Functional Analysis Of HMGR And DXR Genes From Amomum Villosum Lour.on Biosynthesis Of Terpenoids In Tobacco

Fructus Amomi is mainly from Amomum villosum Lour.,which is a Chinese medicine with warm property and pungent taste and has such effects as resolving dampness to improve appetite, warming spleen to arrest diarrhea, regulating Qi to prevent miscarriage etc. The isoprenoids in its volatile oil are the main medicinal compounds of Fructus Amomi.In higher plants, isoprenoids are produced through two universal pathways:one is the mevalonate (MVA) pathway, the other is2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. The enzymes,3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) and1-deoxy-D-xylulose5-phosphate reductase (DXR) are the upstream key-enzymes for MVA and MEP pathway, respectively. Overexpresstion of HMGR and DXR gene can enhance the biosynthesis of isoprenoids in plants.1ObjectiveUsing the upstream key-enzymes in isoprenoid biosynthesis pathway of HMGR and DXR gene from A villosum to analyze the biological function of AvHMGR and AvDXR in transgenic tobacco. Furthermore, using the metabolic engineering method, to manipulate the expresstion of AvHMGR and AvDXR in isoprenoid biosynthesis, can be of value to improve the medicinal quality of A. villosum.2Methods2.1Screening of T1transgenic tobacco plants and phenotypic comparationT1transgenic tobacco seeds were first screened by kanamycin, and then all kanamycin-resistance plants were screened by PCR for the presence of neomycin phosphotransferase Ⅱ (npt Ⅱ) and target genes of AvHMGR and AvDXR. Phenotypic differences were compared between the transgenic plants and untransformed plants.2.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic tobacco plantsRT-PCR (Reverse Transcription-Polymerase Chain Reaction) was used to analyze the expression level of AvHMGR and AvDXR in transgenic tobacco plants. Using the substrate specific method, HMGR and DXR activity were determined in AvHMGR and AvDXR transgenic tobacco plants and wild-type tobacco plants by spectrometer.2.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plantsExtraction and qualitative analysis of isoprenoids contents in transgenic plants and wild-type plants. Chlorophylls (diterpene) and carotenoids (tetaterpene) were extracted by acetone and determined by spectrometer. Volatile isoprenoids, mainly of monoterpene, sesquiterpene and diterpene, were determinated by Head Space-SolidPhase Microextraction (HS-SPME), qualitative analysis was accomplished by GC-MS. Non-volatile isoprenoids, such as triterterpene of sterol, a-tocopherol, and diterpene of phytol, were extracted by dichloromethane and qualitative analysis by GC-MS.3Results3.1Screening of AvHMGR and AvDXR T1transgenic tobacco plants and phenotypic comparationAvHMGR and AvDXR transgenic tobacco plants were screened by Kan and verified by PCR, transgenic lines (H1, H3, H4, H5and D1, D2) were obtained, showed single-copy insertions, probably. Seventeen AvHMGR and twelve AvDXR transgenic tobacco plants were obtained.The height of AvHMGR transgenic plants (line H5) showed obvious phenotypic differences compared with wild-type plants. The stems of AvDXR transgenic plants presented horizontal growth and AvDXR transgenic tobacco suffered from less pests.3.2Expression and enzymes activity analysis in AvHMGR and AvDXR transgenic plantsThe results showed that eleven AvHMGR and eight AvDXR T1transgenic tobacco plants were overexpressed. Compared with wild-type tobacco plants, the HMGR and DXR activity of five AvHMGR and two AvDXR transgenic plants had been significant increased, respectively.3.3Overexpression of AvHMGR and AvDXR genes effect of isoprenoids producion in tobacco plantsCompared with wild-type plants, AvHMGR and AvDXR overexpressing plants can detected more volatile terpenoids, such as monoterpene (m-Mentha-4,8-diene), sesquiterpene (D2-1of AvDXR transgenic tobacco plants showed the production of santalol), diterpene (H3-3of AvHMGR transgenic tobacco plants showed the production of thunbergol).Furthermore, the overexpression of AvHMGR and AvDXR, resulted in elevated levels of photosynthetic pigments (chlorophylls and carotenoids), phytol, stigmasterol and campesterol. However, the contents of β-sitosterol and α-tocopherol had decreased, compared with wild-type plants.4ConclusionThe results verified that regulation of HMGR and DXR can enhance biosynthesis of isoprenoids. AvHMGR and AvDXR genes play important role in isoprenoid biosynthesis pathway. However, all isoprenoids did not increase equally, each isoprenoid biosynthetic pathway could have its own localization in cells. Indicating that the MVA pathway and MEP pathway did not independent, cross-talk between the two pathways had also been documented. The study documented that using metabolic engineering, manipulate upstream key-enzymes of AvHMGR and AvDXR in isoprenoid biosynthesis, can improve the medicinal quality of Amomum villosum Lour.