Scientia Agricultura Sinica ›› 2019, Vol. 52 ›› Issue (10): 1772-1783.doi: 10.3864/j.issn.0578-1752.2019.10.010

• HORTICULTURE • Previous Articles     Next Articles

Screening, Cloning and Functional Research of the Rare Allelic Variation of Caffeine Synthase Gene (TCS1g) in Tea Plants

LIU YuFei1,2,JIN JiQiang1,YAO MingZhe1(),CHEN Liang1()   

  1. 1 Tea Research Institute of the Chinese Academy of Agricultural Sciences/Key Laboratory of Tea Biology and Resource Utilization, Ministry of Agriculture and Rural Affairs, Hangzhou 310008
    2 Graduate School of Chinese Academy of Agricultural Sciences, Beijing 100081
  • Received:2018-12-04 Accepted:2019-02-25 Online:2019-05-16 Published:2019-05-23
  • Contact: MingZhe YAO,Liang CHEN E-mail:yaomz@tricaas.com;liangchen@tricaas.com

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Abstract:

【Objective】 Tea caffeine synthase 1 (TCS1), a key enzyme in the caffeine biosynthesis pathway, shows a wide range of allelic variation within Camellia sect. Thea germplasm. Discovery of the specific alleles of TCS1 as the genetic basis of natural variation in caffeine levels will increase the understanding of the mechanism of caffeine synthesis and accumulation, and provide new genetic resources for improving caffeine content in tea plant. 【Method】 An unique PCR primer set, namely TCS1P InDel F/R, was used to detect TCS1 alleles in 673 accessions of tea germplasm. The primer set (TCS1cDNAF/R) was used to clone the full-length cDNA sequence of novel allele, whose function was subsequently validated by bioinformatics quantitative real-time PCR (qRT-PCR) and prokaryotic expression analysis. 【Result】 The novel rare allele, TCS1g, was identified in the several germplasm of C. taliensis. The TCS1g sequence was obtained by using the accession ‘LL17’, which contained both TCS1a and TCS1g. The CDS of TCS1g was 1098 bp, encoding 365 amino acids with a calculated molecular weight of 40.9 kD and a theoretical isoelectric point 5.1. The sequence similarities between TCS1g and TCS1a, TCS1b, TCS1c, TCS1d, TCS1e, TCS1f ranged from 94.1% to 99.2%. The TCS1g showed a high level (>96%) of amino acid sequence identity with the alleles (TCS1b and TCS1c) which had only theobromine synthase (TS) activity, while it was slightly lower for other alleles (TCS1a, TCS1d, TCS1e and TCS1f ) having both TS and caffeine synthase (CS) activity. The amino acid residue in position 221, located at the active center motif of TCS1, was histidine (His) in the TCS1g, as well as TCS1b and TCS1c, however it was arginine (Arg) for the others. The mutation (His to Arg) would change the isoelectric point and hydrophilicity from 5.05 to 5.06, and from -0.119 to -0.123, respectively. Meanwhile, the 5' upstream regulatory region of TCS1g, TCS1b and TCS1c was 15 bp longer than that of TCS1a, TCS1d, TCS1e and TCS1f. The results of prokaryotic expression analysis indicated that TCS1g had only TS activity (44.3 pkat/mg), and qRT-PCR analysis showed the TCS1g was expressed in the ‘LL17’. The contents of caffeine and theobromine in the ‘LL17’ were 40.3 mg·g -1and 5.4 mg·g -1, respectively. 【Conclusion】 A novel rare allele of TCS1 (TCS1g) was cloned, and the expression was detected in the ‘LL17’. TCS1g had TS activity, but no CS activity, which might be caused by the change of amino acid residue in position 221.

Key words: allelic variation, caffeine synthase, functional analysis, purine alkaloids, tea plant

Fig. 1

The major biosynthetic pathway of caffeine synthesis (1) 7-methylxanthosine synthase (xanthosine N-methyltransferase); (2) N-methylnucleosidase; (3) theobromine synthase (monomethylxanthine N-methyltransferase; TS): ICS1 (TCS1b), PCS1 (TCS1c); (4) caffeine synthase (dimethylxanthine N-methyltransferase; CS); (3-4) dual-functional caffeine synthase: TCS1a, TCS1d, TCS1e, TCS1f. SAM, S-adenosyl-Lmethionine; SAH, S-adenosyl-L-homocysteine. The picture refers to references [10-12]"

Table 1

Primer sequences used in study"

引物名称 Primer name 引物序列Primer sequence (5′-3′)
TCS1P InDel F TATGTCATGTTTCTATTATTT
TCS1P InDel R TACTTTCTCCTTCTCCTCTGT
TCS1cDNAF CACTGCTGTGGCAGCTGGC
TCS1cDNAR CAACTTCTCATTTCTCCCAAC
TCS1gF CGGGATCCATGGGGAAGGTGAACGAAG
TCS1gR CGGAATTCCTATCCAACAATCTTGGAA
18S-F TCTCAACCATAAACGATGCCGACCAG
18S-R TTTCAGCCTTGCGACCATACTCCC
TCS1-CS-F TTACTTTTCTGACGAGGCA
TCS1-CS-R TGCGTTAAGAGCTTGAAGG
TCS1-TS-F ATTACTTTTCTCCGACGAGGCG
TCS1-TS-R TGCAGTAAGAGCTTGAAGAA
TCS1-Total-F AGCTGGCCTCTTTGCYATAA
TCS1-Total-R ACTAYTTTCTCCTTCTCCTC

Fig. 2

Comparison of allelic variations in 5’-untranslated region of TCS1g and other six allelic variants of TCS1"

Table 2

Coding region nucleotide (upper portion of matrix) and amino acid (bottom portion of matrix) sequence comparison (% similarity) between TCS1g and other six allelic variants of TCS1"

TCS1a TCS1b TCS1c TCS1d TCS1e TCS1f TCS1g
TCS1a - 94.6 94.1 97.4 96.4 97.8 95.3
TCS1b 91.0 - 99.2 94.5 95.4 95.3 98.9
TCS1c 89.9 98.6 - 94.1 94.7 94.7 98. 5
TCS1d 94.6 89.6 88.2 - 96.8 98.8 95.4
TCS1e 93.8 91.2 89.9 93.5 - 97.4 96.0
TCS1f 95.4 91.0 89.6 97.3 95.4 - 96.0
TCS1g 92.1 98.1 96.7 91.0 92.3 92.3 -

Fig. 3

Phylogenetic tree of N-methyltransferase CDS sequences (related to caffeine synthesis) from Camellia, Coffea and Theobroma The hollow triangles, hollow circles and hollow squares mark N-methyltransferase genes of Camellia, Coffea, and Theobroma, respectively. TCS1g is highlighted by bold"

Fig. 4

Comparison of amino acid sequences of TCS1g and other six allelic variants of TCS1 The proposed SAM-binding motifs (A, B’, and C) and conserved region that is nominated as ‘‘YFFF-region’’ are shown by red open boxes [15,28-29]. The amino acid residues indicated by a blue open box play a critical role in substrate recognition [15,17,30]. The TCS1b, TCS1c and TCS1 g variant sites compared to TCS1a, TCS1d, TCS1e and TCS1f are indicated by a brownish yellow triangle"

Fig. 5

Predicted tertiary structure homology-modeling of TCS1g gene In the interval 139-287, amino acid residues different from TCS1b, TCS1c, TCS1g and TCS1a, TCS1d, TCS1e, TCS1f are indicated in yellow. Green and red are labeled as amino acid residues that bind to the methyl acceptor and the methyl donor, respectively"

Table 3

Comparison of the activity and substrate selectivity of TCS1g recombinant enzymes"

重组蛋白
Recombinant enzyme		 底物/甲基化位置
Substrate/Methylation position		 Tb/7-mX
(%)
7-mX/3-N
(pkat/mg)		 Tb/1-N
(pkat/mg)
TCS1a 111.9±5.0 27.1±0.1 24.2±1.1
TCS1c 17.9±0.9 ND 0
TCS1g 44.3±0.3 ND 0

Fig. 6

Real-time PCR was used to analyze TCS1g specific expression TCS1-CS, TCS1-TS, and TCS1-Total are the expression levels of allelic variation encoding proteins with both theobromine synthase (TS) and caffeine synthase (CS) activities, only with TS activities, and total TCS1, respectively"

Table 4

Purine alkaloid contents of LL17 containing rare TCS1g (mg·g-1)"

资源
Germplasm		 学名
Scientific name		 等位变异
TCS1 allelic variation		 可可碱
Theobromine (mg·g-1)		 咖啡碱
Caffeine (mg·g-1)
LJ43 Camellia sinensis TCS1a 1.8±0.0 31.2±0.1
CCT C. ptilophylla TCS1c 56.3±0.2 ND
LL17 C. taliensis TCS1a、TCS1g 5.4±0.1 40.3±0.3
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