摘要
高温胁迫可引起植物体内生长素类激素含量和活性变化,也可影响其生理生化过程进而产生胁迫蛋白以提高自身抵抗或适应逆境的能力。根据高温胁迫下花花柴转录组测序分析结果,发现生长激素合成相关基因bHLH显著上调,通过PCR克隆获得该基因碱基序列,命名为KcbHLH71,并利用定量PCR对高温胁迫下该基因的表达模式进行分析。结果显示,花花柴KcbHLH71基因完整ORF为1 038 bp,编码345个氨基酸,分子量大小为38.8 kD,pI为6.8;编码蛋白含有b HLH蛋白家族的特征基序列和保守结构域,有信号肽,无跨膜结构,定位于细胞核。系统发育结果显示花花柴KcbHLH71与菊科的向日葵、莴苣bHLH71蛋白的相似性分别为88%和83%,即认为它们同源性最高,亲缘关系最近。表达分析结果显示,高温胁迫下,花花柴KcbHLH71基因2 h的表达量受到最大抑制,之后逐渐上升,当12 h时达到高峰,12～24 h再次出现下降,但仍高于对照水平。以上研究结果表明,高温可以显著诱导KcbHLH71基因的表达,推测该基因可能参与花花柴响应高温胁迫的正调控。
High temperature stress can cause changes in the content and activity of auxin hormones in plants,and can also affect their physiological and biochemical processes to produce stress proteins to enhance their ability to resist or adapt to stress. According to the transcriptome sequencing analysis results of Karelinia caspia under high temperature stress, it was found that the growth hormone synthesis related gene bHLH was significantly up-regulated. The base sequence of the gene obtained by PCR cloning was named KcbHLH71, and the expression pattern of the gene under high temperature stress was detected by quantitative PCR analysis. The results showed that the complete ORF of KcbHLH71 gene was 1 038 bp, encoding 345 amino acids with a molecular weight of 38.8 kD and a pI of 6.8. The encoded protein contained the characteristic sequence and conserved domain of the bHLH protein family, with signal peptide and no transmembrane structure, and was located in the nucleus. Phylogenetic results showed that the similarity between KcbHLH71 in Karelinia caspia and bHLH71 proteins from Helianthus annuus in Asteraceae and lettuce was 88% and 83%, respectively, which meant that they had the highest homology and the closest relationship. The results of expression analysis showed that under high temperature stress, the expression of KcbHLH71 gene was significantly inhibited at 2 h, then gradually increased, and peaked at 12 h, and decreased again at 12～24 h, but still higher than the control level.The above results indicated that high temperature could significantly induce the expression of KcbHLH71 gene,suggesting that this gene might be involved in the positive regulation of Karelinia caspia in response to high temperature stress.
引文
Dong H.,Zhao H.,Li S.,Li S.,Han Z.,Hu G,Liu C,Yang G.,Wang G.,Xie W.,and Xing Y.,2018,Genome-wide association studies reveal that members of bHLH subfamily 16share a conserved function in regulating flag leaf angle in rice(Oryza sativa),PLo S Genet.,14(4):1-20
Dong Y.,Wang C.,Han X.,Tang S.,Liu S.,Xia X.,and Yin W.,2014,A novl b HLH transcription factor Peb HLH35 from Populus euphratica confers drought tolerance through regulating stomatal development,photosynthesis and growth in Arabidopsis,Biochem.Biophys.Res.Commun.,450(1):453-458
Escandón M.,Caoal M.J.,Pascual J.,Pinto G.,Correia B.,Amaral J.,and Meijón M.,2016,Integrated physiological and hor monal profile of heat-induced hermotolerance in Pinus radiata,Tree Physiol.,36(1):63-77
Feller A.,Machemer K.,Braun E.L.,and Grotewold E.,2011,Evolutionary and comparative analysis of MYB and b HLHplant transcription factors,Plant J.,66(1):94-116
Feng X.M.,2011,Identification and expression analysis of apple basic helix-loop-helix transcription factor famliy,Thesis for M.S.,Shandong Agricultural University,Supervisor:Gao H.,pp.26-30(冯晓明,2011,苹果bHLH基因MdCIbHLH1克隆及功能分析,硕士学位论文,山东农业大学,导师:高华,pp.26-30)
Guang Y.Q.,Song G.C.,Zhang J.F.,Wang X.N.,and Tang C.M.,2014,Molecular cloning and expression analysis of GhbHL-H130,encoding a novel bHLH transcription factor in upland cotton(Gossypium hirsutum L.),Mi anhua Xuebao(Cotton Science),26(4):363-370(光杨其,宋桂成,张金凤,王晓楠,唐灿明,2014,1个新棉花b HLH类基因GhbHLH130的克隆及表达分析,棉花学报,26(4):363-370)
He J.,Gu X.R.,Wei C.H.,Yang X.Z,Li H.,Ma J.X.,Zhang Y.,Yang J.Q.,and Zhang X.,2016,Identification and expression analysis under abiotic stresses of the bHLH transcription factor family genes in Watermelon,Yuanyi Xuebao(Acta Horticulturae Sinica),43(2):281-294(何洁,顾秀容,魏春华,杨小振,李好,马建祥,张勇,杨建强,张显,2016,西瓜bHLH转录因子家族基因的鉴定及其在非生物胁迫下的表达分析,园艺学报,43(2):281-294)
Li H.,Ding Y.,Shi Y.,Zhang X.,Zhang S.,Gong Z.,and Yang S.,2017a,MPK3 and MPK6-mediated ICE1 phosphorylation negatively regulates ICE1 stability and freezing tolerance in Arabidopsis,Dev.Cell.,43(5):630-642
Li J.,Li Y.,Yin Z.,Jiang J.,Zhang M.,Guo X.,Ye Z.,Zhao Y.,Xiong H.,Zhang Z.,Shao Y.,Jiang C.,Zhang H.,An G.,Paek N.C.,Ali J.,and Li Z.,2017b,OsASR5 enhances drought tolerance through a stomatal closure pathway associated with ABA and H2O2signalling in rice,Plant Biotechnol.J.,15(2):183-196
Li N.,2017,Cloning and Function Characterization of CibHLH027 Involved in Leaf Senescence from Caragana intermedia,Thesis for M.S.,Inner Mongolia Agricultural U-niversity,Supervisor:Li G.J.,pp.29-30(李娜,2017,中间锦鸡儿参与叶片衰老基因CibHLH027的克隆和功能研究,硕士学位论文,蒙古农业大学,导师:李国婧,pp.29-30)
Liu X.C.,2017,Cloning and Functional Analysis of Hub HLH1-like from Pitaya(Hylocereus undatus),Thesis for M.S.,Guizhou University,Supervisor:Wen X.P.,pp.45-50(刘小翠,2017,火龙果Hub HLH1-like基因的克隆及功能分析,硕士学位论文,贵州大学,导师:文晓鹏,pp.45-50)
Liu Y.J.,Ji X.Y.,Nie X.G.,Qu M.,Zheng L.,Tan Z.L.,Zhao H.M.,Liu S.N.,Zhang B.,and Wang Y.,2015,Arabidopsis AtbHLH112 regulates the expression of genes involved in abiotic stress tolerance by binding to their E-box and GCG-box motifs,New Phytol.,207(3):692-709
Massari M.E.,and Murre C.,2000,Helix-loop-helix proteins:regulators of transcription in eucaryotic organis ms,Mol.Cell Biol.,20(2):429-440
Pires N.,and Dolan L.,2010,Origin and diversification of basic-helix-loop-helix proteins in plants,Mol.Biol.Evol.,27(4):862-874
Sailsdery J.K.,Atchley W.R.,and Dean R.A.,2012,Phylogenetic analysis and classification of the fungal bHLH domain,Mol.Biol.Evol.,9(5):1301-1318
Sun H.,Fan H.J.,and Ling H.Q.,2015,Genome-wide identification and characterization of the b HLH gene family in tomato,BMC Genomics,16(1):1-16
Vanstraelen M.,and BenkovE.,2012,Hormonal interactions in the regulation of plant development,Annu.Rev.Cell Dev.Biol.,28(28):463-487
Wang Y.Q.,Shi X.J.,and Li Z.J.,2017,An evaluation on heat tolerance of a desert plant Karelinia caspia seedlings(Biotechnol Bull),Shengwu Jishu Tongbao(Biotechnology Bulletin),33(4):157-163(王彦芹,石新建,李志军,2017,沙漠植物花花柴幼苗对高温耐受性评价,生物技术通报,33(4):157-163)
Wang Z.,Libault M.,Joshi T.,Valliyodan B.,Nguyen H.T.,Xu D.,Stacey G.,and Chen J.L.,2010,SoyDB:a knowledge database of soybean transcription factors,BMC Plant Biol.,10(1):14-14
Xu S.C.,Ding H.D.,Lu R.,Shi B.,and Ma F.F.,2008,Study on effects of ABA in antioxidant defense of plant cells,Zhongguo Nongye Daxue Xuebao(Journal of China Agricultural University),13(2):11-19(许树成,丁海东,鲁锐,石犇,马芳芳,2008,ABA在植物细胞抗氧化防护过程中的作用,中国农业大学学报,13(2):11-19)
Zhang Q.Q.,Zhu J.H.,Cai Y.B.,and Zhang Z.L.,2009,Cloning and sequence analysis of HbbHLH1 promoter from Hevea brasiliensis,Fenzi Zhiwu Yuzhong(Molecular Plant Breeding),7(3):531-536(张全琪,朱家红,蔡元保,张治礼,2009,巴西橡胶树HbbHLH1启动子的克隆与序列分析,分子植物育种,7(3):531-536)
Zhao C.,Wang P.,Si T.,Hsu C.,Wang L.,Zayed O.,Yu Z.,Zhu Y.,Dong J.,and Tao W.,2017,MAP kinase cascades regulate the cold response by modulating ICE1 protein stability,Dev.Cell,43(5):618-629
