汉中黑稻花青素合成酶基因克隆及生物信息学分析
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摘要
花青素合成酶(Anthocyanidin Synthase,ANS)是植物花青苷生物合成途径末端的关键酶,催化无色花色素到有色花色素的转变。为研究汉中黑稻的ANS多样性及起源,采用克隆测序的方法对汉中7种品种黑稻的ANS基因序列进行分析。采用生物信息学方法,对该基因序列进行对比,并构建系统进化树和同源树,对其编码蛋白从基本理化性质、亚细胞定位、跨膜结构域、信号肽、导肽、二级结构和三级结构等方面进行预测和分析。结果表明7种汉中黑稻存在黑稻1、黑稻2两种基因序列,开放阅读框均为1 128bp,编码375个氨基酸。进化树表明黑稻1和黑稻2与籼稻、粳稻、浦竹仔、小麦等禾本科植物较近的亲缘关系,同源性分析表明黑稻1和黑稻2与籼稻等植物的ANS具有高度的同源性。氨基酸序列比对发现黑稻1和黑稻2仅有326位氨基酸不同。黑稻1的ANS蛋白含有2OG-FeⅡ_Oxy加氧酶的保守结构域。ANS蛋白三级结构预测,发现黑稻1和籼稻存在差异,推测ANS可能是花青苷合成中起重要作用的关键酶。这些结果表明ANS基因是一个古老的基因,可以作为种属鉴定的参考基因,可能是影响黑稻花青苷生物合成的主要基因之一。
Anthocyanidin synthase(ANS)is one of the key enzymesthat catalyzes the biosynthesis that converts leucoanthocyanidin into colored anthocyanidin in plants.To study the diversity and origin of ANS,the ANS genes from 7varieties of Hanzhong Black Rice were analyzed by PCR cloning and sequencing.Through the bioinformatics,sequences of theANS genes were compared.By constructing the NJ and homology trees,the sequence alignment of ANS protein,basic physical and chemical properties,subcellular localization,transmembrane region,signal peptide,guide peptideas well as the secondary and tertiary structures of ANS protein were studied.The results showed that the ANS genes of theserice varietiescould be divided into two categories,namely,Black Rice 1(BR1)and Black Rice 2(BR2).They had a same length on the open reading frame of 1,128 bp encoded 375 amino acids.Their phylogenetic treessuggested that BR1 and BR2 were genetically close to other gramineous plants,such asIndicaand Japonica,Indosasa hispida,andTriticum aestivum.The sequence of amino acids(AA)revealed that only the 326 th AAwas different between BR1 and BR2.A conserved domain search showed that the ANS protein hada 2OG-FeⅡ_Oxy domain.Its predicted tertiary structure indicated that BR1 differed fromIndica,and suggested that the ANS gene might play a key role in the anthocyanin synthase.It was concluded that the ANS genewas ancient and could be served as a reference gene for species identification,and thatits presence in Hanzhong Black Rice could be one of the main reasons why the anthocyanin synthesis was found in the rice.
Anthocyanidin synthase(ANS)is one of the key enzymesthat catalyzes the biosynthesis that converts leucoanthocyanidin into colored anthocyanidin in plants.To study the diversity and origin of ANS,the ANS genes from 7varieties of Hanzhong Black Rice were analyzed by PCR cloning and sequencing.Through the bioinformatics,sequences of theANS genes were compared.By constructing the NJ and homology trees,the sequence alignment of ANS protein,basic physical and chemical properties,subcellular localization,transmembrane region,signal peptide,guide peptideas well as the secondary and tertiary structures of ANS protein were studied.The results showed that the ANS genes of theserice varietiescould be divided into two categories,namely,Black Rice 1(BR1)and Black Rice 2(BR2).They had a same length on the open reading frame of 1,128 bp encoded 375 amino acids.Their phylogenetic treessuggested that BR1 and BR2 were genetically close to other gramineous plants,such asIndicaand Japonica,Indosasa hispida,andTriticum aestivum.The sequence of amino acids(AA)revealed that only the 326 th AAwas different between BR1 and BR2.A conserved domain search showed that the ANS protein hada 2OG-FeⅡ_Oxy domain.Its predicted tertiary structure indicated that BR1 differed fromIndica,and suggested that the ANS gene might play a key role in the anthocyanin synthase.It was concluded that the ANS genewas ancient and could be served as a reference gene for species identification,and thatits presence in Hanzhong Black Rice could be one of the main reasons why the anthocyanin synthesis was found in the rice.
引文
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[15]邵雅芳,徐非非,唐富福,等.水稻花青素合成相关基因的时空表达研究[J].核农学报,2013,27(1):9-14.
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[17]张羽,冯志峰,吴升华,等.陕西汉中地区水稻主要种质资源的调查及利用[J].安徽农业科学,2008,36(1):159-161.
[18]韩豪,李新生,江海,等.UPLCMS/MS定性分析黑米中花青苷类物质[J].食品工业科技,2014,35(17):289-293.
[19]邓文辉,李新生.黑米色素抗疲劳的生化机理及研究展望[J].食品工业科技,2010,31(6):372-374.
[20]路宏朝,王杨科,李丽霞,等.黑米花青苷复方胶囊对实验性肝损伤的保护作用[J].食品科学,2013,34(3):261-263.
[21]张羽,李新生,冯志峰,等.陕西省有色稻资源的SSR多态性分析[J].植物遗传资源学报,2011,12(5):828-832.
[22]丁锐.黑稻主要栽培品种过氧化物酶同工酶的研究[J].安徽农业科学,2005,33(6):947-948.
[23]SHIH C H,CHU H,TANG L K,et al.Functional characterization of key structural genes in rice flavonoid biosynthesis[J].Planta,2008,228(6):1043-1054.
[24]董娇,周军,辛培尧,等.不同植物LDOX/ANS基因的生物信息学分析[J].基因组学与应用生物学,2010,29(5):815-822.
[2]张学英,张上隆,骆军,等.果实花色素苷合成研究进展[J].果树学报,2004,21(5):456-460.
[3]孙玲,张名位,池建伟,等.黑米资源的黄酮含量及其与粒形性状的相关性[J].湖北农学院学报,2000,20(1):1-5.
[4]曹小勇,李新生.黑米花色素苷类色素研究现状及展望[J].氨基酸和生物资源,2002,24(1):3-6.
[5]SAKAMOTO W,OHMORI T,KAGEYAMA K,et al.The purple leaf(Pl)locus of rice:Pl w allele has a complex organization and includes two genes encoding basic helix-loophelix proteins involved in anthocyanin biosynthesis[J].Plant Cell Physiol,2001,42(9):982-991.
[6]亓希武,帅琴,范丽,等.桑树花青素合成酶(ANS)基因的克隆及在2种果色桑树中的表达特征[J].蚕业科学,2013,39(1):5-13.
[7]MA TINGRUI,ZHANG JINWEN,LIANG HUIGUANG,et al.Plant anthocyanin synthesis and gene regulation[J].Agricultural Science&Technology,2012,13(3):507-511,540.
[8]王芳权,杨杰,范方军,等.水稻紫色果皮的延迟遗传及Pb基因功能标记开发[J].中国水稻科学,2014,28(6):605-611.
[9]HU J P,ANDERSON B,WESSLER S R.Isolation and characterization of rice R genes:Evidence for distinct evolutionary paths in rice and maize[J].Genetics,1996,142(3):1021-1031.
[10]王彩霞,舒庆尧.水稻紫色种皮基因Pb的精细定位与候选基因分析[J].科学通报,2007,52(21):2517-2523.
[11]王冰,王全逸,印敬明,等.野生马铃薯ANS同源基因的克隆与表达分析[J].植物生理学报,2011,47(11):1103-1108.
[12]PELLETIER M K,MURRELL J R,SHIRLEY B W.Characterization of flavonol synthase and leucoanthocyanidin dioxygenase genes in Arabidopsis.Further evidence for differential regulation of“early”and“late”genes.[J].Plant Physiol,1997,113(4):1437-1445.
[13]YAN M L,LIU X J,GUAN C Y,et al.Cloning and expression analysis of an anthocyanidin synthase gene homolog from Brassica juncea[J].Mol Breeding,2011,28(3):313-322.
[14]GONG Z,YAMAZAKI M,SUGIYAMA M,et al.Cloning and molecular analysis of structural genes involved in anthocyanin biosynthesis and expressed in a forma-specific manner[J].Plant Mol Biol,1997,35(6):915-927.
[15]邵雅芳,徐非非,唐富福,等.水稻花青素合成相关基因的时空表达研究[J].核农学报,2013,27(1):9-14.
[16]REDDY A M,REDDY V S,SCHEFFLER B E,et al.Novel transgenic rice overexpressing anthocyanidin synthase accumulates a mixture of flavonoids leading to an increased antioxidant potential[J].Metabolic Engineering,2007,9(1):95-111.
[17]张羽,冯志峰,吴升华,等.陕西汉中地区水稻主要种质资源的调查及利用[J].安徽农业科学,2008,36(1):159-161.
[18]韩豪,李新生,江海,等.UPLCMS/MS定性分析黑米中花青苷类物质[J].食品工业科技,2014,35(17):289-293.
[19]邓文辉,李新生.黑米色素抗疲劳的生化机理及研究展望[J].食品工业科技,2010,31(6):372-374.
[20]路宏朝,王杨科,李丽霞,等.黑米花青苷复方胶囊对实验性肝损伤的保护作用[J].食品科学,2013,34(3):261-263.
[21]张羽,李新生,冯志峰,等.陕西省有色稻资源的SSR多态性分析[J].植物遗传资源学报,2011,12(5):828-832.
[22]丁锐.黑稻主要栽培品种过氧化物酶同工酶的研究[J].安徽农业科学,2005,33(6):947-948.
[23]SHIH C H,CHU H,TANG L K,et al.Functional characterization of key structural genes in rice flavonoid biosynthesis[J].Planta,2008,228(6):1043-1054.
[24]董娇,周军,辛培尧,等.不同植物LDOX/ANS基因的生物信息学分析[J].基因组学与应用生物学,2010,29(5):815-822.