Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T.Chang (Theaceae) from Dayao Mountain

• Published in: Phytochemistry (Oxford) Vol. 199; p. 113167
• Main Authors: Zhou, Meng-zhen, O'Neill Rothenberg, Dylan, Zeng, Wen, Luo, Li, Yan, Chang-yu, Zeng, Zhen, Huang, Ya-hui
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 01.07.2022
• Subjects: Camellia gymnogyna; Cloning and expression; N-methyltransferase genes; Purine alkaloids; Theaceae; Cloning and expression; Cf; Purine alkaloids; Px; 7-mXR; Camellia gymnogyna; Tb; Tc; 1-mX; SDS-PAGE; 3-mX; 7-mX; X; N-methyltransferase genes; KD; XR; SAH; Tp; SAM; PCR; HPLC; Theaceae
• ISSN: 0031-9422; 1873-3700
• DOI: 10.1016/j.phytochem.2022.113167

In the present study, purine alkaloid analysis and transcriptome of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain were performed by high-performance liquid chromatography (HPLC) and RNA-Seq, respectively. The results showed that the major purine alkaloids accumulated in Camellia gymnogyna Hung T. Chang (Theaceae) were theobromine together with a small amount of theacrine and caffeine. Through polymerase chain reaction (PCR), three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Hung T. Chang (Theaceae) and designated GCS1, GCS2, and GCS3. We subsequently expressed GCS1, GCS2, and GCS3 in Escherichia coli and incubated lysates of the bacterial cells with a variety of xanthine substrates in the presence of S-adenosyl-L-methionine as the methyl donor. We found that the recombinant GCS1 proteins catalyzed 1,3,7-trimethyluric acid to produce theacrine, the recombinant GCS3 proteins catalyzed 7-methylxanthine to produce theobromine, while the recombinant GCS2 proteins did not catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Hung T. Chang (Theaceae) and other tea plants provided a reference for further research on the functions of these genes. The transcriptomes of Camellia gymnogyna Chang were analyzed by RNA-Seq. Through transcriptome analysis and PCR, three types of cDNA encoding N-methyltransferases were isolated from the leaves of Camellia gymnogyna Chang and designated GCS1, GCS2, and GCS3. The recombinant GCS1 protein catalyzed 1,3,7-trimethyluric acid to produce theacrine, recombinant GCS3 protein catalyzed 7-methylxanthine to produce theobromine, while recombinant GCS2 protein did no catalyze any xanthine derivatives. Simultaneous analysis of the expressions of GCS1, GCS2, GCS3, and a caffeine synthase gene (TCS1) in Camellia gymnogyna Chang and other tea plants provided a reference for further research on the functions of these genes. N-methyltransferase gene expression patterns in different leaf positions of C. gymnogyna Chang and other tea plants. [Display omitted] •Discovery and Biochemical Characterization of N-methyltransferase Genes Involved in Purine Alkaloid Biosynthetic Pathway of Camellia gymnogyna Hung T. Chang (Theaceae) from Dayao Mountain.•Simultaneous analysis of the expression levels of GCS1, GCS2, GCS3 and TCS1 in Camellia gymnogyna Hung T. Chang (Theaceae) and other three tea plants provides a reference for further research on the functions of these genes.