Caffeine is an important bioactive compound in tea plant, and N-methyltransferase (NMT) is the key enzyme for its biosynthesis. In this research, the promoter of NMT1 was isolated from Camellia sinesis Yinghong 9 by hiTAIL-PCR, and its cis-elements were analyzed by Plant CARE and other online software. According to cis-elements composition, serial 5' shorten promoter were re-amplified from NMT1 promoter and cloned into pBI121 vector fusion with GUS gene respectively. The constructed vectors then transformed into tobaccos. The function of cloned promoter and its response to environmental factors were analyzed by GUS staining and quantitative PCR. The results showed, the isolated NMT1 gene promoter was 767βbp, containing basic cis-acting elements such as TATA-box, CAAT-box and some other response elements involved in the plant abiotic stress. Using recombinant technique, the CaMV35S promoter in pBI121 was replaced by the 5' shorten NMT1 promoters, resulted in 4 new vectors fusion with GUS and marked as pA, pB, pC and pD. The transient expression with the four constructed vectors in tobacco leaves revealed that NMT1 promoters with different lengths all could regulate the expression of GUS, and the activity of GUS enhanced along with promoter length increased. The pA vector which owned full cloned NMT1 promoter was selected for transgenosis and successfully gained transgenic tobacco. Expression of GUS could be detected in different tissues of transgenic tobacco, The expression level followed leaf > stem > root and the expression level in leaves reached 3 times higher than that in roots. When transgenic tobacco treated with light, temperature, simulated drought and abscisic acid, there were significant changes in the expression of GUS in leaves, except at the temperature of 40℃. The results showed that the function of NMT1 promoter was influenced by the environmental stresses.
LIU Ping
,
REN Qiujing
,
KANG Xin
,
ZHANG Yuanyuan
,
LIN Xiaorong
,
LI Bin
,
GAO Xiong
,
CHEN Zhongzheng
. Isolation and Functional Analysis of Promoter for N-methyltransferase Gene Associated with Caffeine Biosynthesis in Tea Plants (Camellia sinensis)[J]. Journal of Tea Science, 2018
, 38(6)
: 569
-579
.
DOI: 10.13305/j.cnki.jts.2018.06.003
[1] 宛晓春. 茶叶生物化学[M]. 北京: 中国农业出版社, 2003.
[2] 吴命燕, 范方媛, 梁月荣, 等. 咖啡碱的生理功能及其作用机制[J]. 茶叶科学, 2010, 30(4): 235-242.
[3] Ashihara H, Sano H, Crozier A.Caffeine and related purine alkaloids: biosynthesis, catabolism, function and genetic engineering[J]. Phytochemistry, 2008, 69(4): 841-856.
[4] Moisyadi S, Neupane K R, Stiles J I.Cloning and characterization of a cDNA encoding xanthosine-N-7-methyltransferase from coffee (Coffea arabica)[J]. Acta Hortic, 1998, 461(42): 367-378.
[5] Kato M, Mizuno K.Caffeine synthase gene from tea leaves[J]. Nature, 2000, 6799(406): 956-957.
[6] Yoneyama N, Morimoto H, Ye C, et al.Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme[J]. Molecular Genetics and Genomics, 2006, 275(2): 125-135.
[7] 金基强, 姚明哲, 马春雷, 等. 合成茶树咖啡碱相关的N-甲基转移酶基因家族的克隆及序列分析[J]. 茶叶科学, 2014, 34(2): 188-194.
[8] 许煜华. 茶叶咖啡碱合成N-甲基转移酶基因克隆和功能鉴定[D]. 广州: 华南农业大学, 2011.
[9] Ross E J H, Stone J M, Elowsky C G, et al. Activation of the Oryza sativa non-symbiotic haemoglobin-2 promoter by the cytokinin regulated transcription factor, ARR1[J]. Journal of Experimental Botany, 2004, 55(403): 1721-1731.
[10] Kasuga M, Miura S, Shinozaki K, et al.A combination of the Arabidopsis DREB1A gene and stress-inducible rd29A promoter improved drought- and low-temperature stress tolerance in tobacco by gene transfer[J]. Plant Cell Physiol, 2004, 45(3): 346-350.
[11] Marraccini P, Courjault C, Caillet V, et al.Rubisco small subunit of Coffea arabica: cDNA sequence, gene cloning and promoter analysis in transgenic tobacco plants[J]. Plant Physiology and Biochemistry, 2003, 41(1): 17-25.
[12] Satyanarayana K V, Kumar V, Chandrashekar A, et al.Isolation of promoter for N-methyltransferase gene associated with caffeine biosynthesis in Coffea canephora[J]. Journal of Biotechnology, 2005, 119(1): 20-25.
[13] Tai Y, Wang H, Wei C, et al. Construction and characterization of a bacterial artificial chromosome library for Camellia sinensis [J]. Tree Genetics & Genomes, 2017, 13: 89. https://doi.org/10.1007/s11295-017-1173-5.
[14] 陈小芳, 许煜华, 陈忠正, 等. 英红9号茶树两可可碱合成酶的亚细胞定位和互作分析[J]. 中国食品学报, 2016, 16(10): 10-18.
[15] Liu Y G, Chen Y.High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences[J]. BioTechniques, 2007, 43(5): 649-656.
[16] 吴英杰, 姜波, 张岩, 等. 农杆菌介导的烟草瞬时表达试验条件优化[J]. 东北林业大学学报, 2010, 38(9): 110-112.
[17] Jefferson R A, Kavanagh T A, Bevan M W.GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants[J]. The EMBO J, 1987, 6(13): 3901-3907.
[18] Mark D. Burow C A.High-frequency generation of transgenic tobacco plants after modified leaf disk cocultivation with Agrobacterium tumefaciens[J]. Plant Molecular Biology Reporter, 1990, 8(2): 124-139.
[19] Yang Y, Costa A, Leonhardt N, et al.Isolation of a strong Arabidopsis guard cell promoter and its potential as a research tool[J]. Plant Methods, 2008, 4(46): 4-6.
[20] Cui X Y, Chen Z Y, Wu L, et al.rbcS SRS4 promoter from Glycine max and its expression activity in transgenic tobacco[J]. Genet. Mol. Res, 2015, 3(14): 7395-7405.
[21] Zhang X, Huang D, Jia X, et al.Functional analysis of the promoter of the molt-inhibiting hormone (mih) gene in mud crab Scylla paramamosain[J]. General and Comparative Endocrinology, 2017, 259(4): 131-140.
[22] Higo K, Ugawa Y, Iwamoto M, et al.Plant cis-acting regulatory DNA elements (PLACE) database: 1999[J]. Nucleic Acids Res, 1999, 27(1): 297-300.
[23] Rombauts S, Dehais P, Van Montagu M, et al.PlantCARE, a plant cis-acting regulatory element database[J]. Nucleic Acids Res, 1999, 27(1): 295-296.
[24] Matys V, Fricke E, Geffers R.Transfac: transcriptional regulation, from patterns to profiles[J]. Nucleic Acids Research, 2003, 31(1): 374-378.
[25] Moon H, Callahan A M.Developmental regulation of peach ACC oxidase promoter-GUS fusions in transgenic tomato fruits[J]. Journal of Experimental Botany, 2004, 55(402): 1519-1528.
[26] Nie L N, Xia L Q, Xu Z S, et al.Progress on cloning and functional study of plant gene promoters[J]. Journal of Plant Genetic Resources, 2008, 9(3): 385-391.
[27] 付永平, 周海涛, 王丕武. 大豆种子特异性启动子的克隆及功能分析[J]. 西北农林科技大学学报(自然科学版), 2009, 37(12): 105-111, 118.
[28] 林文秋. 菠萝AcSERKs启动子克隆及AcSERK1启动子活性的初步分析[D]. 广州: 华南农业大学, 2014.
[29] 敬帆, 罗登攀, 马婧, 等. 蜡梅CpAGL6基因启动子的克隆及功能初步分析[J]. 园艺学报, 2015, 42(6): 1139-1149.
[30] 曹华雯. 甘菊BADH基因启动子功能鉴定及诱导型启动子的分离[D]. 北京: 北京林业大学, 2010.
[31] Li Q, Xie J, Ma X, et al.Molecular cloning of phosphoethanolamine N-methyltransferase (PEAMT) gene and its promoter from the halophyte Suaeda liaotungensis and their response to salt stress[J]. Acta Physiologiae Plantarum, 2016, 38(2): 39-40.
浙公网安备 33019902000101号 
