Functional Characterization of ApCPS Involved in Andrographolides Biosynthesis by Virus-induced Gene Silencing
The ent-copalyl diphosphate synthase gene (ApCPS) with putative invovlment in andrographolides biosynthesis was silenced successfully in the 8-leaf stage of Andrographis paniculata through TRV virus-induced gene silencing (VIGS),which was verified by semiquatitative RT-PCR and quatitative RT-PCR.(1)Gene silencing of ApCPS resulted in decreased accumulation of andrographolides significantly with HPLC analysis.(2)In order to comprehensively investigate gene expression of andrographolides biosynthesis,the uptream genes,HMGR,DXS and GGPS were chosen to be analyzed by qRT-PCR.In VIGS plants,GGPS showed decreased trascript accumulation,which might be resulted from negetive feedback of GGPP accumulated after ApCPS gene silencing.No significant change was observed for HMGR and DXS upon ApCPS gene silencing.(3)HMGR,DXS and GGPS exhibited inducible gene expression with MeJA treatment,as well as ApCPS,indicating pleiotropic regultion of MeJA on andrographolides biosynthesis.It is the first report to characterize ApCPS involved in andrographolides biosynthesis in vivo through VIGS,which lays the foundation for further investigation of other andrographolides biosynthetic genes.
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