山茱萸转录因子CobHLH7基因的克隆及分析
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摘要
目的克隆山茱萸转录因子Cob HLH7的c DNA序列。方法以转录组数据中unigenec15204_g1序列为参考,在其开放阅读框两端设计特异引物,提取山茱萸果实总RNA,并反转录成c DNA,以此为模板,经RT-PCR扩增、连接pMD19-T克隆载体并测序,获得Cob HLH7的cDNA序列。通过Protparatam、Prot Scale、SOPMA等软件,对其进行生物信息学分析。结果 Cob HLH7的cDNA长度为941 bp,编码266个氨基酸,其相对分子质量为29 220,等电点为6.32。经SOPMA在线软件预测,该蛋白为不稳定蛋白,其高级结构以α-螺旋和无规卷曲为主,含有少量的β-转角和延伸带。多序列比对和亲缘关系比较显示,Cob HLH7氨基酸序列与马铃薯和模式植物烟草的bHLH类转录因子亚家族的ILR3同源性较高。结论在首次对山茱萸叶片和果实转录组测序的基础上,对筛选并预测的可能与环烯醚萜苷合成相关的转录因子Cob HLH7进行克隆与生物信息学分析,为进一步研究其功能奠定基础。
Objective Cornus officinalis is a commonly used medicinal material in our country. In the study, the CobHLH7 was cloned and analyzed, which might be closely related to the iridoid glycosides synthesis, based on the transcriptome sequencing of Cornus officinalis fruits. Methods The special primers were designed from the both sides of open reading frame of unigene c15204_g1. The total RNA was extracted and reversed transcribed into cDNA. The transcription factor CobHLH7 was cloned through RT-PCR method,and the pMD19-T cloning vector were used for sequencing. A series of bioinformatics analysis of Cob HLH7 was performed by software Protparatam, ProtScale, and SOPMA etc. Results The cDNA of CobHLH7 gene was 941 bp in length, encoding 266 amino acids with a molecular weight of 29 220 and isoelectric point of 6.32. The analysis of bioinformatics through SOPMA showed that the protein was a neutral unstable protein. Its advanced structure mainly was alpha helix and random coil, and the content of beta turn and extended strand were less. Multiple sequence alignments and phylogenetic trees showed that CobHLH7 protein has high homology with ILR3 of Solanum tuberosum, ILR3-like of model organism Nicotiana attenuate. Conclusion For the first time, the CobHLH7 cDNA sequence was cloned and analyzed successfully from C. officinalis, which would lay the foundation for studying its biological functions deeply.
Objective Cornus officinalis is a commonly used medicinal material in our country. In the study, the CobHLH7 was cloned and analyzed, which might be closely related to the iridoid glycosides synthesis, based on the transcriptome sequencing of Cornus officinalis fruits. Methods The special primers were designed from the both sides of open reading frame of unigene c15204_g1. The total RNA was extracted and reversed transcribed into cDNA. The transcription factor CobHLH7 was cloned through RT-PCR method,and the pMD19-T cloning vector were used for sequencing. A series of bioinformatics analysis of Cob HLH7 was performed by software Protparatam, ProtScale, and SOPMA etc. Results The cDNA of CobHLH7 gene was 941 bp in length, encoding 266 amino acids with a molecular weight of 29 220 and isoelectric point of 6.32. The analysis of bioinformatics through SOPMA showed that the protein was a neutral unstable protein. Its advanced structure mainly was alpha helix and random coil, and the content of beta turn and extended strand were less. Multiple sequence alignments and phylogenetic trees showed that CobHLH7 protein has high homology with ILR3 of Solanum tuberosum, ILR3-like of model organism Nicotiana attenuate. Conclusion For the first time, the CobHLH7 cDNA sequence was cloned and analyzed successfully from C. officinalis, which would lay the foundation for studying its biological functions deeply.
引文
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[23]Zhang H T,Hedhili S,Montiel G,et al.The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus[J].Plant J,2011,67(1):61-71.
[24]van Moerkercke A,Steensma P,Schwezer F,et al.The bHLH transcription factor BIS1 controls the irodoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus[J].PNAS,2015,112(26):8130-8135.
[25]刘芳朋.小麦转录因子基因TabHLH1的分子特征和生物学功能[D].保定:河北农业大学,2012.
[26]Zhang J,Liu B,Li M,et al.The bHLH transcription factor b HLH104 interacts with IAA-LEUCINERESISTANT3 and modulates iron homeostasis in Arabidopsis[J].Plant Cell,2015,27(3):787-805.
[27]Hou D Y,Shi L C,Yang M M,et al.De novo transcriptomic analysis of leaf and fruit tissue of Cornus officinalis using Illumina platform[J].PLoS One,2018,13(2):e0192610.
[28]Zhang W,Sun Y,Timofejeva L,et al.Regulation of Arabidopsis tapetum development and function by Dysfunctional Tapetum1(DYT1)encoding a putative bHLH transcription factor[J].Development,2006,133(16):3085-3095.
[29]Liang G,Zhang H,Li X,et al.bHLH transcription factor b HLH115 regulates iron homeostasis in Arabidopsis thaliana[J].J Exp Bot,2017,68(7):1743-1755.
[30]LI X,Zhang H,Ai Q,et al.Two bHLH transcription factors,bHLH34 and bHLH104,regulate iron homeostasis in Arabidopsis thaliana[J].Plant Physiol,2016,170(4):2478-2493.
[31]张凯伦,罗祖良,郭玉华,等.b HLH转录因子调控药用植物萜类化合物生物合成的研究进展[J].中国现代中药,2017,19(1):142-147.
[2]Ma W,Wang K J,Cheng C S,et al.Bioactive compounds from Cornus officinalis fruits and their effects on diabetic nephropathy[J].J Ethnopharmacol,2014,153(3):840-845.
[3]Park J Y,Han A R,Kil Y S,et al.A new secoiridoid glycoside from the fruits of Cornus officinalis(Cornaceae)[J].Nat Prod Res,2016,30(13):1504-1510.
[4]李红,任莹莹,司俊玲,等.山茱萸籽多糖的提取工艺研究[J].食品工业,2013,34(7):46-49.
[5]中国药典[S].一部.2015.
[6]贾羲,苏成福,董诚明.山茱萸提取物抗肿瘤作用及机制探讨[J].中国实验方剂学杂志,2016,22(20):117-121.
[7]邹品文,赵春景,李攀,等.山茱萸多糖的抗肿瘤作用及其免疫机制[J].中国医院药学杂志,2012,32(1):20-22.
[8]Jiang W L,Zhang S M,Tang X X,et al.Protective roles of cornuside in acute myocardial ischemia and referfusion injury in rats[J].Phytomedicine,2011,18:266-271.
[9]Jiang W L,Zhang S P,Jenssen T.Effects of loganin on experiemental diabetic nephropathy[J].Phytomedicine,2012,19:217-222.
[10]郭洁,张晓双,刘继平.山茱萸环烯醚萜总苷对晚期糖基化终末产物诱导的肾系膜炎症反应的调节[J].中成药,2013,35(10):2067-2072.
[11]Ma W,Wang K J,Cheng C S,et al.Bioactive compounds from Comus officinalis fruits and their effects on diabetic nephropathy[J].J Ethnopharmacol,2014,153(3):840-845.
[12]Hwang E S,Kim H B,Lee S,et al.Loganin enhances long-term potentiation and recovers scopolamine-induced learning and memory impairments[J].Physiol Behav,2017,171(1):243-248.
[13]Lee K Y,Sung S H,Kim S H,et al.Cognitive-enhancing activity of loganin isolated from Cornus officinalis in scopolamine-induced amnesic mice[J].Arch Pharm Res,2009,32(5):677-683.
[14]周力,马齐兵,陈哲,等.山茱萸籽栲胶制备工艺的研究[J].食品工业,2016,37(8):35-40.
[15]Dong Y,Wang C P,Han X,et al.A novel b HLHtranscription factor Peb HLH35 from Populus euphratica confers drought through regulating stomatal development,photosynthesis and growth in Arabidopsis[J].Biochem Biophys Res Commun,2014,450(1):453-458.
[16]Anjum S A,Wang L,Farooq M,et al.Methyl jasmonate-induced alteration in lipid peroxidation,antioxidative defence system and yield in soybean under drought[J].J Agron Crop Sci,2011,197(4):296-301.
[17]Xu M,Dong J,Zhu M.NO mediates fungal elicitor puerarin in kudzu suspension cell biosynthesis by salicylic acid(SA)or jasmonic acid(JA)signaling pathways[J].Sci Sin Vit,2006,36(1):66-75.
[18]Gremski K,Ditta G,Yanofsky M F,et al.The HECATEgenes regulate female reproductive tract development in Arabidopsis thaliana[J].Development,2007,134:3593-3601.
[19]Hong G J,Xue X Y,Mao Y B,et al.Arabidopsis MYC2interacts with DELLA proteins in regulating sesquiterpene synthase gene expression[J].Plant Cell,2011,24(6):2635-2648.
[20]Kiribuchi K,Suginoro M,Takeda M,et al.RERJ1 a jasmonic acid-responsive gene from rice,encodes a basic helix-loop-helix protein[J].Biochem Biophys Res Commun,2004,325:857-863.
[21]Ji Y P,Xiao J W,Shen Y L,et al.Cloning and characterization of AabHLH,a bHLH transcription factor that positively regulates artemisinin biosynthesis in Artemisia annua[J].Plant Cell Physiol,2014,55(9):1592-1604.
[22]Pre M,Siberil Y,Memelink J,et al.Isolation by the yeast one-hybrid System of c DNAs encoding transcription factor that bind to the G-Box element of the strictosidine synthase gene promoter from Catharanthus roseus[J].JExp Bot,2003,54(392):2587-2588.
[23]Zhang H T,Hedhili S,Montiel G,et al.The basic helix-loop-helix transcription factor CrMYC2 controls the jasmonate responsive expression of the ORCA genes that regulate alkaloid biosynthesis in Catharanthus roseus[J].Plant J,2011,67(1):61-71.
[24]van Moerkercke A,Steensma P,Schwezer F,et al.The bHLH transcription factor BIS1 controls the irodoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus[J].PNAS,2015,112(26):8130-8135.
[25]刘芳朋.小麦转录因子基因TabHLH1的分子特征和生物学功能[D].保定:河北农业大学,2012.
[26]Zhang J,Liu B,Li M,et al.The bHLH transcription factor b HLH104 interacts with IAA-LEUCINERESISTANT3 and modulates iron homeostasis in Arabidopsis[J].Plant Cell,2015,27(3):787-805.
[27]Hou D Y,Shi L C,Yang M M,et al.De novo transcriptomic analysis of leaf and fruit tissue of Cornus officinalis using Illumina platform[J].PLoS One,2018,13(2):e0192610.
[28]Zhang W,Sun Y,Timofejeva L,et al.Regulation of Arabidopsis tapetum development and function by Dysfunctional Tapetum1(DYT1)encoding a putative bHLH transcription factor[J].Development,2006,133(16):3085-3095.
[29]Liang G,Zhang H,Li X,et al.bHLH transcription factor b HLH115 regulates iron homeostasis in Arabidopsis thaliana[J].J Exp Bot,2017,68(7):1743-1755.
[30]LI X,Zhang H,Ai Q,et al.Two bHLH transcription factors,bHLH34 and bHLH104,regulate iron homeostasis in Arabidopsis thaliana[J].Plant Physiol,2016,170(4):2478-2493.
[31]张凯伦,罗祖良,郭玉华,等.b HLH转录因子调控药用植物萜类化合物生物合成的研究进展[J].中国现代中药,2017,19(1):142-147.