黑果枸杞中调控花青素合成代谢的bHLH基因克隆与序列分析
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摘要
以黑果枸杞为研究对象,利用RT-PCR方法从黑果枸杞cDNA中克隆一个bHLH的转录因子——LrJAF13,通过生物信息学的分析,发现该基因的全长为1 890 bp,编码624个氨基酸的亲水蛋白,其分子式是C3012H4843N853O978S24,分子量为6.94 kD,脂肪指数(Aliphatic index)为85.45,等电点pI为5.24,亲水性(Grand average of hydropathicity)为-0.497,不稳定指数(Instability index)为50.13,推断出该基因编码的蛋白位于细胞核中,蛋白质三维结构复杂,包含多个α-螺旋和β-折叠结构,与矮牵牛拟南芥bHLH基因编码的蛋白序列的一致性为88%,进化树的分析表明黑果枸杞的LrJAF13与矮牵牛的bHLH的转录因子petunia_JAF13的亲缘关系比较接近。通过上述分析,为解析黑果枸杞果实中高花青素含量的分子遗传机理、优异资源筛选和新品种选育提供了理论依据。
T his study took the Lycium ruthenicum as a research object, and cloned a new bHLH transcription factor named LlJAF13 by RT-PCR technology from Lycium ruthenicum. This study found the full length of the gene was 1 890 bp, encoding 624 amino acids of hydrophilic protein by bioinformatics analysis. The molecular formula was C3012 H4843 N853 O978 S24. The molecular weight was 6.94 kD. The fat index was 85.45. The isoelectric point pI was 5.24. The Grand average of hydropathicity was-0.497, and the instability index was 50.13. This study suggested that the protein encoded by the gene was located in the nucleus. The three-dimensional structure of the protein was complex and contained a number of α-helix and β-folded structures. The consistency of the protein sequence with the protein sequence of Arabidopsis thaliana bHLH gene was 88%. The analysis of the phylogenetic tree showed that the LrJAF13 of the Lycium ruthenicum was close to that of petunia_JAF13, the transcription factor of b HLH. The above analysis might provide the theoretical basis for the analysis of the molecular genetic mechanism of high anthocyanin content in the fruit of Lycium ruthenicum, the selection of excellent resources and the breeding of new varieties.
T his study took the Lycium ruthenicum as a research object, and cloned a new bHLH transcription factor named LlJAF13 by RT-PCR technology from Lycium ruthenicum. This study found the full length of the gene was 1 890 bp, encoding 624 amino acids of hydrophilic protein by bioinformatics analysis. The molecular formula was C3012 H4843 N853 O978 S24. The molecular weight was 6.94 kD. The fat index was 85.45. The isoelectric point pI was 5.24. The Grand average of hydropathicity was-0.497, and the instability index was 50.13. This study suggested that the protein encoded by the gene was located in the nucleus. The three-dimensional structure of the protein was complex and contained a number of α-helix and β-folded structures. The consistency of the protein sequence with the protein sequence of Arabidopsis thaliana bHLH gene was 88%. The analysis of the phylogenetic tree showed that the LrJAF13 of the Lycium ruthenicum was close to that of petunia_JAF13, the transcription factor of b HLH. The above analysis might provide the theoretical basis for the analysis of the molecular genetic mechanism of high anthocyanin content in the fruit of Lycium ruthenicum, the selection of excellent resources and the breeding of new varieties.
引文
Borevit z J.O.,Xia Y.J.,Blount J.,Dixon R.A.,and Lamb C.2000,Activation tagging identifies a conserved MYB regulator of phenylpropanoid biosynthesis,The Plant Cell,12(12):2383-2394
Dunlop D.S.,and Curtis W.R.,1991,Synergistic response of plant hairy-root cultures to phosphate limitation and funga elicitation,Biotechnology Progress,7(5):434-438
Goff S.A.,Cone K.C.,and Chandler V.L.,1992,Functional analysis of the transcriptional activator encoded by the maize Bgene:evidence for a direct functional interaction between two classes of regulatory proteins,Genes&Development,6(5):864-875
Jia Z.D.,Ma P.Y.,Bian X.F.,Yang Q.,Guo X.D.,and Xie Y.Z.2014,Biosynthesis metabolic pathway and molecular regulation of plants anthocyanin,Xibei Zhiwu Xuebao(Journa of Northwest Botanical Sciences),34(7):1496-1506(贾赵东,马佩勇,边小峰,杨清,郭小丁,谢一芝,2014,植物花青素合成代谢途径及其分子调控,西北植物学报,34(7)1496-1506)
Kastell A.,Smetanska I.,Ulrichs C.,Cai Z.Z.,and Mewis I.2013,Effects of phytohormones and jasmonic acid on glucosinolate content in hairy root cultures of Sinapis alba and Brassica rapa,Appl.Biochem.Biotechnol.,169(2):624-635
Kobayashi S.,Yamamoto N.G.,and Hirochika H.,2005,Association of VvmybA1 gene expression with anthocyanin production in grape(Vitis vinifera)skin-color mutants,Journa of the Japanese Society for Horticultural Science,74(3):196-203
Li C.B.,Shi Q.S.,Shu X.L.,Ouyang Y.S.,and Chen Y.B.,2006Plant antimicrobial proteins nsLTPs,Zhiwu Shenglixue Tongxun(Chinese Journal of Plant Physiology),42(3):539-544(李诚斌,施庆珊,疏秀林,欧阳友生,陈仪本,2006,植物抗菌蛋白nsLTPs,植物生理学通讯,42(3):539-544)
Liu W.W.,and Li W.X.,2013,Progress of plant bHLH transcription factor,Shengwu Jishu Jinzhan(Current Biotechnology)3(1):7-11(刘文文,李文学,2013,植物bHLH转录因子研究进展,生物技术进展,3(1):7-11)
Liu X.Y.,Wang W.S.,and Fu B.Y.,2011,Research progress of plant bHLH transcription factor family,Shengwu Jishu Jinzhan(Current Biotechnology),1(6):391-397(刘晓月,王文生,傅彬英,2011,植物bHLH转录因子家族的功能研究进展,生物技术进展,1(6):391-397)
Petroni K.,Cominelli E.,Consonni G.,Gusmaroli G.,Gavazzi G.and Tonelli C.,2000,The developmental expression of the maize regulatory gene Hopi determines germination-dependent anthocyanin accumulation,Genetics Society of America,155(1):323-336
Porret N.,Cousins P.,and Owens C.,2006,DNA sequence variation within the promoter of VvmybA1 associates with flesh pigmentation of intensely colored grape varieties,Hort Science,41(4):1049
Shoeva O.Y.,Gordeeva E.I.,and Khlestkina E.K.,2014,The regulation of anthocyanin synthesis in the wheat pericarp,Molecules,19(12):20266-20279
Sujatha G.,Zdravkovic-Korac S.,Calic D.,Flamini G.,and Ranjitha Kumari B.D.,2013,High-efficiency Agrobacterium rhizogenes-mediated genetic,transformation in Artemisia vulgaris:Hairy root production and essential oil analysis,Industrial Crops and Products,44:643-652
Sweeney M.T.,Thomson M.J.,Pfeil B.E.,and McCouch S.,2006,Caught red-handed:Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice,The Plant Cell,18(2):283-294
This P.,Lacombe T.,Cadle-Davidson M.,and Owens C.L.,2007,Wine grape(Vitis vinifera L.)color associates with allelic variation in the domestication gene VvmybA1,Theor.Appl.Genet.,114(4):723-730
Yan Y.M.,Dai Y.M.,Ran L.W.,Cao Y.L.,Luo Q.,Li X.Y.,Qin K.,Dai G.L.,and Zeng X.X.,2014,Composition and in vitro antioxidant activity of anthocyanins extracted from Lycium ruthenicum Murr.,different fruits and vegetables,Shipin Gongye Keji(Food Industries),35(16):133-136(闫亚美,代彦满,冉林武,曹有龙,罗青,李晓莺,秦垦,戴国礼,曾晓雄,2014,黑果枸杞与5种果蔬中花色苷组成及体外抗氧化活性比较,食品工业科技,35(16):133-136)
Yang P.C.,Zhou B.,and Li Y.H.,2012,The bHLH transcription factors involved in anthocyanin biosynthesis in plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(8):747-758(杨鹏程,周波,李玉花,2012,植物花青素合成相关的bHLH转录因子,植物生理学报,48(8):747-758)
Zhang X.,Song J.Y.,Hu Y.L.,Xu J.,Xu Z.C.,Ji A.J.,Luo H.M.,and Chen S.L.,2014,Research progress of the regulation on active compound biosynthesis by the bHLH transcription factors in plants,Yaoxue Xuebao(Journal of Pharmaceutical Sciences),49(4):435-442(张鑫,宋经元,胡鸢雷,徐江,徐志超,季爱加,罗红梅,陈士林,2014,bHLH转录因子调控植物活性成分生物合成的研究进展,药学学报,49(4):435-442)
Zhou H.,Zhu Q.,Yang Y.F.,Liu H.W.,Yu F.X.,and Qiu D.Y.,2015,Cloning and sequence analysis of bHLH gene from Taxus chinensis var.mairei,Zhiwu Yanjiu(Bulletin of Botanical Research),35(1):52-59(周华,朱祺,杨艳芳,刘洪伟,余发新,邱德有,2015,南方红豆杉bHLH基因克隆与序列分析,植物研究,35(1):52-59)
Zhu Z.X.,and Lu Y.Q.,2016,Plant color mutants and the anthocyanin pathway,Zhiwu Xuebao(Chinese Bulletin of Botany),51(1):107-119(祝志欣,鲁迎青,2016,花青素代谢途径与植物颜色变异,植物学报,51(1):107-119)
Dunlop D.S.,and Curtis W.R.,1991,Synergistic response of plant hairy-root cultures to phosphate limitation and funga elicitation,Biotechnology Progress,7(5):434-438
Goff S.A.,Cone K.C.,and Chandler V.L.,1992,Functional analysis of the transcriptional activator encoded by the maize Bgene:evidence for a direct functional interaction between two classes of regulatory proteins,Genes&Development,6(5):864-875
Jia Z.D.,Ma P.Y.,Bian X.F.,Yang Q.,Guo X.D.,and Xie Y.Z.2014,Biosynthesis metabolic pathway and molecular regulation of plants anthocyanin,Xibei Zhiwu Xuebao(Journa of Northwest Botanical Sciences),34(7):1496-1506(贾赵东,马佩勇,边小峰,杨清,郭小丁,谢一芝,2014,植物花青素合成代谢途径及其分子调控,西北植物学报,34(7)1496-1506)
Kastell A.,Smetanska I.,Ulrichs C.,Cai Z.Z.,and Mewis I.2013,Effects of phytohormones and jasmonic acid on glucosinolate content in hairy root cultures of Sinapis alba and Brassica rapa,Appl.Biochem.Biotechnol.,169(2):624-635
Kobayashi S.,Yamamoto N.G.,and Hirochika H.,2005,Association of VvmybA1 gene expression with anthocyanin production in grape(Vitis vinifera)skin-color mutants,Journa of the Japanese Society for Horticultural Science,74(3):196-203
Li C.B.,Shi Q.S.,Shu X.L.,Ouyang Y.S.,and Chen Y.B.,2006Plant antimicrobial proteins nsLTPs,Zhiwu Shenglixue Tongxun(Chinese Journal of Plant Physiology),42(3):539-544(李诚斌,施庆珊,疏秀林,欧阳友生,陈仪本,2006,植物抗菌蛋白nsLTPs,植物生理学通讯,42(3):539-544)
Liu W.W.,and Li W.X.,2013,Progress of plant bHLH transcription factor,Shengwu Jishu Jinzhan(Current Biotechnology)3(1):7-11(刘文文,李文学,2013,植物bHLH转录因子研究进展,生物技术进展,3(1):7-11)
Liu X.Y.,Wang W.S.,and Fu B.Y.,2011,Research progress of plant bHLH transcription factor family,Shengwu Jishu Jinzhan(Current Biotechnology),1(6):391-397(刘晓月,王文生,傅彬英,2011,植物bHLH转录因子家族的功能研究进展,生物技术进展,1(6):391-397)
Petroni K.,Cominelli E.,Consonni G.,Gusmaroli G.,Gavazzi G.and Tonelli C.,2000,The developmental expression of the maize regulatory gene Hopi determines germination-dependent anthocyanin accumulation,Genetics Society of America,155(1):323-336
Porret N.,Cousins P.,and Owens C.,2006,DNA sequence variation within the promoter of VvmybA1 associates with flesh pigmentation of intensely colored grape varieties,Hort Science,41(4):1049
Shoeva O.Y.,Gordeeva E.I.,and Khlestkina E.K.,2014,The regulation of anthocyanin synthesis in the wheat pericarp,Molecules,19(12):20266-20279
Sujatha G.,Zdravkovic-Korac S.,Calic D.,Flamini G.,and Ranjitha Kumari B.D.,2013,High-efficiency Agrobacterium rhizogenes-mediated genetic,transformation in Artemisia vulgaris:Hairy root production and essential oil analysis,Industrial Crops and Products,44:643-652
Sweeney M.T.,Thomson M.J.,Pfeil B.E.,and McCouch S.,2006,Caught red-handed:Rc encodes a basic helix-loop-helix protein conditioning red pericarp in rice,The Plant Cell,18(2):283-294
This P.,Lacombe T.,Cadle-Davidson M.,and Owens C.L.,2007,Wine grape(Vitis vinifera L.)color associates with allelic variation in the domestication gene VvmybA1,Theor.Appl.Genet.,114(4):723-730
Yan Y.M.,Dai Y.M.,Ran L.W.,Cao Y.L.,Luo Q.,Li X.Y.,Qin K.,Dai G.L.,and Zeng X.X.,2014,Composition and in vitro antioxidant activity of anthocyanins extracted from Lycium ruthenicum Murr.,different fruits and vegetables,Shipin Gongye Keji(Food Industries),35(16):133-136(闫亚美,代彦满,冉林武,曹有龙,罗青,李晓莺,秦垦,戴国礼,曾晓雄,2014,黑果枸杞与5种果蔬中花色苷组成及体外抗氧化活性比较,食品工业科技,35(16):133-136)
Yang P.C.,Zhou B.,and Li Y.H.,2012,The bHLH transcription factors involved in anthocyanin biosynthesis in plants,Zhiwu Shengli Xuebao(Plant Physiology Journal),48(8):747-758(杨鹏程,周波,李玉花,2012,植物花青素合成相关的bHLH转录因子,植物生理学报,48(8):747-758)
Zhang X.,Song J.Y.,Hu Y.L.,Xu J.,Xu Z.C.,Ji A.J.,Luo H.M.,and Chen S.L.,2014,Research progress of the regulation on active compound biosynthesis by the bHLH transcription factors in plants,Yaoxue Xuebao(Journal of Pharmaceutical Sciences),49(4):435-442(张鑫,宋经元,胡鸢雷,徐江,徐志超,季爱加,罗红梅,陈士林,2014,bHLH转录因子调控植物活性成分生物合成的研究进展,药学学报,49(4):435-442)
Zhou H.,Zhu Q.,Yang Y.F.,Liu H.W.,Yu F.X.,and Qiu D.Y.,2015,Cloning and sequence analysis of bHLH gene from Taxus chinensis var.mairei,Zhiwu Yanjiu(Bulletin of Botanical Research),35(1):52-59(周华,朱祺,杨艳芳,刘洪伟,余发新,邱德有,2015,南方红豆杉bHLH基因克隆与序列分析,植物研究,35(1):52-59)
Zhu Z.X.,and Lu Y.Q.,2016,Plant color mutants and the anthocyanin pathway,Zhiwu Xuebao(Chinese Bulletin of Botany),51(1):107-119(祝志欣,鲁迎青,2016,花青素代谢途径与植物颜色变异,植物学报,51(1):107-119)