Clonging Analyses Of GGPS And Biosynthetic Pathway Analysis Of Double Ester Type Alkaloidin Aconitum Carmichaelii Debx.

Fuzi(Monkshood)is the sub root processed products of Ranunculaceae plant Wuiou(Aconitum carmichaelii Debx.).with a lot of effects such as return Yang and save the inverse, fill the fire and help the Yang, dispel pathogenic cold and relieving pain.It has a long history with medicinal and artificial cultivation, it is often used in collapse due to depletion of Yang, cold limbs vein, deficiency of vomiting and diarrhea and abdominal cold abdominal pain like, but it also with a strong toxicity.Monkshood has great value in medicinal, although it has a long time in cultivation history, but the traditional mode of production that "mountain breeding, the dam cultivation" is still used in the current production. cultivated area per year introduction of provenance are not fixed, resulting in Provenances of confusion and instability. So far, there is no a new varieties that effective composition containing significantly increased in the cultivation.The main bioactive components of Monkshood is Aconitum alkaloids, belongs to ditepenoid alkaloids in the classification of alkaloids.In this research, Fuzi geranyl geranyl diphosphate synthase (GGPS) gene which is the key enzymes in the biosynthesis of ditepenoid alkaloids gene was cloned and tissue expression analysised, at the same time, the main medicinal activity of aconitine:diester alkaloids (mesaconitine, aconitine, hypaconitine) in the different growth period and plant tissue was tested through the HPLC, predicted the function of the GGPS gene, to investigate the effect of GGPS gene in plants for aconite aconite aconitine biosynthesis, lay a solid foundation for studying the aconitine biosynthetic pathway, provides a way for by means of biological technology to cultivate new varieties with high alkali content of aconite aconite. The following results were obtained in this study:1. Cloning of GGPS gene:aconite first by homologous cloning from aconite root tissue culture material, combined with the RACE transcriptome and cloned a 1420bp long sequence of cDNA, NCBI and its homologous alignment showed Salvia species such as tobacco GGPS gene homology, and Adonis aestivalis homology the highest, up to 80%; ORF Finder showed that the CDS region of 861bp, encoding 287 amino acids; homologous sequence multiple alignment and phylogenetic analysis showed that the GGPS gene has shared twe polyisoprene synthase specific sequences "LMHDDLPCMDNDDLRRG" and "IGLLFQVVDDILD", and all the 5 conserved domain of GGPS protein; prediction the molecular weight was 30.95kDa, isoelectric point 5.24; subcellular localization prediction showed that the protein mainly expressed in chloroplast, mitochondria and other sites also have a small amount of expression Multi dimensional structure analysis showed that there was no regular curl in its structure, which accounted for 30.56%, alpha helix accounted for 65.28%, and beta folding was 4.17%. The structure comparison showed that the 3D structure was highly conservative.2. The GGPS gene expression in different growth periods:fluorescence quantitative PCR data of all the materials for the longitudinal comparison of different growth periods, the results show, different growth periods of plant gene has different degrees of expression. Specific to the plant tissue, different plant tissues differ in different periods of expression, expression peak period of Inter Organizational differences, but overall, its expression quantity and plant life of metabolic activity intensity has obvious positive correlation.3. The expression of GGPS gene in different tissues:the GGPS gene was expressed in all tissues, but the expression quantity was different. In the vigorous growth period between organizations expressed as root> leaf> stem> root.4. The effective components in different tissues of content:in different tissue samples of each batch were detected in aconitine, in plants of aconite aconitine site of synthesis is not limited to the roots, but all tissues of the plant are synthetic, but the content is not the same, from the point of view of the results of this study, the plants among different organizations, the content of aconitine level followed by fibrous root> root> root> root> leaf> stem.5. The active ingredient in different growth periods of content:in different growth period Fuzi aconitine content detection results in different tissues in different content in different growth period and linear relationship is not the same, in leaves and roots of the content with time was first increased and then decreased trend, stem content of aconitine in has been increased, and fibrous roots in the content of aconitine with time for a little change.6. The relationship between GGPS gene and aconitine biosynthesis:GGPS gene expression and the content of aconitine in analysis, it is found that the aconitine synthesis and gene expression is highly consistent in the different parts of the plants, and the expression intensity with the time trend and the content of aconitine dynamic changes with time is consistent with the trend, indicating that the aconitine synthesis and GGPS gene expression is closely related with other reported studies can come to a conclusion:Fuzi GGPS gene aconite alkaloid biosynthetic pathway key enzyme genes and pathways for the biosynthesis of aconitine for from diterpenes and diterpenoid alkaloids.