| Caffeine is an important secondary metabolite of tea plant (Camellia sinensis), whosecontent is affected by multiple factors during its synthesis. In the present study,a series ofexperiments on different tea plant varieties were carried out to investigate the followingfactors that influence content of caffeine: theobromine content by the high performanceliquid chromatography (HPLC), cSNPs by direct sequencing method and gene relativeexpressions by real-time fluorescent quantitative PCR (qRT-PCR) of three key enzymegenes (TCS1, TIDH, and SAMS). In summary a systematic study of the influence factors ofcaffeine biosynthesis of tea plant varieties grown under natural conditions were carried outand provided theoretical and practical guidance for tea plant molecular marker assistedbreeding. Conclusions are as follows:1. HPLC method was used to measure the caffeine and theobromine contents ofdifferent varieties of tea plant. The caffeine contents of the25tea plant varieties rangedfrom2.74%to5.28%with the variation of14.37%. Theobromine content ranged from0.04%to0.34%with the variation of36.37%. Significant differences were observed in thecontents of theobromine and caffeine among different tea plant varieties (P <0.01).However, the difference was greater for theobromine content than that of caffeine.Regression analysis showed that the correlation coefficient of theobromine and caffeinecontents was0.72, and there existed a significant positive correlation (P <0.01), whichindicated that the precursor theobromine contents had significant influence on thebiosynthesis of caffeine in tea plants.2. In spring, the TCS1gene relative expressions of10tea varieties were between0.54and1.78, with the range of1.24, and with the variation coefficient of24.13%; TIDH werebetween0.49and1.02, with the range of0.52, and with the variation coefficient of21.84%;SAMS were between0.43and1.38, with the range of0.95, and with the variationcoefficient of26.20%. Then in autumn, TCS1gene relative expressions were between1.00and1.94, with range of0.94, and with the variation coefficient of24.82%; TIDH werebetween0.57and1.37, with range of0.80, and with the variation coefficient of23.97%;SAMS were between0.61and1.49, with range of0.88, and with the variation coefficient of30.31%. The correlation analysis between the gene expressions and the contents of caffeineand theobromine indicated that TCS1were significantly and positively correlated only tothe caffeine contents but not with the theobromine contents. But TIDH and SAMS had no significant correlation with the caffeine and theobromine contents.3. TIDH and SAMS, their lengths of the open reading frame were1512bp and1182bp,encoding504and393amino acids, respectively. The predicting molecular weights were53.5kDa and53.5kDa, and theoretical isoelectric point values were5.9and5.8,respectively. The two genes both belonged to the hydrophilic protein, and existed neithersignal peptide nor transmembrane structure. TIDH and SAMS had8,7cSNP sites,respectively, whose cSNP frequency were1/189bp and1/169bp, respectively.Meanwhile the cSNPs were all biallelic, but some cSNP sites within the same tea plantvarieties presented heterozygous alleles. The numbers of nonsynonymous mutation cSNPswere6and4, respectively, the rest were synonymous mutation cSNPs. These two genesalso had3and4transition cSNPs, respectively, in addition to the transversion cSNPs.There were no insertion/deletion mutations. The associated analysis between genotypeand caffeine contents were carried out, the result was that TIDH T573A and SAMS C393Twere found to be significantly correlated to the caffeine contents (P <0.05). Therefore, itcan be presumed that there existed closely hereditary linkage mark between TIDH andSAMS to tea plant caffeine content.Therefore, it could be concluded from the study findings that the following threefactors had significant influence on the biosynthesis of caffeine in tea plants: the precursortheobromine content; cSNPs in TCS1, TIDH, and SAMS; and TCS1expression level. |
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