| Camellia gymnogyna Chang belongs to the family of Genaceae Camellia Linn,Camellia Sect.Thea(L.)Dyer,and Ser.Gymmogynae Chang.It is mainly distributed in South China tea area and Yunnan-Guizhou plateau area.Studying the biochemical quality and regulation of secondary metabolite expression are helpful to understand the metabolic characteristics and genetic influence of Camellia gymnogyna Chang,and it will has very important scientific and practical significance.Based on these situation,We have used the methods of ultraviolet spectrophotometry and high performance liquid chromatography(HPLC)to analyzed the constituents and contents of purine alkaloids in different leaf positions of Camellia gymnogyna Chang,and also inclued tea polyphenols,catechin monomers,free amino acids,flavonoids,theanine,et al.,At the same time,we compared the tea plants(Camellia sinensis)with Camellia gymnogyna Chang,and used high-throughput sequencing technology to construct the differential expression data of the purine alkaloids metabolism pathway in the tea tree transcriptome,combined with real-time fluorescence quantification(q RT-PCR)and gene cloning of key genes.In addition,we also researched on the subcellular localization,protein expression(Western-blot),isolation and purification of enzyme protein,and enzymatic properties of the key protein of caffeine synthase(CS)in the purine alkaloid synthesis pathway,the main purpose were to explore the biochemical quality characteristics of the Camellia gymnogyna Chang and the molecular mechanism of the synthesis of purine alkaloids.The research results of the thesis are as follows:1.Quality components of Camellia gymnogyna ChangThe composition and ratio of purine alkaloids in Camellia gymnogyna Chang were significantly different from those in tea plants(Camellia sinensis).There were three components of theobromine,caffeine and theacrine,and the content of theobromine was 13.46~39.72 mg·g-1,the content of caffeine was 0.51~2.02 mg·g-1(the lowest),and the content of theacrine was intermediate and increases with the maturity of the leaves,while the Camellia sinensis only contained caffeine and theobromine,and the content changes were 22.22~53.13 mg·g-1 and 0.47~12.82 mg·g-1,respectively.In the same leaf position,the content of tea catechins monomer in the Camellia gymnogyna Chang was EGCG>C>ECG>EGC>EC>GC>CG>GCG,and the total catechin content and ester catechin content were lower than Camellia sinensis,and total contents of not ester catechins was close to 40~50 mg·g-1.Except for the change of flavonoid content in each leaf position,the changes of other quality components basically conformed to the first leaf > bud > the second leaf > the third leaf > the fourth leaf,and the bud and two leaf was between the first leaf and the second leaf,and the other quality components such as tea polyphenols,flavonoids,theanine were lower than the Camellia sinensis.2.Transcriptome sequencing analysisThe transcriptome sequencing analysis was carried out on two different alkaloid metabolites of Camellia gymnogyna Chang and Yinghong 9.After sequencing and filtering,clean reads obtained 327,505 unigenes by de novo assembly,and 215,450 pairs were compared with public databases.Unigene was annotated,accounting for approximately 65.79% of the total,among them,in the GO function annotation,the catalytic activity and binding in molecular function were the most commented.In the KEGG annotation,the genes involved in singal transduction were the most,followed by carbohydrate metabolism,et al.,at the same time,we obtained a total of 9778 up-regulated expression and 5575 down-regulated genes,which focused on the purine nucleotide pathway and the major synthetic pathways of alkaloids,and obtained some differential genes for further study of purine alkaloid anabolism-related genes.In order to explore the mining and functional verification of purine alkaloid anabolism related genes,and a large amount of data was provided.3.Cloning and expression of key genes in the purine alkaloid pathwayAnalyzed of the Camellia gymnogyna Chang's Cgc CS gene using the NCBI ORF Finder contains a 1095 bp open reading frame(ORF)encoding 365 amino acids.The homology alignment of the amino acid sequence of the registered N-methyltransferase in Gene Bank revealed that the Cgc CS gene has the highest homology with Camellia irrawadiensis(BAE79729.1)of 98%,with Camellia sinensis(ABP98983.1)was 93%.Combined with transcriptome sequencing data,we used q RT-PCR to quantitatively analyzed the expression of SAMS,CS1,CS2,IMPDH,AMP and XMT genes in the pathway using the differences in the metabolism patterns of purine alkaloids from Camellia gymnogyna Chang,Yinghong 9 and Fenghuangdancong.The genes expression in the Camellia gymnogyna Chang were found to be significantly different from others tea leaves.There was a positive correlation between theobromine and the expression levels of SAMS,CS1,CS2,IMPDH,AMP and XMT.The correlation coefficient(R2)ranged from 0.047 to 0.874,and was significantly correlated with SAMS,CS1,CS2 and AMP genes(p<0.01);There was no significant correlation between theacrine and each gene,but the correlation coefficient with theobromine content was as high as 0.989(p<0.01),the correlation coefficient with caffeine was-0.876,which was extremely negatively correlated.The correlation coefficients of caffeine with SAMS and XMT genes were significantly positively correlated with 0.586 and 0.673,respectively,and were significantly positively correlated with AMP,while the correlation coefficients with CS1 and CS2 genes were 0.345 and 0.324,which showed a significant negative correlation with AMP gene.Caffeine was negatively correlated with theobromine and theacrine,and the correlation coefficients were-0.497(p<0.05)and-0.876(p<0.01),respectively.The fusion protein was prepared by using Cgc CS gene as template.By optimizing the induction conditions,the optimal expression conditions of the recombinant protein were as follows: 30 °C,4 h,the total protein,soluble protein,and inclusion body protein after induction showed an obvious foreign source band.The antibody was tested by ELISA and the titer was 1: 256000.The Western-blot assay showed that the antibody had relatively good specificity.The polyclonal antibody against Cgc CS was successfully prepared,and the first leaf and the fourth of 'Camellia gymnogyna Chang' were prepared.The expression of Western-blot expression in leaves showed that the expression of caffeine synthase protein in different leaf positions was different,and the relative protein expression in the first leaf position was higher than that in the fourth leaf position,these results were positively correlated with theobromine,caffeine,gene expression and total protein extraction.4.Subcellular localization of Cgc CS gene in Camellia gymnogyna ChangThe Cgc CS gene fragment was cloned by PCR from the fresh leaves of the Camellia gymnogyna Chang,the PCR product was ligated with the p MD20-T Vector and transferred into DH5? competent cells.Selected the correct positive clones and double-digested(Sma I and Bam H I),then p C1301-GFP plasmid form a subcellular localization fusion expression protein.The Agrobacterium tumefaciens was inoculated by vacuum infiltration,and the target gene and GFP fluorescent protein tag fusion protein were observed by laser confocal microscopy after inoculation.The transient expression in Nicotiana benthamiana showed localization in the nucleus.5.Isolation and purification of caffeine synthase from Camellia gymnogyna ChangUsed tissue homogenate extraction,ammonium sulfate,dialysis,Q-Sepharose Fast Flow strong-anion exchange chromatography,Sephadex G-75 gel filtration chromatography and ultrafiltration to separate and purify the caffeine synthase electrophoresis.The results showed that the stability of the caffeine synthase was poor in vitro,the order of the substrate affinity was theobromine > xanthosine > 7-dimethylxanthine,but no reaction to caffeine,and the Michaelis constant Km values ranged from 41 to 197 ?mol·L-1.There were significant differences in the recognition of key sites between caffeine synthase and methyl receptors from different sources.The optimal reaction time of the enzymewas 6 h,the optimum p H was 8.0,the optimum reaction temperature was 35 °C,and the relative enzyme activity was only 27.16 % after treatment at 60 °C for 20 min.The enzyme was completely inactivated with the further increase of temperature. |
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