| Lonicera macranthoides(Lonicera macranthoides Hand.-Mazz), also registered as “mountain honeysuckle”, is a medicinal herb that is widely distributed in southern China. Polyphenols are important secondary metabolites in L. macranthoides, of which, chlorogenic acid(CGA) is a core of biologically active substance. Hence, it's always desirable to increase the yield of CGA. Recently it there has became a hot topic in the study of CGA content,biosynthetic pathways and molecular mechanisms. There have been few reports on the molecular mechanisms of CGA biosynthesis in Lonicera plants, and the regulatory mechanisms underlying CGA biosynthesis remain a mystery in L. macranthoides. In this study, "Yu Lei 1#" was used as plant materials. Key genes and regulatory factors associated with CGA biosynthesis were identified by transcriptomics analysis. And the effects of different growing environments on the accumulation of polyphenols, especially CGA, were assessed. The main results were as follows:1. A detection methods for polyphenols determination including CGA, ferulic acid, coumaric acid, caffeic acid, cinnamic acid, rutin and luteoloside using high performance liquid chromatography(HPLC) were established based on optimization of sample preparation, mobile phase and detection wavelength in L. macranthoides. The contents of these polyphenols were analyzed in flowers at different developmental stages. And the results showed that CGA accounted for greatest proportion, followed by rutin and luteoloside.We also found that six compounds were detectable except for coumaric acid, and the contents of the total compounds were the highest in white flowers, while lowest in the young buds. In terms of CGA, the content was in highest level in young leaves followed by young stems. However, the contents were in rapid decline during tissue aging. Interestingly, the concentration of luteoloside was extremely higher in aging l e a v e s t h a n t h a t i n o t h e r t i s s u e s a t d i f f e r e n t d e v e l o p m e n t a l s t a g e s.2. CGA content was quantified by HPLC, and CGA levels differed significantly among three tissues. Specifically, the CGA content in young leaves(YL) was greater than that in young stems(YS), which was greater than that in mature fl owers(MF). Transcriptome analysis of L. macranthoides yielded a total of 53,533,014 clean reads and 76,453 unigenes. A total of 447 unigenes were involved in biosynthetic pathways of phenylpropanoids. Of these unigenes, 80 were possibly related to CGA biosynthesis. RNA-Seq showed that 6831 genes were differentially expressed between MF and YL, and 691 genes were differentially expressed among MF, YL and YS. In this study, two possible pathways associated with CGA biosynthesis were identified and key enzymes were screened, including phenylalanine ammonia lyase(PAL), 4-coumarate coenzyme A l i g a s e( 4 C L), c i n n a m a t e 4- H yd r o x yl a s e( C 4 H), h yd r o x yc i n n a m o yl transferase/hydroxycinnamoyl-Co A quinate transferase(HCT/HQT) and coumarate 3-hydroxylase(C3H).Furthermore, several genes including one PAL, three C4 H and five HCT / HQT were considered to be key genes involved in CGA biosynthesis based on the correlations between gene expression and CGA content. Also, 14 differentially expressed transcription factors including AP2, ERF, MYB, NAC, and zinc finger protein were indicated as potential regulatory factors associated with CGAbiosynthesis.3. This study successfully established the rapid reproduction technology of L. macranthoides, and optimization of genetic transformati on system of L. macranthoides mediated by agrobacterium was performed. Based on nucleotide sequences from transcriptome data, we isolated Lm HQT1 gene. Bioinformatics analysis showed that Lm HQT1 protein is highly homologous to Ns HQT and Sl HQT homologous. QRT-PCR results showed that Lm HQT1 displayed highest expression in young leaves, which was significantly higher than that in other tissues. And it expressed positively correlation between Lm HQT1 transcripts and CGA content in different tissues. To furthe r characterize the function of genes, Lm HQT1 overexpression vector were constructed and successfully transformed to L. macranthoides. It found that CGA content was i n c r e a s e d b y a b o u t 5 0 % i n t r a n s g e n i c p l a n t s.4. In this study, the effects of different light quality on plant growth and photosynthesis were evaluated.Results showed that the index of plant growth and photosynthesis including biomass, stem growth and several parameters(net photosynthetic rate, intercellular CO2 concentration, transpiration rate and stomatal conductance) reached the highest levels under red light, indicating that the red light positively regulates plant growth and photosynthesis. Furthermore, the effects of combinations of different hormones based on basic medium on plant growth and accumulation of secondary metabolites were assessed. And the optimal medium for growth was screened, that is MB medium+ 1.0mg / L BA + 1.0mg / L IBA, also did the optimal medium for the accumulation of secondary metabolites, that is MB medium+2.0mg / L BA + 0.2mg / L IBA. In addition, we investigated the effects of combined hormones and light on the accumulation of polyphenols. The results showed that the accumulations of phenolic compounds were promoted under red light on the best metabolic medium, but white light has opposite effects. Whereas, white and blue light promoted the accumulation of phenolic compounds on the best growth medium, but it was retarded under green light.In summary, the important genes and regulatory factors associated with CGA biosynthetic pathway by transcriptome analysis were identified. Fuctional characterization of HQT gene was performed based on the establishment of genetic transformation technology. The regulatory mechanism of different culture conditions on the accumulation of polyphenols, especially CGA, was explored. Our results provide important insights on biosynthetic pathways and regulatory mechanism of CGA in Lonicera macranthoides in this study. |
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