GhTLP1基因特征分析、cDNA克隆及组织表达特性
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摘要
为了获取和验证类甜蛋白功能基因,并了解其基本的理化性质和组织表达特性,本研究在前期的海岛棉转录组高通量测序基础上,得到一个类甜蛋白功能基因的完整开放阅读框,命名为GhTLP1,通过全长特异引物,对GhTLP1进行了T-A克隆,测序结果符合已知序列。对GhTLP1基因序列特征分析结果表明,GhTLP1基因ORF序列全长为912 bp,共编码303个氨基酸残基,分子量为32.18 kD,等电点为4.36,第1～22位存在信号肽,无跨膜结构,预测属于亲水性蛋白;GhTLP1基因与其它植物的TLP基因的相似度达到50.15,具有完整的16个保守的半胱氨酸残基位点;GhTLP1基因与同源序列的聚类分析结果表明,GhTLP1基因与Gossypium hirsutum (XM_016846902.1)和Gossypium arboreum (XM_017769653.1)的TLP基因聚在同一分支上。本研究结果支持GhTLP1归为类甜蛋白基因的定性和功能判断。RT-PCR试验表明,GhTLP1在正常植株和受病原胁迫植株的根、茎、叶中均有表达,相对表达量顺序为根>叶>茎。GhTLP1可作为棉花抗病育种和材料筛选的调控靶标基因。
In order to obtain and verify the functio nal genes of thaumatin-like protein, and clarify its basic physicochemical properties and tissue expression characteristics, this study obtained a complete open reading frame of a functional gene of thaumatin-like protein based on the previous high throughput sequencing of Gossypium barbadense, which was named as GhTLP1. T-A cloning was performed on GhTLP1 by full length specific primers, and the sequencing results were consistent with the known sequence. The sequence characteristic analysis of the GhTLP1 gene showed that the total length of the GhTLP1 gene ORF sequence was 912 bp, encoding a total of 303 amino acid residues. The molecular weight was 32.18 kD, and the isoelectric point was 4.36. Signal peptides were existed on the 1～22 sites. The protein was predicted to be hydrophilic without transmembrane structure. The similarity between the GhTLP1 gene and the TLP gene of other plants reached 50.15, with 16 intact conserved cysteine residues. The cluster analysis of GhTLP1 gene and homologous sequences showed that the GhTLP1 gene was clustered on the same branch with the TLP gene of Gossypium hirsutum(XM_016846902.1)and Gossypium arboreum(XM_017769653.1). The above results supported the qualitative and functional judgement of GhTLP1 as a thaumatin-like protein gene. RT-PCR test showed that GhTLP1 was expressed in roots, stems and leaves of normal plants and pathogeny stressed plants, and the relative expression level was root >leaf >stem.GhTLP1 could be used as a target gene for cotton disease resistance breeding and material selection.
In order to obtain and verify the functio nal genes of thaumatin-like protein, and clarify its basic physicochemical properties and tissue expression characteristics, this study obtained a complete open reading frame of a functional gene of thaumatin-like protein based on the previous high throughput sequencing of Gossypium barbadense, which was named as GhTLP1. T-A cloning was performed on GhTLP1 by full length specific primers, and the sequencing results were consistent with the known sequence. The sequence characteristic analysis of the GhTLP1 gene showed that the total length of the GhTLP1 gene ORF sequence was 912 bp, encoding a total of 303 amino acid residues. The molecular weight was 32.18 kD, and the isoelectric point was 4.36. Signal peptides were existed on the 1～22 sites. The protein was predicted to be hydrophilic without transmembrane structure. The similarity between the GhTLP1 gene and the TLP gene of other plants reached 50.15, with 16 intact conserved cysteine residues. The cluster analysis of GhTLP1 gene and homologous sequences showed that the GhTLP1 gene was clustered on the same branch with the TLP gene of Gossypium hirsutum(XM_016846902.1)and Gossypium arboreum(XM_017769653.1). The above results supported the qualitative and functional judgement of GhTLP1 as a thaumatin-like protein gene. RT-PCR test showed that GhTLP1 was expressed in roots, stems and leaves of normal plants and pathogeny stressed plants, and the relative expression level was root >leaf >stem.GhTLP1 could be used as a target gene for cotton disease resistance breeding and material selection.
引文
Chen H.,Tan J.H.,Xu H.L.,Tang S.S.,Feng Y.H.,and Yang Z.Q.,2017,Cloning and expression analysis of PmCYP77A1gene on Pinus massoniana,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(3):985-992(陈虎,谭健晖,徐慧兰,唐生森,冯源恒,杨章旗,2017,马尾松PmCY P77A1基因的克隆及表达分析,基因组学与应用生物学,36(3):985-992)
Ganger M.T.,Dietz G.D.,and Ewing S.J.,2017,A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments,BMC Bioinformatics,18(1):534
Guo S.D.,Wang Y.,Sun G.Q.,Jin S.Q.,Zhou T.,Meng Z.G.,and Zhang R.,2015,Twenty years of research and application of transgenic cotton in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(17):3372-3387(郭三堆,王远,孙国清,金石桥,周焘,孟志刚,张锐,2015,中国转基因棉花研发应用二十年,中国农业科学,48(17):3372-3387)
Jiang X.L.,Huang Q.X.,Li H.,and Zhao J.P.,2012,A review of advances in plant thaumatin-like proteins,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),29(2):279-287(姜晓玲,黄秋娴,李虹,赵嘉平,2012,植物类甜蛋白基因家族研究进展,浙江农林大学学报,29(2):279-287)
Kuwabara C.,Arakawa K.,and Yoshida S.,1999,Abscisic acidinduced secretory proteins in suspension-cultured cells of winter wheat,Plant Cell Physiol.,40(2):184-191
Ma L.,Yuan J.H.,Sun W.C.,Liu Z.G.,Zeng X.C.,Wu J.Y.,Fang Y.,Li X.C.,Chen Q.,Xu Y.Z.,Pu Y.Y.,Liu H.Q.,Yang G.,and Liu L.B.,2017,Selection and cloning of thaumatin-like protein(TLP)gene from winter Brassica rapa and its expression under low temperature stress,Zuowu Xuebao(Acta Agronomica Sinica),43(4):620-628(马骊,袁金海,孙万仓,刘自刚,曾秀存,武军艳,方彦,李学才,陈奇,许耀照,蒲媛媛,刘海卿,杨刚,刘林波,2017,白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达,作物学报,43(4):620-628)
Munis M.F.,Tu L.,Deng F.,Tan J.,Xu L.,Xu S.,Long L.,and Zhang X.,2010,A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahlia and other stresses in transgenic tobacco,Biochem.Biophys.Res.Commun.,393(1):38-44
Piggott N.,Ekramoddoullah A.K.M.,Liu J.J.,and Yu X.S.,2004,Gene cloning of a thaumatin-like(PR-5)protein of western white pine(Pinus monticola D.Don)and expression studies of members of the PR-5 group,Physiol.Mol.Plant Pathol.,64(1):1-8
Singh N.K.,Kumar K.R.,Kumar D.,Shukla P.,and Kirti P.B.,2013,Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi,a wild peanut,PLoS One,8(12):e83963
Song Y.Q.,Chen Q.X.,Hu Z.J.,Li W.L.,Chen Y.W.,and Dong J.F.,2018,Cloning,identification and expression of the YsigOrco gene in Yemma signatus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(5):1900-1906(宋月芹,陈庆霄,胡镇杰,李文亮,陈应武,董钧锋,2018,锤胁跷蝽YsigOrco基因的克隆、鉴定与表达,基因组学与应用生物学,37(5):1900-1906)
Sun S.M.,Zhu J.,Ge X.P.,Shi L.Y.,Cheng L.,Zhang W.X.,and Su Y.L.,2017,Cloning of caspase 3 gene in Megalobrama amblycephala and analysis of its expression in high temperature stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(10):4090-4099(孙盛名,朱健,戈贤平,石连玉,程磊,张武肖,苏艳丽,2017,团头鲂(Megalobrama amblycephala)caspase基因克隆及其在高温胁迫中的表达分析,基因组学与应用生物学,36(10):4090-4099)
Wang Q.H.,Li F.G.,Zhang X.,Zhang Y.A.,Hou Y.X.,Zhang S.R.,and Wu Z.X.,2011,Purification and characterization of a CkTLP protein from Cynanchum komarovii seeds that confers antifungal activity,PLoS One,6(2):e16930
Zhao Z.Q.,Sun G.Q.,Zhang G.L.,Ma P.P.,Wang Z.J.,Ye C.X.,and Xie Z.M.,2017,Cloning and expression analysis of the GbRLCK10 gene in Gossypium barbadense L.,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(11):2130-2138(赵曾强,孙国清,张国丽,马盼盼,王志军,叶春秀,谢宗铭,2017,海岛棉BbRLCK10基因克隆及表达分析,西北植物学报,37(11):2130-2138)
Ganger M.T.,Dietz G.D.,and Ewing S.J.,2017,A common base method for analysis of qPCR data and the application of simple blocking in qPCR experiments,BMC Bioinformatics,18(1):534
Guo S.D.,Wang Y.,Sun G.Q.,Jin S.Q.,Zhou T.,Meng Z.G.,and Zhang R.,2015,Twenty years of research and application of transgenic cotton in China,Zhongguo Nongye Kexue(Scientia Agricultura Sinica),48(17):3372-3387(郭三堆,王远,孙国清,金石桥,周焘,孟志刚,张锐,2015,中国转基因棉花研发应用二十年,中国农业科学,48(17):3372-3387)
Jiang X.L.,Huang Q.X.,Li H.,and Zhao J.P.,2012,A review of advances in plant thaumatin-like proteins,Zhejiang Nonglin Daxue Xuebao(Journal of Zhejiang A&F University),29(2):279-287(姜晓玲,黄秋娴,李虹,赵嘉平,2012,植物类甜蛋白基因家族研究进展,浙江农林大学学报,29(2):279-287)
Kuwabara C.,Arakawa K.,and Yoshida S.,1999,Abscisic acidinduced secretory proteins in suspension-cultured cells of winter wheat,Plant Cell Physiol.,40(2):184-191
Ma L.,Yuan J.H.,Sun W.C.,Liu Z.G.,Zeng X.C.,Wu J.Y.,Fang Y.,Li X.C.,Chen Q.,Xu Y.Z.,Pu Y.Y.,Liu H.Q.,Yang G.,and Liu L.B.,2017,Selection and cloning of thaumatin-like protein(TLP)gene from winter Brassica rapa and its expression under low temperature stress,Zuowu Xuebao(Acta Agronomica Sinica),43(4):620-628(马骊,袁金海,孙万仓,刘自刚,曾秀存,武军艳,方彦,李学才,陈奇,许耀照,蒲媛媛,刘海卿,杨刚,刘林波,2017,白菜型冬油菜类甜蛋白的筛选、克隆及其在低温胁迫下的表达,作物学报,43(4):620-628)
Munis M.F.,Tu L.,Deng F.,Tan J.,Xu L.,Xu S.,Long L.,and Zhang X.,2010,A thaumatin-like protein gene involved in cotton fiber secondary cell wall development enhances resistance against Verticillium dahlia and other stresses in transgenic tobacco,Biochem.Biophys.Res.Commun.,393(1):38-44
Piggott N.,Ekramoddoullah A.K.M.,Liu J.J.,and Yu X.S.,2004,Gene cloning of a thaumatin-like(PR-5)protein of western white pine(Pinus monticola D.Don)and expression studies of members of the PR-5 group,Physiol.Mol.Plant Pathol.,64(1):1-8
Singh N.K.,Kumar K.R.,Kumar D.,Shukla P.,and Kirti P.B.,2013,Characterization of a pathogen induced thaumatin-like protein gene AdTLP from Arachis diogoi,a wild peanut,PLoS One,8(12):e83963
Song Y.Q.,Chen Q.X.,Hu Z.J.,Li W.L.,Chen Y.W.,and Dong J.F.,2018,Cloning,identification and expression of the YsigOrco gene in Yemma signatus,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),37(5):1900-1906(宋月芹,陈庆霄,胡镇杰,李文亮,陈应武,董钧锋,2018,锤胁跷蝽YsigOrco基因的克隆、鉴定与表达,基因组学与应用生物学,37(5):1900-1906)
Sun S.M.,Zhu J.,Ge X.P.,Shi L.Y.,Cheng L.,Zhang W.X.,and Su Y.L.,2017,Cloning of caspase 3 gene in Megalobrama amblycephala and analysis of its expression in high temperature stress,Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(10):4090-4099(孙盛名,朱健,戈贤平,石连玉,程磊,张武肖,苏艳丽,2017,团头鲂(Megalobrama amblycephala)caspase基因克隆及其在高温胁迫中的表达分析,基因组学与应用生物学,36(10):4090-4099)
Wang Q.H.,Li F.G.,Zhang X.,Zhang Y.A.,Hou Y.X.,Zhang S.R.,and Wu Z.X.,2011,Purification and characterization of a CkTLP protein from Cynanchum komarovii seeds that confers antifungal activity,PLoS One,6(2):e16930
Zhao Z.Q.,Sun G.Q.,Zhang G.L.,Ma P.P.,Wang Z.J.,Ye C.X.,and Xie Z.M.,2017,Cloning and expression analysis of the GbRLCK10 gene in Gossypium barbadense L.,Xibei Zhiwu Xuebao(Acta Botanica Boreali-Occidentalia Sinica),37(11):2130-2138(赵曾强,孙国清,张国丽,马盼盼,王志军,叶春秀,谢宗铭,2017,海岛棉BbRLCK10基因克隆及表达分析,西北植物学报,37(11):2130-2138)