红花玉兰MwZFP10基因的克隆及功能分析
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摘要
该研究选取了湖北五峰地区红花玉兰新品种‘娇红1号’为研究材料,基于实验室前期对红花玉兰转录组测序差异显著表达基因中筛选到的645bp花发育相关的核心片段,采用RACE技术克隆得到1个SUPERMAN类基因MwZFP10(GenBank登录号为MH037314)。生物信息学分析显示,MwZFP10基因全长为1 117bp,开放阅读框726bp,编码241个氨基酸,蛋白分子式为C_(1128)H_(1760)N_(322)O_(383)S_(14),分子量26.4kD,理论等电点4.42,含有一个C2H2型锌指结构域。对比分析发现MwZFP10属于SUPERMAN类锌指蛋白,具有N端的QALGGH和C端富含亮氨酸的L(I)DLXLR(K)L保守结构域。α螺旋(Hh)、延伸链(Ee)和β转角(Bt)构成红花玉兰MwZFP10蛋白的基本结构,蛋白的三级结构具有典型的锌指空间构型。MwZFP10蛋白为亲水性蛋白,可能定位于细胞核中。半定量和实时荧光定量结果显示,MwZFP10在花器官、叶片和苞片中均有表达,且花器官的雌蕊中表达量较高。过表达MwZFP10转基因拟南芥表现为植株矮小且不育,叶片卷缩,花器官发育畸形等特征。研究认为,MwZFP10基因在植物花器官的生长和发育中起重要作用。
In this study,the new species Magnolia wufengensis ‘Jiaohong No.1'was selected as the research material in Wufeng,Hubei.A SUPERMAN like gene MwZFP10(GenBank accession MH037314)was cloned with 3′RACE method,based on the 645 bp core fragment related with floral development and the fragment was filtered out from differentially expressed genes based on pre-Laboratory cDNA library of Magnolia wufengensis.Bioinformatics analysis of MwZFP10 gene showed that the full length of MwZFP10 cDNA was 1 117 bp with an open reading frame(ORF)of 726 bp encoding 241 amino acids.The protein molecular formula is C_(1128)H_(1760)N_(322)O_(383)S_(14),molecular weight is 26.4 kD and isoelectric point is4.42.The protein contains a conserved domain:the C2 H2-type zinc finger.Comparative analysis found that MwZFP10 belongs to the SUPERMAN-like zinc finger protein,with the"QALGGH"in the N termi-nal and"L(I)DLXLR(K)L"in the C terminal.α-helix(Hh),extended chain(Ee)andβ-turn(Bt)constitute are the basic structure of the protein.The tertiary structure of the protein has a typical zinc finger spatial configuration.The protein is hydrophilic and mainly localized in the nucleus.The semi-quantitative PCR and quantitative real-time PCR results showed that MwZFP10 was expressed in floral organs,leaf buds and bracts,and the pistil expression was higher in floral organs.MwZFP10 transgenic Arabidopsis thalianais characterized by short and sterile plants,curling leaves,malformation of flower organs,etc.The gene MwZFP10 play an important role in the floral growth and development.
In this study,the new species Magnolia wufengensis ‘Jiaohong No.1'was selected as the research material in Wufeng,Hubei.A SUPERMAN like gene MwZFP10(GenBank accession MH037314)was cloned with 3′RACE method,based on the 645 bp core fragment related with floral development and the fragment was filtered out from differentially expressed genes based on pre-Laboratory cDNA library of Magnolia wufengensis.Bioinformatics analysis of MwZFP10 gene showed that the full length of MwZFP10 cDNA was 1 117 bp with an open reading frame(ORF)of 726 bp encoding 241 amino acids.The protein molecular formula is C_(1128)H_(1760)N_(322)O_(383)S_(14),molecular weight is 26.4 kD and isoelectric point is4.42.The protein contains a conserved domain:the C2 H2-type zinc finger.Comparative analysis found that MwZFP10 belongs to the SUPERMAN-like zinc finger protein,with the"QALGGH"in the N termi-nal and"L(I)DLXLR(K)L"in the C terminal.α-helix(Hh),extended chain(Ee)andβ-turn(Bt)constitute are the basic structure of the protein.The tertiary structure of the protein has a typical zinc finger spatial configuration.The protein is hydrophilic and mainly localized in the nucleus.The semi-quantitative PCR and quantitative real-time PCR results showed that MwZFP10 was expressed in floral organs,leaf buds and bracts,and the pistil expression was higher in floral organs.MwZFP10 transgenic Arabidopsis thalianais characterized by short and sterile plants,curling leaves,malformation of flower organs,etc.The gene MwZFP10 play an important role in the floral growth and development.
引文
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[15]HUANG F,CHI Y,MENG Q,et al.GmZFP1encoding a single zinc finger protein is expressed with enhancement in reproductive organs and late seed development in soybean(Glycine max)[J].Molecular Biology Reports,2006,33(4):279-285.
[16]杨郁文,倪万潮,张保龙,等.陆地棉SUPERMAN类似锌指蛋白基因的克隆与表达分析[J].遗传,2006,28(4):443-448.YANG Y W,NI W C,ZHANG B L,et al.Molecular cloning and expression analysis of a SUPERMAN-like zinc finger protein gene in upland cotton[J].Hereditas,2006,28(4):443-448.
[17]许轲,刘娜,谢璇,等.苹果SUPERMAN家族基因的组织特异表达及MdSUP3的功能分析[J].园艺学报,2016,43(3):419-430.XU K,LIU N,XIE X,et al.Expression analysis of the SUPERMAN family genes in apple(Malus×domestica Borkh.)and function analysis of MdSUP3[J].Acta Horticulturae Sinica,2016,43(3):419-430.
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[19]贺随超,马履一,陈发菊.红花玉兰种质资源遗传多样性初探[J].西北植物学报,2007,27(12):2 421-2 428.HE S C,MA L Y,CHEN F J.Genetic diversity of Magnolia wufengensis based on AFPL[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(12):2 421-2 428.
[20]桑子阳,马履一,陈发菊.干旱胁迫对红花玉兰幼苗生长和生理特性的影响[J].西北植物学报,2011,31(1):109-115.SANG Z Y,MA L Y,CHEN F J.Growth and physiological characteristics of Magnolia wufengensis seedlings under drought stress[J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(1):109-115.
[21]YANG Y,JIA Z K,CHEN F J,et al.Comparative analysis of natural cold acclimation and deacclimation of two Magnolia,species with different winter hardiness[J].Acta Physiologiae Plantarum,2015,37(7):1-11.
[22]景丹龙,马江,张博,等.红花玉兰MwAG基因在花发育不同时期的表达[J].植物学报,2013,48(2):145-150.JING D L,MA J,ZHANG B,et al.Expression analysis of MwAGin different organs and developmental stages of Magnolia wufengensis[J].Chinese Bullletin of Botany,2013,48(2):145-150.
[23]CHEN L Y,CHEN F J,HE S C,et al.High genetic diversity and small genetic variation among populations of Magnolia wufengensis(Magnoliaceae),revealed by ISSR and SRAP markers[J].Electronic Journal of Biotechnology,2014,17(6):268-274.
[24]WANG L,XIAO A H,MA L Y,et al.Identification of Magnolia wufengensis(Magnoliaceae)cultivars using phenotypic traits,SSR and SRAP markers:insights into breeding and conservation[J].Genetics&Molecular Research,2017,16(1).doi:10.4238/gmr16019473.
[25]CLOUGH S J,BENT A F.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant Journal,1998,16:735-743.
[2]DINKINS R D,PFLIPSEN C,THOMPSON A,et al.Ectopic expression of an Arabidopsis single zinc finger gene in tobacco results in dwarf plants[J].Plant&Cell Physiology,2002,43(7):743.
[3]JIANG C J,AONO M,TAMAOKI M,et al.SAZ,a new SUPERMAN-like protein,negatively regulates a subset of ABA-responsive genes in Arabidopsis[J].Molecular Genetics&Genomics,2008,279(2):183.
[4]翟晓霏,靳永胜,张文,等.SUPERMAN类单锌指蛋白的研究进展[J].分子植物育种,2010,8(3):563-566.ZHAI X F,JIN Y S,ZHANG W,et al.Progresses in the SUPERMAN-like single zinc finger protein[J].Molecular Plant Breeding,2010,8(3):563-566.
[5]ENJLBRECHT C C,SCHOOF H,BOHM S.Conservation,diversification and expansion of C2H2zinc finger proteins in the Arabidopsis thaliana genome[J].BMC Genomics,2004,5(1):39.
[6]COEN E S,MEYEROWITZ E M.The war of the whorls:genetic interactions controlling flower development[J].Nature,1991,353:31-37.
[7]YUN J Y,WEIGEL D,LEE I.Ectopic expression of SUPERMAN suppresses development of petals and stamens[J].Plant&Cell Physiology,2002,43(1):52-57.
[8]BOWMAN J L,SAKAI H,JACK T,et al.SUPERMAN,a regulator of floral homeotic genes in Arabidopsis[J].Development,1992,114(3):599.
[9]SAKAI H,MEDRANO L J,MEYEROWITZ E M.Role of SUPERMAN in maintaining Arabidopsis floral whorl boundaries[J].Nature,1995,378(6 553):199-203.
[10]TAGUE B W,GALLANT P,GOODMAN H M.Expression analysis of an Arabidopsis C2H2zinc finger protein gene[J].Plant Molecular Biology,1996,32(5):785-796.
[11]TAKEDA S,MATSUMOTO N,OKADA K.RABBIT EARS,encoding a SUPERMAN-like zinc finger protein,regulates petal development in Arabidopsis thaliana[J].Development,2004,131(2):425-434.
[12]KRIZEK B A,LEWIS M W,FLETCHER J C.RABBIT EARS is a second-whorl repressor of AGAMOUS that maintains spatial boundaries in Arabidopsis flowers[J].Plant Journal for Cell&Molecular Biology,2006,45(3):369-383.
[13]PAYNE T,JOHNSON S D,KOLTUNOW A M.KNUCKLES(KNU)encodes a C2H2zinc-finger protein that regulates development of basal pattern elements of the Arabidopsis gynoecium[J].Development,2004,131(15):3 737-3 749.
[14]NAKAGAWA H,JIANG C J,SAKAKIBARA H,et al.Overexpression of a petunia zinc-finger gene alters cytokinin metabolism and plant forms:Petunia zinc-finger protein alters cytokinin metabolism and branching[J].Plant Journal,2004,41(4):512-523.
[15]HUANG F,CHI Y,MENG Q,et al.GmZFP1encoding a single zinc finger protein is expressed with enhancement in reproductive organs and late seed development in soybean(Glycine max)[J].Molecular Biology Reports,2006,33(4):279-285.
[16]杨郁文,倪万潮,张保龙,等.陆地棉SUPERMAN类似锌指蛋白基因的克隆与表达分析[J].遗传,2006,28(4):443-448.YANG Y W,NI W C,ZHANG B L,et al.Molecular cloning and expression analysis of a SUPERMAN-like zinc finger protein gene in upland cotton[J].Hereditas,2006,28(4):443-448.
[17]许轲,刘娜,谢璇,等.苹果SUPERMAN家族基因的组织特异表达及MdSUP3的功能分析[J].园艺学报,2016,43(3):419-430.XU K,LIU N,XIE X,et al.Expression analysis of the SUPERMAN family genes in apple(Malus×domestica Borkh.)and function analysis of MdSUP3[J].Acta Horticulturae Sinica,2016,43(3):419-430.
[18]MA L Y,WANG L R,HE S C,et al.A new variety of Magnolia(Magnoliaceae)from Hubei,China[J].Bulletin of Botanical Research,2006,26(1):4-7.
[19]贺随超,马履一,陈发菊.红花玉兰种质资源遗传多样性初探[J].西北植物学报,2007,27(12):2 421-2 428.HE S C,MA L Y,CHEN F J.Genetic diversity of Magnolia wufengensis based on AFPL[J].Acta Botanica Boreali-Occidentalia Sinica,2007,27(12):2 421-2 428.
[20]桑子阳,马履一,陈发菊.干旱胁迫对红花玉兰幼苗生长和生理特性的影响[J].西北植物学报,2011,31(1):109-115.SANG Z Y,MA L Y,CHEN F J.Growth and physiological characteristics of Magnolia wufengensis seedlings under drought stress[J].Acta Botanica Boreali-Occidentalia Sinica,2011,31(1):109-115.
[21]YANG Y,JIA Z K,CHEN F J,et al.Comparative analysis of natural cold acclimation and deacclimation of two Magnolia,species with different winter hardiness[J].Acta Physiologiae Plantarum,2015,37(7):1-11.
[22]景丹龙,马江,张博,等.红花玉兰MwAG基因在花发育不同时期的表达[J].植物学报,2013,48(2):145-150.JING D L,MA J,ZHANG B,et al.Expression analysis of MwAGin different organs and developmental stages of Magnolia wufengensis[J].Chinese Bullletin of Botany,2013,48(2):145-150.
[23]CHEN L Y,CHEN F J,HE S C,et al.High genetic diversity and small genetic variation among populations of Magnolia wufengensis(Magnoliaceae),revealed by ISSR and SRAP markers[J].Electronic Journal of Biotechnology,2014,17(6):268-274.
[24]WANG L,XIAO A H,MA L Y,et al.Identification of Magnolia wufengensis(Magnoliaceae)cultivars using phenotypic traits,SSR and SRAP markers:insights into breeding and conservation[J].Genetics&Molecular Research,2017,16(1).doi:10.4238/gmr16019473.
[25]CLOUGH S J,BENT A F.Floral dip:A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[J].Plant Journal,1998,16:735-743.