domain of plant dehydrase cofactor binding-domain in N-terminal. Phylogenetic analysis demonstrated that ApPDS was more related to PDS of Sesamum indicum and Pogostemon cablin. The semi-quantitative RT-PCR analysis revealed that ApPDS expressed in whole aboveground tissues with the highest expression in leaves.4. Virus induced gene silencing (VIGS) was performed to characterize the functional of ApPDS in planta. The VIGS vector pTRV2-PDS was constructed and transformed into A. paniculata leaves with Agrobacterium infiltration. Slightly photobleaching was observed in infiltrated leaves, and the PDS gene was down-regulated at the yellowish area, indicating that VIGS technology can be used in A.paniculata.5. The expression of ApCPS was decreased 49.08% in A.paniculata leaves by VIGS. In VIGS plants, GGPS also showed decreased trascript accumulation, which might be resulted from negetive feedback of GGPP accumulated after ApCPS gene silencing, but no effect was observed on the expression of HMGR and DXS. After 15 days of ApCPS silencing, the accumulation of andrographolides was decreased 53.52% with HPLC anlaysis.Through the study in vitro and in planta, we characterized the function of ApCPS involved in andrographolides biosynthetic pathway, which catalyzed GGPP to form the core structure of andrigrapholides, ent-CPP. And ApCPS was silenced successfully in A paniculata, resulted in significant reduction of andrographolides accumulation, which lays the foundation for further investigation of the function of new genes in andrographolides biosynthetic pathway. |
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