Cloning Of AMP Deaminase Gene And CSNP Analysis Of TCS1in Tea Plant (Camellia Sinensis)

Caffeine is one of the most important purine alkaloids and it is secondarymetabolites in tea plant, as it plays an important role in the flavor of tea. It’s ofsignificance to study the metabolic pathway of caffeine.Adenine nucleotide pathway was the main way of caffeine biosynthesis, the keyenzymes were AMP deaminase, TCS, IMPDH and SAM synthetase, except AMPdeaminase, the other three genes of enzyme have been cloned; the cloning of AMPdeaminase gene was still in a blank state. TCS was a key enzyme that catalyzed thefinal two methylation and regulation of caffeine biosynthesis.This experiment mainly had two aspects to determine, one was to screen an ESTfrom the whole organic transcriptomic library of tea, which has high homology withAMP deaminase gene from other organisms, and amplified through RACE technologyto obtain the cDNA full-length of AMP deaminase gene, biological information ofamino acid encoded by AMP deaminase gene was predicted and analysed. Another,the content of caffeine in leaves of different tea plant cultivars was investigated byHPLC, and cSNPs of TCS1was detected by direct sequencing, and the correlationanalysis of phenotype and genotype was carried out to select the cSNPs, which hadsignificant association with the caffeine content. It will lay a foundation for geneticimprovement of caffeine content in tea. The main results are as follow:1. The full length cDNA of AMP deaminase gene has been cloned. The cDNAfull-length was3215bp with a single2571bp opening reading frame that predicted toencode856amino acids, and its3′untranslated region has an obvious polyadenylationsignal. The deduced protein molecular weight was97.6kD and its theoreticalisoelectric point was6.31. The gene has been submitted to the GenBank and theaccession number was AGJ84350.1. Multiple sequence alignment showed that theAMP deaminase gene has a number of highly conserved regions in its3′end; thephylogenetic tree indicated that AMP deaminase gene closed to dicotyledon.2.7cSNPs were found in TCS1, including5transitions (2A/G and3C/T),2transversions (1C/G and1A/C), and no insertion/deletion mutations; there also existed5nonsynonymous mutation cSNPs and2synonymous mutation cSNPs.3. The linkage disequilibrium analysis showed that there were21r~2values;5highly significant linkage disequilibriums existed in TCS1. Besides,385and898cSNP locus in a state of complete linkage disequilibrium (r~2=1). On the basis of linkage cSNPs disequilibrium7haplotypes were detected.4. The association analysis between cSNPs and caffeine content showed that onlythe caffeine content of AA and CC genotype was significantly correlated in995cSNPlocus.