辣椒尿黑酸植基转移酶基因(HPT)的克隆及生物信息学分析
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摘要
尿黑酸植基转移酶(HPT)是控制植物中维生素E合成的一个关键限速酶,本实验利用RACE方法成功克隆辣椒尿黑酸植基转移酶基因并对其进行生物信息学分析,结果表明,HPT开放阅读框长为1 227 bp,共编码408个氨基酸,属于异戊烯转移酶PT_UbiA超家族。理化性质分析显示HPT蛋白分子量为4.61 kD,半衰期为30 h,理论等电点为9.52,含35个磷酸化位点,无信号肽,是一个疏水性蛋白质,存在9个跨膜结构域,亚细胞定位在类囊体膜上。二级结构预测结果表明HPT蛋白α-螺旋占59.07%、延伸链占10.54%、无规则卷曲占30.39%。进化树分析显示辣椒HPT与番茄HPT同源性较高,为今后利用辣椒尿黑酸植基转移酶通过基因工程技术提高辣椒维生素E的含量提供了重要依据。
Homogentisate phytyltransferase(HPT) is a key rate-limiting enzyme for vitamin E biosynthesis in pants.In this study, the HPT gene from pepper was successfully cloned by RACE method, and the bioinformatics analysis of it was carried out. The results indicated that the open reading frame of HPT was 1 227 bp, encoding 408 amino acids,which belonged to the PT_UbiA superfamily. The physicochemical properties analysis showed that the molecular weight of HPT protein was 4.61 kD, the half-life was 30 h and the theoretical isoelectric point was 9.52. There were35 phosphorylation sites in amino acid and no signal peptide. It was a hydrophobic protein and had nine transmembrane domains, with the subcellular localization on thylakoid membrane. Prediction results of secondary structure suggested that alpha-helix, elongation chain, random coil in HPT protein accounted for 59.07%, 10.54%,30.39%, respectively. The phylogenetic tree analysis showed the pepper HPT had higher similarity with tomato HPT,which could provide an important basis for using the HPT to improve the content of vitamin E in pepper by genetic engineering technology in the future.
Homogentisate phytyltransferase(HPT) is a key rate-limiting enzyme for vitamin E biosynthesis in pants.In this study, the HPT gene from pepper was successfully cloned by RACE method, and the bioinformatics analysis of it was carried out. The results indicated that the open reading frame of HPT was 1 227 bp, encoding 408 amino acids,which belonged to the PT_UbiA superfamily. The physicochemical properties analysis showed that the molecular weight of HPT protein was 4.61 kD, the half-life was 30 h and the theoretical isoelectric point was 9.52. There were35 phosphorylation sites in amino acid and no signal peptide. It was a hydrophobic protein and had nine transmembrane domains, with the subcellular localization on thylakoid membrane. Prediction results of secondary structure suggested that alpha-helix, elongation chain, random coil in HPT protein accounted for 59.07%, 10.54%,30.39%, respectively. The phylogenetic tree analysis showed the pepper HPT had higher similarity with tomato HPT,which could provide an important basis for using the HPT to improve the content of vitamin E in pepper by genetic engineering technology in the future.
引文
Cai N.N.,Chen Y.H.,and Li W.,2010,Prediction of protein tertiary structure based on neural network,Jisuanji Gongcheng(Computer Engineering),36(9):176-177(蔡娜娜,陈月辉,李伟,2010,基于神经网络的蛋白质三级结构预测,计算机工程,36(9):176-177)
Collakova E.,and DellaPenna D.,2003a,Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis,Plant Physiol.,131(2):632-642
Collakova E.,and DellaPenna D.,2003b,The role of homogentisate phytyltransferaseand and other tocopherol pathway enzymes in regulation of toeopherol biosynthesis during abiotic stress,Plant Physiol.,133(2):930-940
Feng D.,Liu B.L.,Wang Y.L.,Zhang N.,Wen Y.K.,Si H.J.,and Wang D.,2013,Cloning,bioinformatics and expression analysis of ethylene response factor gene of potato,Gansu Nongye Daxue Xuebao(Journal of Gansu Agricultural University),2(1):80-86(冯丹,刘柏林,王永林,张宁,文义凯,司怀军,王蒂,2013,马铃薯乙烯响应因子基因的克隆表达及生物信息学分析,甘肃农业大学学报,2(1):80-86)
Gálvez-Valdivieso G.,Carde觡osa R.,Pineda M.,and Aguilar M.,2015,Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii,J.Plant Physiol.,188:80-88
Gon g Y.Y.,Guo S.Q.,Shu H.M.,and Ni W.C.,2013,Cloning and expression of GhZIP4 gene from Gossypium hirsutum,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(1):27-33(巩元勇,郭书巧,束红梅,倪万潮,2013,棉花GhZIP4基因的克隆及表达分析,西北植物学报,33(1):27-33)
Gui L.Z.,Cui P.W.,Pan Q.P.,Tang J.F.,and Lu Y.B.,2013,Cloning and sequence analysis of LaCOMT in the biosynthesis pathway of galanthamine in Lycoris aurea,Hunan Z-hongyiyao Daxue Xuebao(Journal of Traditional Chinese Medicine University of Hunan),33(3):35-40(桂柳姿,崔培梧,潘清平,唐金凤,鲁耀邦,2013,忽地笑加兰他敏生物合成代谢途径中COMT酶基因克隆及序列分析,湖南中医药大学学报,33(3):35-40)
Hou Y.X.,Tang H.R.,Lin Y.X.,Chen Q.,Zhang Y.,and Guo Y.,2016,Cloning and expression of Faapx-c gene and bioinformatics analysis in Fragaria×ananassa based on RACE,Beifang Yuanyi(Northern Horticulture),(11):88-92(侯艳霞,汤浩茹,林源秀,陈清,张勇,郭艳,2016,基于RACE方法的草莓Faapx-c基因克隆表达及其生物信息学分析,北方园艺,(11):88-92)
Huang Y.Z.,Zeng Y.R.,Zhou Q.,Xia G.H.,and Huang Y.J.,2015,Analysis of ESTs associated with oil metabolism in seeds of Carya cathayensis,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),32(2):229-236(黄银芝,曾燕如,周秦,夏国华,黄有军,2015,山核桃种子脂肪代谢期EST序列的初步分析,浙江农林大学学报,32(2):229-236)
Li H.,Wang M.W.,Shao Y.,and You W.D.,2001,A rapid method for cloning candidated homologenes by combinating RACE technology with bioinformatics,Shengwu Jishu Tongxun(Letters in Biotechnology),12(4):257-259(李红,王孟薇,邵勇,尤纬缔,2001,利用RACE技术和生物信息学资源快速钓取多个侯选同源基因,生物技术通讯,12(4):257-259)
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Li Y.,2009,Engineeringand regulating plant thcopherol biosynthetic pathway,Dissertation for Ph.D.,Fudan University,Supervisor:Tang K.X.,pp.1-24(李殷,2009,植物维生素E生物合成途径及调控,博士学位论文,复旦大学,导师:唐克轩,pp.1-24)
Meng X.P.,Li X.X.,Li Y.X.,Cai M.H.,and Hu Y.K.,2007,In silico cloning of homogentisate phytyltransferaseand gene from soybean,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),22(4):14-18(孟宪萍,李晓晓,李雅轩,蔡民华,胡英考,2007,大豆尿黑酸叶绿基转移酶基因的克隆与进化分析,华北农学报,22(4):14-18)
Pan W.D.,Li X.F.,Chen S.Y.,and Liu G.S.,2006,Progress in vitamin E synthesis-related enzyme genes and in vivo functions in plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(1):68-77(潘卫东,李晓峰,陈双燕,刘公社,2006,植物维生素E合成相关酶基因的克隆及其在体内功能研究进展,植物学通报,23(1):68-77)
Wang J.J.,Guo Q.Q.,and Wang S.Y.,2012,SWOT analysis and development countermeasures of hot pepper industry in Guizhou,Lajiao Zazhi(Journal of China Capsicum),(3):39-42(汪建军,郭启琼,王社英,2012,贵州辣椒产业的SWOT分析与发展对策,辣椒杂志,(3):39-42)
Wu X.Y.,Ren J.H.,and Cao M.Y.,2004,Effect of vitamine Ewith tissue factor and tissue factor pathway inhibitor on coronary heart disease,Linchuang Xinxueguanbing Zazhi(Journal of Clinical Cardiology),20(10):616-618(吴晓燕,任江华,曹茂银,2004,维生素E对组织因子及其抑制物在冠心病中的干预作用,临床心血管病杂志,20(10):616-618)
Ye C.Y.,Liu N.B.,and Zheng S.P.,2009,Cloning of key enzyme genes involved in vitamin E biosynthesis and acquirement of transgenic plants,Xiandai Shipin Keji(Modern Food Science and Technology),25(9):1020-1023(叶从勇,刘南波,郑穗平,2009,维生素E合成途径关键酶基因的克隆及转基因植株的构建,现代食品科技,25(9):1020-1023)
Zhang G.Y.,Liu R.R.,Zhang P.,Xu G.,Zhu J.,Liang G.H.,and Liu Q.Q.,2012,Increase of tocotrienol content by overexpression of OsHGGT gene in transgenic rice,Shengwu Jishu Tongbao(Biotechnology Bulletin),(8):64-70(张桂云,刘如如,张鹏,徐根,朱姜,梁国华,刘巧泉,2012,转基因水稻中过表达OsHGGT基因提高种子三烯生育酚含量的研究,生物技术通报,(8):64-70)
Zhang X.M.,and Li P.L.,2014,Bioinformatics analysis of the plnBCD gene from Lactobacillus paraplantarum L-XM1,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),19(3):175-179(张香美,李平兰,2014,类植物乳杆菌L-XM1 plnBCD基因的生物信息学分析,中国农业大学学报,19(3):175-179)
Collakova E.,and DellaPenna D.,2003a,Homogentisate phytyltransferase activity is limiting for tocopherol biosynthesis in Arabidopsis,Plant Physiol.,131(2):632-642
Collakova E.,and DellaPenna D.,2003b,The role of homogentisate phytyltransferaseand and other tocopherol pathway enzymes in regulation of toeopherol biosynthesis during abiotic stress,Plant Physiol.,133(2):930-940
Feng D.,Liu B.L.,Wang Y.L.,Zhang N.,Wen Y.K.,Si H.J.,and Wang D.,2013,Cloning,bioinformatics and expression analysis of ethylene response factor gene of potato,Gansu Nongye Daxue Xuebao(Journal of Gansu Agricultural University),2(1):80-86(冯丹,刘柏林,王永林,张宁,文义凯,司怀军,王蒂,2013,马铃薯乙烯响应因子基因的克隆表达及生物信息学分析,甘肃农业大学学报,2(1):80-86)
Gálvez-Valdivieso G.,Carde觡osa R.,Pineda M.,and Aguilar M.,2015,Homogentisate phytyltransferase from the unicellular green alga Chlamydomonas reinhardtii,J.Plant Physiol.,188:80-88
Gon g Y.Y.,Guo S.Q.,Shu H.M.,and Ni W.C.,2013,Cloning and expression of GhZIP4 gene from Gossypium hirsutum,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),33(1):27-33(巩元勇,郭书巧,束红梅,倪万潮,2013,棉花GhZIP4基因的克隆及表达分析,西北植物学报,33(1):27-33)
Gui L.Z.,Cui P.W.,Pan Q.P.,Tang J.F.,and Lu Y.B.,2013,Cloning and sequence analysis of LaCOMT in the biosynthesis pathway of galanthamine in Lycoris aurea,Hunan Z-hongyiyao Daxue Xuebao(Journal of Traditional Chinese Medicine University of Hunan),33(3):35-40(桂柳姿,崔培梧,潘清平,唐金凤,鲁耀邦,2013,忽地笑加兰他敏生物合成代谢途径中COMT酶基因克隆及序列分析,湖南中医药大学学报,33(3):35-40)
Hou Y.X.,Tang H.R.,Lin Y.X.,Chen Q.,Zhang Y.,and Guo Y.,2016,Cloning and expression of Faapx-c gene and bioinformatics analysis in Fragaria×ananassa based on RACE,Beifang Yuanyi(Northern Horticulture),(11):88-92(侯艳霞,汤浩茹,林源秀,陈清,张勇,郭艳,2016,基于RACE方法的草莓Faapx-c基因克隆表达及其生物信息学分析,北方园艺,(11):88-92)
Huang Y.Z.,Zeng Y.R.,Zhou Q.,Xia G.H.,and Huang Y.J.,2015,Analysis of ESTs associated with oil metabolism in seeds of Carya cathayensis,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),32(2):229-236(黄银芝,曾燕如,周秦,夏国华,黄有军,2015,山核桃种子脂肪代谢期EST序列的初步分析,浙江农林大学学报,32(2):229-236)
Li H.,Wang M.W.,Shao Y.,and You W.D.,2001,A rapid method for cloning candidated homologenes by combinating RACE technology with bioinformatics,Shengwu Jishu Tongxun(Letters in Biotechnology),12(4):257-259(李红,王孟薇,邵勇,尤纬缔,2001,利用RACE技术和生物信息学资源快速钓取多个侯选同源基因,生物技术通讯,12(4):257-259)
Li X.J.,Liu H.B.,Lin X.Q.,Wu Z.D.,Xu C.H.,and Liu X.L.,2015,In silico cloning and bioinformatics analysis of KNOXgene in sugarcane(Sckn1),Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),34(1):136-142(李旭娟,刘洪博,林秀琴,吴转娣,徐超华,刘新龙,2015,甘蔗KNOX基因(Sckn1)的电子克隆及生物信息学分析,基因组学与应用生物学,34(1):136-142)
Li Y.,2009,Engineeringand regulating plant thcopherol biosynthetic pathway,Dissertation for Ph.D.,Fudan University,Supervisor:Tang K.X.,pp.1-24(李殷,2009,植物维生素E生物合成途径及调控,博士学位论文,复旦大学,导师:唐克轩,pp.1-24)
Meng X.P.,Li X.X.,Li Y.X.,Cai M.H.,and Hu Y.K.,2007,In silico cloning of homogentisate phytyltransferaseand gene from soybean,Huabei Nongxuebao(Acta Agriculturae Boreali-Sinica),22(4):14-18(孟宪萍,李晓晓,李雅轩,蔡民华,胡英考,2007,大豆尿黑酸叶绿基转移酶基因的克隆与进化分析,华北农学报,22(4):14-18)
Pan W.D.,Li X.F.,Chen S.Y.,and Liu G.S.,2006,Progress in vitamin E synthesis-related enzyme genes and in vivo functions in plants,Zhiwuxue Tongbao(Chinese Bulletin of Botany),23(1):68-77(潘卫东,李晓峰,陈双燕,刘公社,2006,植物维生素E合成相关酶基因的克隆及其在体内功能研究进展,植物学通报,23(1):68-77)
Wang J.J.,Guo Q.Q.,and Wang S.Y.,2012,SWOT analysis and development countermeasures of hot pepper industry in Guizhou,Lajiao Zazhi(Journal of China Capsicum),(3):39-42(汪建军,郭启琼,王社英,2012,贵州辣椒产业的SWOT分析与发展对策,辣椒杂志,(3):39-42)
Wu X.Y.,Ren J.H.,and Cao M.Y.,2004,Effect of vitamine Ewith tissue factor and tissue factor pathway inhibitor on coronary heart disease,Linchuang Xinxueguanbing Zazhi(Journal of Clinical Cardiology),20(10):616-618(吴晓燕,任江华,曹茂银,2004,维生素E对组织因子及其抑制物在冠心病中的干预作用,临床心血管病杂志,20(10):616-618)
Ye C.Y.,Liu N.B.,and Zheng S.P.,2009,Cloning of key enzyme genes involved in vitamin E biosynthesis and acquirement of transgenic plants,Xiandai Shipin Keji(Modern Food Science and Technology),25(9):1020-1023(叶从勇,刘南波,郑穗平,2009,维生素E合成途径关键酶基因的克隆及转基因植株的构建,现代食品科技,25(9):1020-1023)
Zhang G.Y.,Liu R.R.,Zhang P.,Xu G.,Zhu J.,Liang G.H.,and Liu Q.Q.,2012,Increase of tocotrienol content by overexpression of OsHGGT gene in transgenic rice,Shengwu Jishu Tongbao(Biotechnology Bulletin),(8):64-70(张桂云,刘如如,张鹏,徐根,朱姜,梁国华,刘巧泉,2012,转基因水稻中过表达OsHGGT基因提高种子三烯生育酚含量的研究,生物技术通报,(8):64-70)
Zhang X.M.,and Li P.L.,2014,Bioinformatics analysis of the plnBCD gene from Lactobacillus paraplantarum L-XM1,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),19(3):175-179(张香美,李平兰,2014,类植物乳杆菌L-XM1 plnBCD基因的生物信息学分析,中国农业大学学报,19(3):175-179)