Prokaryotic Expression of Caffeine Synthase Gene(TCS1),Its Polyclonal Antibody Preparation and Identification

doi:10.7525/j.issn.1673-5102.2015.03.003

Abstract

Abstract: Cloing and prokaryotic expressing of TCS1(Caffeine synthase 1) gene, preparing the antibody are the preliminary works for further research of the expression of TCS1 in tea plant. According to the cDNA fulllength sequence of TCS1 from GenBank, we found out the complete ORF(Open Reading Frame). The open reading frame(ORF) of TCS1 gene from the cDNA of leaves of tea plant was amplified. The ORF was then ligated into the expression vector pGEX-4T-2. The recombinant protein was expressed by induction with IPTG. Then, we tested in vitro enzyme activity, and purified the recombinant protein subsequently by affinity chromatography. The TCS1 antibody was further refined by immunizing white rabbits with the purified protein. The antibody titer and specificity of the polyclonal antibody were confirmed by ELISA and Western blot. Induced by optimizing the conditions, the best expression condition of the recombinant protein was at 30℃ for 4 h. After induction of total protein, soluble protein and inclusion body protein showed an obvious foreign proteins stripes. ELISA detection of the antibody recommended dilution ratio of 1∶2 000. We verified the favourable specific of the antibodies by western blot. The prokaryotic expression plasmid pGEX-4T-2-TCS1 was constructed, and the prepared polyclonal antibody had a high titer and good specificity.

Key words: Camellia sinensis, caffeine, tea caffeine synthase, prokaryotic expression, polyclonal antibody

CLC Number:

DENG Wei-Wei；JIN Yang；LI Min；MA Lin-Long；ZHANG Zheng-Zhu. Prokaryotic Expression of Caffeine Synthase Gene(TCS1),Its Polyclonal Antibody Preparation and Identification[J]. Bulletin of Botanical Research, 2015, 35(3): 333-339.

References

1.宛晓春.茶叶生物化学［M］.中国农业出版社,2006.

2.Ashihara H,Shimizu H,Takeda Y,et al.Caffeine matabolism in high and low caffeine containing cultivars of Camellia sinensis［J］.Z.Naturforsch,1995,50:602-607.

3.Ashihara H.Crozier A.Caffeine:a well known but little mentioned compound in plant science［J］.Trends in Plant Science,2001,6(9):407-413.

4.阮宇成.茶叶咖啡碱与人体健康［J］.茶叶通讯,1997.

5.Smith A.Effects of caffeine on human behavior［J］.Food and Chemical Toxicology,2002(9):1243-1255.

6.Wen W Q,Shu X O,et al.The associations of maternal caffeine consumption and nausea with spontaneous abortion［J］.Epidemiology,200l,(1):38-42.

7.Kato A,Crozier A,Ashihara H.Subcellular localization of Nmethyltransferase involved in caffeine biosynthesis in tea［J］.Phytochemistry,1998,48(5):777-779.

8.Ashihara H,Gillies F M,et al.Metabolism of caffeine and related purine alkaloids in leaves of tea(Camellia sinensis L.)［J］.Plant Cell Physiology,1997,38(4):413-419.

9.Koshiish C,Kato A,et al.A new caffeine biosynthetic path way in tea leaves:utilization of adenosine released from the S-adenesyl-L-methionine cycle［J］.FEBS Letters,2001,449 (1-2):50-54.

10.Mosli Waldhauser S S.Compartmentation of caffeine and related purine alkaloids depends exclusively on the physical chemistry of their vacuolar complex formation with chlorogenic acids［J］.Phytochemistry,1996,42:985-996.

11.Kato M,Kanehara T,et al.Caffeine biosynthesis in young leaves of Camellia sinensis:In vitro studies on N-methyltransferase activity involved in the conversion of xanthosine to caffeine［J］.Physiologia Plantarom,1996,98:629-636.

12.Ashihara H,Monteiro A M,Gillies F M,et al.Biosynthesis of caffeine in leaves of coffee［J］.Plant Physiol,1996,111(3):747-753.

13.Kato M,Mizuno K.Caffeine synthase gene from tea leaves［J］.Nature,2000,406:956-957.

14.方堃，白丁平，陈玉林.大肠杆菌cysE和cysM基因的克隆、原核表达及多克隆抗体的制备［J］.西北农林科技大学学报，2011，39（11）：35-40.

15.谷瑞升,刘群录,陈雪梅.木本植物蛋白提取和SDS-PAGE分析方法的比较和优化［J］.植物学通报,1999,16(2):171-177.

[1]	Heming LI, Hongtao QIN, Deqiang WEI, Xu LI, Xingguo LAN. Prokaryotic Expression, Preparation Polyclonal Antibody and Protein Expression of BoMAPK4 of Ornamental Kale(Brassica oleracea var. acephala) [J]. Bulletin of Botanical Research, 2022, 42(4): 584-591.
[2]	LI Yang, SHI Ya-Kun, GAO Shi-Ke, Li Xiao-Yu, LAN Xing-Guo. Gene Cloning and Expression Analysis of PDIL1-2 of Ornamental Kale(Brassica oleracea var. acephala) [J]. Bulletin of Botanical Research, 2020, 40(2): 293-300.
[3]	LI Wen-Jing, FENG Yu, HOU Xiao-Qiang, Sun Yan-Xiang, Han Mei-Ling, LI Xiao-Xue, WANG Yong, XIANG Bei-Bei. Cloning, Bioinformatics and Expression of SmSLS2 Gene in Swertia mussotii [J]. Bulletin of Botanical Research, 2019, 39(3): 431-440.
[4]	ZHANG Ji, ZHANG Li-Li, ZHANG Bei-Bei, ZHANG Fu-Chun. Prokaryotic Expression of Halostachys caspica HcDMC1 and Preparation of Its Antiserum [J]. Bulletin of Botanical Research, 2018, 38(3): 399-405.
[5]	LIAO Xu, HE Ming-Liang, ZHANG Su-Yan, MA Hai-Yan, WANG Xu-Hui, LUO Qiu-Xiang, GUAN Qing-Jie. Prokaryotic Expression,Activity Assay of Chlorella Cyclophilin A and Salt Tolerance Analysis of Arabidopsis Over-expressing CsCyp1A [J]. Bulletin of Botanical Research, 2017, 37(5): 722-729.
[6]	CHEN Qing1；JIANG Lei-Yu1；WANG Yan1；ZHU Fang-Li1；TANG Hao-Ru1*；WANG Xiao-Rong1,2. Optimization of Prokaryotic Expression Factors for Blackberry UGT78H2 Using Responsive Surface Methodology and Protein Purification [J]. Bulletin of Botanical Research, 2015, 35(5): 724-729.
[7]	LI Shuang;ZHENG Tang-Chun;DAI Li-Juan;ZANG Li-Na;QU Guan-Zheng. Cloning and Prokaryotic Expression of PsnAP1-1 and PsnAP1-2Genes in Poplar(Populus simonii×Populus nigra*) [J]. Bulletin of Botanical Research, 2014, 34(4): 498-504.
[8]	DENG Wei-Wei;JIN Yang;YUAN Yan;ZHANG Zheng-Zhu. Profiles of Purine Metabolism and Purine Alkaloid Biosynthesis in Schima* and Eurya Plants [J]. Bulletin of Botanical Research, 2013, 33(4): 410-415.
[9]	WU Qi-Yao;WEI Chuan-Bao*;LI Jin-Hua. Prokaryotic Expression of TFMV,FVY and LMoV CP Gene,Antiserum Preparation and Virus Detection [J]. Bulletin of Botanical Research, 2013, 33(1): 73-79.
[10]	CAO Ying;HU Shang-Lian;ZHANG Hui-Ying;TANG Xiao-Feng;LIU Yong-Sheng. Cloning,Sequence Analysis and Prokaryotic Expression of an Alcohol Acyltransferase(AAT) Gene in Tomato(Solanum lycopersicum) [J]. Bulletin of Botanical Research, 2012, 32(6): 731-736.
[11]	GUO Zheng-Hong;WEI Lan-Zhen;WANG Quan-Xi. Antibody Preparation and Protein Expression of NdhK in Nostoc flagelliforme* [J]. Bulletin of Botanical Research, 2012, 32(2): 204-207.
[12]	GAO Bin;ZHANG Hai-Yan;FAN Hai*. Expression,Purification and Immunoreactivity of Hygromycin-B-Phosphotransferase [J]. Bulletin of Botanical Research, 2011, 31(1): 56-60.
[13]	LI Dong-Mei;PENG Shao-Lin. Molecular Cloning,Sequence Analysis of a Glycolate oxidase* Gene from Mikania micrantha and Its Expression in Escherichia coli [J]. Bulletin of Botanical Research, 2010, 30(4): 434-440.
[14]	ZHANG Teng-Guo;LIU Yu-Bing;SUN Kun;YANG Ning;AN Li-Zhe. Cloning and Prokaryotic Expression of Chorispora bungeana* CbMAPK3 Gene [J]. Bulletin of Botanical Research, 2009, 29(6): 692-695.
[15]	ZU Yuan-Gang;FANG Si-Liang;NIE Ming-Zhu. The Vector Construction and Prokaryotic Expression of Catharanthus roseus metallothionein(CrMT) in E.coli [J]. Bulletin of Botanical Research, 2007, 27(5): 588-592.