Study On Triterpenoid Biosynthesis Regulation In Birch Saplings By MeJA And SA And The Key Enzyme Gene FPS Cloning

In the thesis, the triterpenoid synthetic physiological and biochemical characteristics of early response in3years old birch(Betula platyphylla Suk.) saplings by different concentrations of methyl jasmonate and salicylic acid treatment was explored, and the role of elicitor in birch specific organization of the triterpenoid key gene expression changes by real time quantitative PCR was detected. At the same time, full-length cDNA sequence of the birch triterpenoid biosynthesis pathway key enzyme gene FPS by homologous sequence RT-PCR method combined with RACE technology was got in birch suspension culture. The main results were as follows:The influence of the the birch physiological characteristics and photosynthetic capacity by MeJA and SA treatments.Different concentrations of MeJA and SA promoted the height growth of birch saplings, but diameter at breast height, diameter thickening were inhibited. Photosynthesis system of MeJA and SA in the induction of the medium-term (14d), the promotion of a birch sapling leaf photosynthetic indexes of Pn, Gs and Tr, and increased by varying degrees of birch saplings chlorophyll content, chlorophyll a and b content. The results show that the birch leaf photosynthetic capacity could improved though MeJA and SA treatment.The response of MeJA and SA treatment on birch resistant physiological indexes.(1)SOD, POD and CAT activity were significantly higher than those of the control in the early phase(12h、2d and7d), medium(14d) and late(28d)by MeJA treatment.(2)After SA treatment, the effect time of three kinds of enzymes are also significantly different, high concentrations of SA to make POD display high activity in2-7d, while CAT was significantly higher than those of the control after the two concentrations of SA treatment in14-28d.(3)There are two accumulation peak in1and14d by treatment for MDA content; under Elicitor treatment, SOD, POD and CAT complement and coordination each other scavenging active oxygen was a reason of increasing the resistance and the stimulation of secondary metabolite accumulation.(4)Birch leaf soluble sugar content of secondary metabolites first increased and then decreased slowly with induction time in MeJA and SA treatment. Soluble proteins accumulate a maximum of1.16mg/g by0.5mmol·L-1MeJA treatment on the5d.Birch leaf soluble protein content was higher than that of the control except for12h and2d, in the induction of5d reached the maximum by50mmol·L-1SA treatment. The results showed that MeJA and SA treatment could increased the content of soluble sugar and soluble protein content, it made a foundation for production of compound.The influence of birch triterpene content accumulation by MeJA and SA treatment. MeJA and SA treatment on birch saplings total triterpene accumulation induced in the early.(1)The different concentration of MeJA were promoted birch leaves total triterpene accumulation, the accumulation in the induction of the5d,0.5mmol·L-1MeJA reached a maximum of107.27mg/g, in the medium and late stage of induction, treatment group were higher than those in controls.1mmol·L-1MeJA disposed total triterpene accumulation of birch stems in the initial stage, triterpene content of stem bark reached maximum81.86mg/g on the1d of induction,146%higher than control.(2)In the50mmol·L-1SA processing section12h-5d, birch leaf total triterpene accumulation was higher than that of the control, and the12h reached the maximum90.72mg/g. Total triterpene accumulation of stems peaks in the5mmol·L-1SA processing section14d, content of91.4mg/g. Visible, MeJA and SA treatment on total triterpene accumulation of birch young leaves mainly manifested in the induction of early (12h-5d), while the stem bark of induced peak respectively in1d and14d.Birch triterpene biosynthesis pathway of key enzyme genes on MeJA and SA induced response. Fluorescence quantitative PCR techniquation was applicated for detecting the leaf and stem bark of birch saplings sesquiterpene synthase gene (FPS), cycloartenol synthase gene (BPX and BPX2), P-Amyrin synthase gene (BPY) and lupeol synthase gene(BPW) expression changes after MeJA and SA induction. The results show that,(1)in the leaves, FPS in MeJA and SA processing at different time were induced upregulation expression,1mmol·L-1, MeJA and50mmol·L-1SA was the best treatment. BPX, BPX2were induced upregulation of expression in early12h, and in the Id and14d were significantly downregulated expression by MeJA and SA treatment. BPY upregulates the expression on the0.5mmol·L-1MeJA in1d and3d, BPW in early12h, the genes upregulation significantly,and downregulated at the rest of the time. BPY of leaves in12h-3d upregulates expression, and are downregulated expression by SA treatment.(2)The stem bark, FPS significantly increased through MeJA and SA treatment in1～14d induced and decreases with increasing concentration, the more obvious effect. BPX gene of Stem bark after MeJA and SA treated, showed1～14d significantly inhibited, but BPX2gene were upregulated expression. BPY and BPW of stem bark with MeJA and SA induced were expressed, expect reduce the expression at12h,1～14d were upregulated expression.(3)SA and MeJA processing FPS of birch leaves and stem bark upregulates expression was one of the main factors of total triterpene content increased in the corresponding tissue, and under SA and MeJA treatment on1～14d, BPX gene of stem bark significantly downregulated and BPY and BPW reduced significantly upregulated expression will facilitate oleanotic acid and betulinic the efficient synthesis of alcohols.FPS gene has cloned and analysised of sequence in the birch triterpenoid biosynthetic pathway. Birch suspension cells as material, through homologous sequence RT-PCR method combined with RACE technology cloned and obtained firstly birch farnesyl pyrophosphate synthase FPS cDNA full-length sequence. Bioinformatics analysis showed, birch FPS gene cDNA sequence total length was1357bp, contains a1029bp open reading frame encoding342amino acid residues, the deduced protein molecular weight39.59kDa. Protein secondary structure is mainly composed of a-helix, and hydrophilic proteins. FPS amino acid sequence contains a30-340isoprene synthase conserved region of Trans-IPPS-HT. Protein multiple sequence alignment showed that the Cyclocarya paliurus, Panax ginseng and Panax notoginseng FPS amino acid sequence homology in more than80%, phylogenetic tree analysis showed the closest relationship with the woody plants Cyclocarya paliurus, and herbaceous plants Arabidopsis thaliana and other distantly related.