Cloning And Functional Analysis Of The Genes Related To The Metabolism Of The Terpenoids From Nicotiana Tobacco

Aroma is an important indicator for measuring the qualities of tobacco leaves and their products, the less flavor content is serious impacting on the qualities of tobacco, with the development of the tobacco industry, improving the quality of tobacco aroma has become one of the urgent problems. Terpenoid compounds are the tobacco precursors, 1-deoxy-D-xylulose-5-phosphate reductase(DXR) 、 1-Deoxy-D-xylulose-5-phosphate synthase(DXS) and 3-Hydroxy-3-methyglutary-co A reductase(HMGR1) gene were the key genes from the Methylerythritol phosphate(MEP) pathway and Mevalonate(MVA) pathway, the gene expression of DXS、DXR and HMGR1 changes will directly or indirectly affect the metabolic pathways of the compounds, which affect the synthesis of tobacco aroma precursors, and ultimately affect the aroma of tobacco. Also the tobacco is a model plant, the study of the enzyme of terpenoid metabolic pathway may provide a basical theory for other plants? research. In this paper tobacco cultivar “honghuadajinyuan” as the experimental materials to change the DXR and HMGR1 gene expression which used gene engineering technology, in order to study the function of DXR and HMGR1 in the isoprenoid metabolic pathway, which will provide a guidance for the further study of the molecular mechanism of tobacco aroma components and the improvement of tobacco flavor quality, as well which will provide a basical theory for promoting other plants? research of terpene metabolic pathway. The main resuls of this paper are as follows:(1) We successfully cloned Nt DXS/Nt DXR gene of MEP pathway and Nt HMGR1/Nt HMGR2 gene of MVA pathway from tobacco cultivar “honghuadajiinyuan” and analysised its bioinformatics. Functional domain structure prediction results show that Nt DXS protein has DXP_synthase_N、 Transketolase_C and Transket_pyr domain; Nt DXR protein has DXP_reductoisom and DXP_redisom_C functional domains which are highly conserved; both Nt HMGR1 and Nt HMGR2 protein have HMG-Co A_red conserved domain, which is the binding site of NADPH. Function prediction results show that the function of Nt DXS and Nt DXR protein may be signal transduction, transport, transcription and transcriptional regulation, and Nt DXR protein may be involved in stress response; The function of Nt HMGR1 protein and Nt HMGR2 protein may participate in signal transduction, voltage gate ion channel, transcription and transcriptional regulation. Subcellular localization prediction results show that Nt DXS and Nt DXR in the chloroplast, and Nt HMGR1 and Nt HMGR2 in the cytoplasm. Phylogenetic trees and multiple sequence alignment results show that the similarities between genes of tobacoo and genes of tomato are hignest, also we found that DXS and DXR of tobacco and other palnts both contain a plastid transit peptide which is not in bacteria.(2) We detected the expression of Nt DXS/Nt DXR/Nt HMGR1/Nt HMGR2 gene in different tissues and different periods of “Honghuadajinyuan”, the results show that Nt DXS and Nt DXR have similar expression patterns, which express high in seedling and flower, less in seedling roots, stems and stamen; Nt HMGR1 and Nt HMGR2 have a similar expression pattern in mature stage which have a high expression in flower, but in seeding stage their expression pattern have a lot differences. Also we constructed the vector of subcellular localization of Nt DXR, and transfected in tobacco protoplast, the result shows that Nt DXR plays a major role in the chloroplast. These results suggest that Nt DXS and Nt DXR may have the same fuction in plant, but the Nt HMGR1 and Nt HMGR2 which are homologous genes, may involve differently activities in plant.(3) We successfully constructed plants? overexpression and knockout vector of Nt DXR and Nt HMGR1, transformed into plant leaf tissues by means of leaf discs method. PCR and q RT-PCR were used to detect the overexpression lines, lately we obtained Nt DXR and Nt HMGR1 overexpression transgenic T1 generation plants each 10 beads and their knockout transgenic T0 generation plants each 6 beads, which three of the Nt DXR knockout transgenic plants showed obviously etiolated phenotypes.(4) We detected the content of pigments、sterols、monoterpenoids、diterpenoids、triterpenoids or other terpenoids in Nt HMFR1 and Nt DXR overexpression transgenic T1 generation plants by GC-MS/MS and HPLC. The content of Chlorophyll a 、campesterol and stigmasterol of Nt DXR and Nt HMGR1 overexpression transgenic tobacco lines respectively incresed compared to the control, also the content of lanostero increased in Nt DXR overexpression lines, the content of carotene and beta-sitosterol increased in Nt HMGR1 overexpression lines compared to the control. Higer quantity of alpha-4,8,13-cembratrie, beta-4,8,13-cembratrie, abienol, sclareol, phytol and beta-amyrin was detected in plants overexpression Nt DXR transformed lines compared to wild type. Differently, in Nt HMGR1 overexpression transformed lines less quantity of abienol and sclareol compared with wild type. But the content of t,t-farnesol, phytol, beta-amyrin and squalene increased in Nt HMGR1 overexpression transgenic plants. We also detected the content of pigments in the Nt DXR knockout transgenic plants, the content of Chlorophyll a 、Chlorophyll b and neoxanthin were less than control. This results suggest that Nt DXR and Nt HMGR1 may have a common promoting influence in chlorophyll a, campesterol, stigmasterol synthesis; and Nt DXR mainly affects diterpenoid synthesis, but Nt HMGR1 mainly affects the synthesis of sesquiterpene and triterpene substances.(5) q RT-PCR was used to detected other gene expression of MVA pathway and MEP pathway in Nt DXR and Nt HMGR1 transgenic plants, the results show that: the expression of HMGR1,HMGR2,MK which from MVA pathway reduced for Nt DXR overexpression transgenic tobacco lines compared with the control. Inversely, the expression of HMGR1, HMGR2, MK and PMD increased in Nt DXR knockout transgenic plants. The expression of genes of DXR and HDR which from MEP pathway in Nt HMGR1 overexpression transgenic tobacco lines decreased. Conversely, the expression of DXR,HDS and HDR increased in Nt HMGR1 konckout transgenic tobacco lines. This result suggest that MVA pathway and MEP pathway may have an inhibitory effect on each other.