茶树咖啡碱合成酶基因稀有等位变异TCS1g的筛选、克隆及功能
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摘要
【目的】茶树咖啡碱合成酶1(TCS1)是山茶属(Camellia)茶组植物咖啡碱合成的关键酶,TCS1具有丰富的等位变异。从我国丰富的茶树种质资源中发掘TCS1稀有等位变异并研究其功能,深入解析茶树咖啡碱合成机制,为低咖啡碱育种提供新的基因资源。【方法】利用特异引物TCS1P InDel F/R对673份茶树资源中的TCS1等位变异进行鉴定;使用引物TCS1cDNAF/R,从含有新稀有等位变异的茶树资源中克隆基因的cDNA全长序列;利用生物信息学、实时荧光定量PCR(qRT-PCR)和原核表达等方法研究新发现稀有等位基因的功能。【结果】在部分大理茶(C.taliensis)资源中鉴定到一个新的TCS1稀有等位变异,命名为TCS1g。从大理茶资源‘龙陵17’(LL17,含有的TCS1等位变异为TCS1a和TCS1g)中克隆了TCS1g。TCS1g的编码区序列全长为1 098 bp,编码365个氨基酸,编码蛋白的分子量和理论等电点分别为40.9 kD和5.1。序列比对结果表明,TCS1g与TCS1a、TCS1b、TCS1c、TCS1d、TCS1e、TCS1f的相似度在94.1%—99.2%;与具有可可碱合成酶活性(TS)的TCS1b和TCS1c编码蛋白序列相似度均大于96.7%,而与同时具有TS和咖啡碱合成酶(CS)活性的TCS1a、TCS1d、TCS1e、TCS1f相似度都小于95.4%,且TCS1g第221位氨基酸残基与TCS1b、TCS1c同为组氨酸(His),而TCS1a、TCS1d、TCS1e、TCS1f为精氨酸(Arg)。第221位氨基酸残基位于TCS1g蛋白活性中心,该位点的突变(His突变为Arg),可以导致TCS1g的等电点(5.05变为5.06)和亲水性(-0.119变为-0.123)发生改变。此外,5′上游调控区域的比对结果显示TCS1g、TCS1b、TCS1c起始密码子(ATG)比TCS1a、TCS1d、TCS1e、TCS1f延后了15 bp。原核表达发现TCS1g具有TS活性,活性为44.3 pkat/mg,但未检测到CS活性。qRT-PCR的结果表明LL17中等位变异TCS1g有表达。LL17的咖啡碱和可可碱的含量分别为40.3 mg·g~(-1)和5.4 mg·g~(-1)。【结论】鉴定并克隆了一个新的TCS1稀有等位变异TCS1g,其在LL17中具有一定的表达水平,且其表达蛋白具有TS活性,而未检测到CS活性;推测决定TCS1g仅具有TS活性的关键位点是第221位氨基酸残基。
【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 from94.1%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 5upstream 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~(-1 )and 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.
【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 from94.1%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 5upstream 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~(-1 )and 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.
引文
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[2]PARE W.The effect of caffeine and seconal on a visual discrimination task.Journal of Comparative and Physiological Psychology,1961,54(5):506-509.
[3]SMITH A.Effects of caffeine on human behavior.Food and Chemical Toxicology,2002,40(9):1243-1255.
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[7]HALLSTROM H,WOLK A,GLYNN A,MICHA?LSSON K.Coffee,tea plants and caffeine consumption in relation to osteoporotic fracture risk in a cohort of Swedish women.Osteoporos International,2006:17(7):1055-1064.
[8]陈盛相,齐桂年,李建华,夏建冰.低咖啡因茶树育种研究进展.福建茶叶,2009,32(1):2-3.CHEN S X,QI G N,LI J H,XIA J B.Research progress in low caffeine content germplasm breeding of tea plants.Tea in Fujian,2009,32(1):2-3.(in Chinese)
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[10]NEGISHI O,OZAWA T,IMAGAWA H.Conversion of xanthosine into caffeine in tea plants.Agricultural Biology and Chemistry,1985,49(1):251-253.
[11]ASHIHARA H,SANO H,CROZIER A.Caffeine and related purine alkaloids:Biosynthesis,catabolism,function and genetic engineering.Phytochemistry,2008,69(4):841-856.
[12]MOHANPURIA P,KUMAR V,YADAV S K.Tea caffeine:metabolism,functions,and reduction strategies.Food Science and Biotechnology,2010,19(2):275-287.
[13]SUZUKI T.The participation of S-adenosylmethionine in the biosynthesis of caffeine in the tea plants.FEBS Letters,1972,24(1):18-20.
[14]KATO M,MIZUNO K,CROZIER A,FUJIMURA T,ASHIHARA H.Caffeine synthase gene from tea leaves.Nature,2000,406(6799):956-957.
[15]YONEYAMA N,MORIMOTO H,YE C X,ASHIHARA H,MIZUNO K,KATO M.Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme.Molecular Genetics and Genomics,2006,275(2):125-135.
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[17]JIN J Q,YAO M Z,MA C L,MA J Q,CHEN L.Natural allelic variations of TCS1 play a crucial role in caffeine biosynthesis of tea plant and its related species.Plant Physiology and Biochemistry,2016,100(1):18-26.
[18]OGAWA M,HERAI Y,KOIZUMI N,KUSANO T,SANO HIROSHI.7-Methylxanthine methyltransferase of coffee plants.Gene isolation and enzymatic properties.The Journal of Biological Chemistry,2001,276(11):8213-8218.
[19]PICHERSKY E,LEWINSOHN E.Convergent evolution in plant specialized metabolism.Annual Review of Plant Biology,2011,62(1):549-566.
[20]XIA E H,ZHANG H B,SHENG J,LI K,ZHANG Q J,KIM C,ZHANG Y,LIU Y,ZHU T,LI W,HUANG H,TONG Y,NAN H,SHIC,SHI C,JIANG J J,MAO S Y,JIAO J Y,ZHANG D,ZHAO Y,ZHAO Y J,ZHANG L P,LIU Y L,LIU B Y,YU Y,SHAO S F,NI D J,EICHLER E E,GAO L Z.The tea tree genome provides insights into tea flavor and independent evolution of caffeine biosynthesis.Molecular Plant,2017,10(6):866-877.
[21]WEI C,YANG H,WANG S,ZHAO J,LIU C,GAO L,XIA E,LU Y,TAI Y,SHE G,SUN J,CAO H,TONG W,GAO Q,LI Y,DENG W,JIANG X,WANG W,CHEN Q,ZHANG S,LI H,WU J,WANG P,LIP,SHI C,ZHENG F,JIAN J,HUANG B,SHAN D,SHI M,FANG C,YUE Y,LI F,LI D,WEI S,HAN B,JIANG C,YIN Y,XIA T,ZHANG Z,BENNETZEN J L,ZHAO S,WAN X.Draft genome sequence of Camellia sinensis var.sinensis provides insights into the evolution of the tea genome and tea quality.Proceedings of the National Academy of Sciences of the United States of America,2018,115(18):4151-4158.
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[23]ASHIHARA H,KATO M,YE C X.Biosynthesis and metabolism of purine alkaloids in leaves of Cocoa tea(Camellia ptilophylla).Journal of Plant Research,1998,111(4):599-604.
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