Expressional And Functional Analysis Of HMGR, DXS And DXR Genes From Amomum Villosum Lour

Plant produces diverse amount of isoprenoids compounds, which not only play essential for plant growth and development but also important for the interaction of plant with environment. These compounds are also used in medicines and industrial field by human beings. Essential oil, a composition of different isoprenoids, is active compound Chinese medicine. In higher plants, isoprenoids are produced through two universal pathways:one is the mevalonate pathway (MVA), which located in the cytosol (endoplasmic reticulum); another is mevalonate-independent pathway, methyl erythritol 4-phosphate pathway (MEP), located in the plastids. In plant, the first committed step of MVA pathway is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) where 1-deoxy-D-xylulose 5-phosphate synthase (DXS) and 1-deoxy-D-xylulose 5-phosphate reductase (DXR) are key enzymes in MEP pathway.The fruits of Amomum villosum Lour.(Fructus Amomi) are used for dissipating dampness and appetizing, warming spleen and stopping diarrhea, regulating Qi flow and preventing miscarriage. Essential oil is the main medicinal material of Fructus Amomi. Three cultivars, Yuanguo, Changguo and Chunxuan of A.villosum are found in Yangchun, China. The HMGR, DXS and DXR from A. villosum were named AvHMGR, AvDXS and AvDXR. We analyzed the expressional profile of HMGR, DXS and DXR genes in different plant parts in different cultivars. We also studied the function of AvHMGR, AvDXS and AvDXR protein in E. coli cell by color complementation methods. Beside this, we analyzed the function of HMGR, DXS and DXR by developing transgenic tobacco plant.1. Expressional analysis of AvHMGR, AvDXS and AvDXR.Total RNA was extracted from different plant part of A.villosum and subjected for semi-quantitative RT-PCR. The result showed these three genes were expressed in all cultivars of A. villosum but at different levels. DXS expressed more strongly but HMGR and DXR expressed nearly at same level. All genes expressed at high level in stems, roots, pericarps and seeds. But the situation was different in leaves. DXS expressed more highly but HMGR and DXR expressed at low level.We tried to regulate the three genes expression by UV-light irradiation. The genes showed downregulation under UV-light exposure. 2. Functional analysis of AvHMGR, AvDXS and AvDXR.The full-length cDNA of HMGR, DXS and DXR genes had already been cloned by Dr. Yang Jinfen from A. villosum and then coding fragments of AvHMGR, AvDXS and AvDXR were amplified by PCR to construct recombinant plasmids and co-transformed to E. coli TOP 10 cells along with pAC-BETA plasmid containing gene clusters for (3-carotene synthesis. The result showed that orange yellow color of bacteria was enhanced after transformation of our target gene AvHMGR and AvDXS to E. coli cells, but AvDXR transformed cells could not give the clear result. It revealed that except AvDXR, AvHMGR and AvDXS proteins accelerated the isoprenoids production with enhancing the orange yellow color ofβ-carotene in E. coli cells.3. Generation of transgenic tobacco plant.Recombinant plasmids pBI-AvHMGR and pBI-AvDXR were constructed and transformed into Agrobacterium tumefaciens cells. These Agrobacterium cells harboring recombinant plasmids of respective genes were transformed to tobacco plant to generate the transgenic tobacco plants. The transformed plants were screened by polymerse chain reaction (PCR). Expressional profile of these genes was observed by semi-quantitative RT-PCR method in transgenic plant. The result showed that both AvHMGR and AvDXR genes were expressed more in transgenic plant showing higher transcript level than control plants.It is the first study about AvHMGR, AvDXS and AvDXR in A. villosum. This study helps us to understand about these genes function at molecular level.