水稻锌指蛋白基因OsBBX6的克隆、表达及生物信息学分析
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摘要
锌指蛋白作为植物体内一类重要的转录因子,对植物生长发育、基因调控以及响应外界环境变化方面发挥重要作用。Os BBX6基因属于水稻锌指蛋白B-Box基因家族成员,启动子元件分析发现其含有高温应答元件(HSE)、干旱应答元件(MBS)及非生物胁迫响应元件(TC-rich repeats)等逆境相关元件。组织特异性定量表达分析表明,Os BBX6在叶片中表达最高,根其次,茎和幼穗中表达最低。胁迫处理后的荧光定量PCR发现其受低温诱导上调,受高温、干旱、盐胁迫等抑制表达,表明其正向响应低温胁迫,负向响应高温、干旱、盐胁迫等。另外,本研究还克隆了OsBBX6基因,并对其进行了系统进化、蛋白跨膜、蛋白亚细胞定位及OsBBX6基因共表达等分析,为进一步研究其生物学功能奠定基础。
Zinc finger proteins play an important role in plant growth and development, gene regulation and response to environmental change as a kind of important transcription factor. OsBBX6 is a member of the B-box(BBX) family in rice. The promoter component analysis of OsBBX6 revealed that it contained stress responsive elements such as high temperature response element(HSE), drought responsive element(MBS) and abiotic stress response element(TC-rich repeats). Tissue specific quantitative analysis showed that OsBBX6 expressed the highest in leaves, followed by roots, and the lowest in stems and young panicles. By Fluorescence quantitative PCR after different stress treatments, OsBBX6 was found to be up-regulated by low temperature, and inhibited by high temperature, drought and salt stress, which indicated that it was positively related to low temperature stress and negatively to high temperature, drought and salt stress. In addition, the Os BBX6 gene was cloned, and its phylogenetic analysis, protein transmembrane analysis, protein subcellular localization and OsBBX6 gene co-expression analysis were all carried out, which laid the foundation for further study of its biological function.
Zinc finger proteins play an important role in plant growth and development, gene regulation and response to environmental change as a kind of important transcription factor. OsBBX6 is a member of the B-box(BBX) family in rice. The promoter component analysis of OsBBX6 revealed that it contained stress responsive elements such as high temperature response element(HSE), drought responsive element(MBS) and abiotic stress response element(TC-rich repeats). Tissue specific quantitative analysis showed that OsBBX6 expressed the highest in leaves, followed by roots, and the lowest in stems and young panicles. By Fluorescence quantitative PCR after different stress treatments, OsBBX6 was found to be up-regulated by low temperature, and inhibited by high temperature, drought and salt stress, which indicated that it was positively related to low temperature stress and negatively to high temperature, drought and salt stress. In addition, the Os BBX6 gene was cloned, and its phylogenetic analysis, protein transmembrane analysis, protein subcellular localization and OsBBX6 gene co-expression analysis were all carried out, which laid the foundation for further study of its biological function.
引文
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Rao L.Q.,Liu L.L.,Wang Q.M.,Shuai J.,Peng P.,Li M.Y.,and Tang S.W.,2015,Clone and expression of heat induced OsBBX30 in rice,Hunan Daxue Xuebao(Zikeban)(Journal of Hunan University(Natural Science Edition)),42(6):101-106(饶力群,刘兰兰,汪启明,帅进,彭澎,李梦云,唐世伟2015,热诱导表达的水稻OsBBX30基因克隆和表达分析,湖南大学学报(自科版),42(6):101-106)
Robson F.,Costa M.M.,Hepworth S.R.,Vizir I.,Pixeiro M.,Reeves P.H.,Putterill J.,and Coupland G.,2001,Functional importance of conserved domains in the flowering-time gene constants demonstrated by analysis of mutant alleles and transgenic plants,Plant Journal for Cell&Molecular Biology,28(6):619-631
Shen Q.S.,Chen F.,Ye Q.,Li T.,Chen X.B.,and Zhang X.W.,2012,Cloning and transient expression of the three stress-related rice promoters,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),32(11):29-34(申秋硕,陈凤,叶青,李涛,陈信波,张先文,2012,三个逆境相关的水稻启动子的克隆与瞬时表达分析,中国生物工程杂志,32(11):29-34)
Valverde F.,Mouradov A.,Soppe W.,Ravenscroft D.,Samach A.,and Coupland G.,2004,Photoreceptor regulation of constants protein in photoperiodic flowering,Science,303(5660):1003
Xiang J.H.,Li L.Z.,and Chen X.B.,2012,Progress in the study of abiotic stress related zinc finger protein genes in plants,Henong Xuebao(Acta Agriculturae Nucleatae Sinica),26(4):666-672(向建华,李灵之,陈信波,2012,植物非生物逆境相关锌指蛋白基因的研究进展,核农学报,26(4):666-672)
Zhang C.,Luo Y.,Xie M.,Wang W.P.,Li N.,Rao L.Q.,and Wang Q.M.,2017,The research of zinc finger family gene OsBBX24 in response to heat stress in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2035-2041(张超,骆鹰,谢旻,王伟平,黎妮,饶力群,汪启明,2017,水稻锌指蛋白基因OsBBX24响应热胁迫的研究,分子植物育种,15(6):2035-2041)
Chu Z.,Wang X.,Li Y.,Yu H.,Li J.,Lu Y.,Li H.X.,and Ouyang B.,2016,Genomic organiza-tion,phylogenetic and expression analysis of the b-box gene family in tomato,Fronti-ersin Plant Science,2016:7
Huang J.,Zhao X.,Weng X.,Lei W.,and Xie W.,2012,The rice b-box zinc finger gene family:genomic identification,characterization,expression profiling and diurnal analysis,PLo SOne,7(10):e48242
Khanna R.,Kronmiller B.,Maszle D.R.,Coupland G.,Holm M.Mizuno T.,and Wu S.H.,2009,The arabidopsis b-box zinc finger family,Plant Cell,21(11):3416-3420
Klug A.,and Schwabe J.W.,1995,Protein motifs 5.zinc fingers FASEB J.,9(8):597-604
Liu Y.,Xing L.J.,Li J.H.,and Dai S.J.,2012,Rice B-box zinc finger protein OsBBX25 is involved in the abiotic response Zhiwu Xuebao(Bulletin of Botany),47(4):366-378(刘焱邢立静,李俊华,戴绍军,2012,水稻含有B-box锌指结构域的OsBBX25蛋白参与植物对非生物胁迫的响应,植物学报,47(4):366-378)
Luo Y.,Xie M.,Zhang C.,Wang W.P.,Zhu J.H.,Wan X.Y.Wang Q.M.,and Rao L.Q.,2017,Function analysis of rice zinc finger protein gene OsBBX22 in response to heat stress Jiyinzuxue Yu Yingyong Shengwuxue(Genomics and Applied Biology),36(7):2940-2946(骆鹰,谢旻,张超,王伟平,朱建华,万向元,汪启明,饶力群,2017,水稻锌指蛋白基因OsBBX22响应热胁迫的功能分析,基因组学与应用生物学,36(7):2940-2946)
Rao L.Q.,Liu L.L.,Wang Q.M.,Shuai J.,Peng P.,Li M.Y.,and Tang S.W.,2015,Clone and expression of heat induced OsBBX30 in rice,Hunan Daxue Xuebao(Zikeban)(Journal of Hunan University(Natural Science Edition)),42(6):101-106(饶力群,刘兰兰,汪启明,帅进,彭澎,李梦云,唐世伟2015,热诱导表达的水稻OsBBX30基因克隆和表达分析,湖南大学学报(自科版),42(6):101-106)
Robson F.,Costa M.M.,Hepworth S.R.,Vizir I.,Pixeiro M.,Reeves P.H.,Putterill J.,and Coupland G.,2001,Functional importance of conserved domains in the flowering-time gene constants demonstrated by analysis of mutant alleles and transgenic plants,Plant Journal for Cell&Molecular Biology,28(6):619-631
Shen Q.S.,Chen F.,Ye Q.,Li T.,Chen X.B.,and Zhang X.W.,2012,Cloning and transient expression of the three stress-related rice promoters,Zhongguo Shengwu Gongcheng Zazhi(China Biotechnology),32(11):29-34(申秋硕,陈凤,叶青,李涛,陈信波,张先文,2012,三个逆境相关的水稻启动子的克隆与瞬时表达分析,中国生物工程杂志,32(11):29-34)
Valverde F.,Mouradov A.,Soppe W.,Ravenscroft D.,Samach A.,and Coupland G.,2004,Photoreceptor regulation of constants protein in photoperiodic flowering,Science,303(5660):1003
Xiang J.H.,Li L.Z.,and Chen X.B.,2012,Progress in the study of abiotic stress related zinc finger protein genes in plants,Henong Xuebao(Acta Agriculturae Nucleatae Sinica),26(4):666-672(向建华,李灵之,陈信波,2012,植物非生物逆境相关锌指蛋白基因的研究进展,核农学报,26(4):666-672)
Zhang C.,Luo Y.,Xie M.,Wang W.P.,Li N.,Rao L.Q.,and Wang Q.M.,2017,The research of zinc finger family gene OsBBX24 in response to heat stress in rice,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),15(6):2035-2041(张超,骆鹰,谢旻,王伟平,黎妮,饶力群,汪启明,2017,水稻锌指蛋白基因OsBBX24响应热胁迫的研究,分子植物育种,15(6):2035-2041)