棉花GhFAD2-1基因5’UTR内含子的克隆与序列分析
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摘要
【目的】对棉花~(Δ12)-油酸去饱和酶基因Gh FAD2-1 5'UTR区的内含子进行克隆和序列分析,为研究Gh FAD2-1的表达调控奠定基础。【方法】利用5'RACE技术,克隆Gh FAD2-1的5'UTR序列,结合棉花基因组序列,克隆Gh FAD2-1 5'UTR内含子,并利用PLACE等生物信息学软件对其顺式作用原件进行分析。【结果】棉花A、D基因组的Gh FAD2-1 5'UTR中各含一内含子序列,全长分别为1 103 bp、1 111 bp;Gh FAD2-1成熟mRNA的5'UTR为77bp,转录的起点碱基为T;5'UTR内含子两个剪切位点分别AA-GG、CA-GC。该内含子包括一些典型的与光响应相关的作用元件,以及与激素和胁迫因素相关的应答元件等。【结论】克隆获得了棉花A、D基因组的Gh FAD2-1基因5'UTR内含子序列;明确其转录的起点碱基及5'UTR内含子的剪切位点,为进一步在分子水平上研究Gh FAD2-1功能及其表达调控规律,为植物的遗传改良奠定了基础。
【Objective】Cloning and sequence analysis of the 5 'UTR intron of the delta 12-oleic acid desaturase gene Gh FAD2-1 would lay the foundation for the study of the expression and regulation of Gh FAD2-1. 【Method】In this study,the 5'UTR sequence of Gh FAD2-1 was cloned by 5 'RACE technology. And then,5'UTR intron of Gh FAD2-1 was cloned in combination with the cotton genome sequence. Cis elements were also analyzed by PLACE and other bioinformatics software.【Result】The Gh FAD2-1 5'UTR sequence 77 bp was obtained from Gossypium hirsutum using 5 'RACE technique. The result showed that the transcription start site is T based on a combination of 5'UTR and genome sequence analysis. Gh FAD2-1 5'UTR contained a full-length 1,103 bp intron sequence in the A genome of cotton. The full-length sequence of Gh FAD2-1 5'UTR intron was 1,111 bp in D genome. The cleavage sites of 5 ' UTR intron were AA-GG,CA-GC,respectively. Cis element analysis showed that the intron included some typical function and optical response related elements and elements related to hormone regulation or stress response. 【Conclusion】5 'UTR intron sequences of Gh FAD2-1 gene were cloned from the A and D genome,respectively. The transcription start site and the cleavage sites of 5' UTR intron were also identified. The results have laid a foundation to further study expression and regulation of Gh FAD2-1 at the molecular level.
【Objective】Cloning and sequence analysis of the 5 'UTR intron of the delta 12-oleic acid desaturase gene Gh FAD2-1 would lay the foundation for the study of the expression and regulation of Gh FAD2-1. 【Method】In this study,the 5'UTR sequence of Gh FAD2-1 was cloned by 5 'RACE technology. And then,5'UTR intron of Gh FAD2-1 was cloned in combination with the cotton genome sequence. Cis elements were also analyzed by PLACE and other bioinformatics software.【Result】The Gh FAD2-1 5'UTR sequence 77 bp was obtained from Gossypium hirsutum using 5 'RACE technique. The result showed that the transcription start site is T based on a combination of 5'UTR and genome sequence analysis. Gh FAD2-1 5'UTR contained a full-length 1,103 bp intron sequence in the A genome of cotton. The full-length sequence of Gh FAD2-1 5'UTR intron was 1,111 bp in D genome. The cleavage sites of 5 ' UTR intron were AA-GG,CA-GC,respectively. Cis element analysis showed that the intron included some typical function and optical response related elements and elements related to hormone regulation or stress response. 【Conclusion】5 'UTR intron sequences of Gh FAD2-1 gene were cloned from the A and D genome,respectively. The transcription start site and the cleavage sites of 5' UTR intron were also identified. The results have laid a foundation to further study expression and regulation of Gh FAD2-1 at the molecular level.
引文
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[3]牛俊奇,吴朝兴,杨丽涛,等.甘蔗So NIN1基因结构及其内含子信息分析[J].生物技术通报,2014,(12):147-152.Niu Jun-qi,Wu Chao-xing,Yang Li-tao,et al.(2014).Analysis of Genomic Structure and Introns of So NIN1 Gene from Sugarcane[J].Biotechnology Bulletin,(12):147-152.(in Chinese)
[4]李秀兰,姜曰水.高等植物细胞器Ⅱ类内含子剪接的研究进展[J].植物生理学报,2017,53(8):1 365-1 371.LI Xiu-Lan,JIANG Yue-Shui.(2017).Research progress of group II intron splicing in higher plant organelles[J].Plant Physiology Journal,53(8):1,365-1,371.(in Chinese)
[5]Zhang,J.,Liu,H.,Sun,J.,Li,B.,Zhu,Q.,&Chen,S.,et al.(2012).Arabidopsis fatty acid desaturase fad2 is required for salt tolerance during seed germination and early seedling growth.Plos One,7(1):e30355.
[6]Matteucci,M.,D'Angeli,S.,Errico,S.,Lamanna,R.,Perrotta,G.,&Altamura,M.M.(2011).Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of olea europaea l.genotypes with different cold hardiness.Journal of Experimental Botany,62(10):3,403-3,402.
[7]Kargiotidou,A.,Deli,D.,Galanopoulou,D.,Tsaftaris,A.,&Farmaki,T.(2008).Low temperature and light regulate delta 12 fatty acid desaturases(fad2)at a transcriptional level in cotton(gossypium hirsutum).Journal of Experimental Botany,59(8):2,043-2,056.
[8]Donath,M.,Mendel,R.,Cerff,R.,&Martin,W.(1995).Intron-dependent transient expression of the maize gapa1,gene.Plant Molecular Biology,28(4):667-676.
[9]Callis,J.,Fromm,M.,&Walbot,V.(1987).Introns increase gene expression in cultured maize cells.Genes&Development,1(10):1,183-1,200.
[10]Maas,C.,Laufs,J.,Grant,S.,Korfhage,C.,&Werr,W.(1991).The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron1 enhances reporter gene expression up to 1,000-fold.Plant Molecular Biology,16(2):199-207.
[11]Morello,L.,Bardini,M.,Cricrì,M.,Sala,F.,&Breviario,D.(2006).Functional analysis of dna sequences controlling the expression of the rice oscdpk2,gene.Planta,223(3):479-491.
[12]Jeon Jong Seong,Lee Si Chul,Jung Ki Hong,Jun Sung Hoon,Kim Chan Hong,&An Gyn Heung.(2000).Tissue-preferential expression of a riceα-tubulin gene,ostuba1,mediated by the first intron.Plant Physiology,123(3):1,005-1,014.
[13]Dean,C.,Favreau,M.,Bond-Nutter,D.,Bedbrook,J.,&Dunsmuir,P.(1989).Sequences downstream of translation start regulate quantitative expression of two petunia rbcs genes.Plant Cell,1(2):201-208.
[14]Leon,P.,&Walbot,V.(1991).Transient gene expression in protoplasts of phaseolus vulgaris isolated from a cell suspension culture.Plant Physiology,95(3):968-972.
[15]Bourdon,V.,Harvey,A.,&Lonsdale,D.M.(2001).Introns and their positions affect the translational activity of mrna in plant cells.Embo Reports,2(5):394-398.
[16]Vasil,V.,Clancy,M.,Ferl,R.J.,Vasil,I.K.,&Hannah,L.C.(1989).Increased gene expression by the first intron of maize shrunken-1 locus in grass species.Plant Physiology,91(4):1,575-1,579.
[2]Liu,Q.,Brubaker,C.L.,Green,A.G.,Marshall,D.R.,Sharp,P.J.,&Singh,S.P.(2001).Evolution of the fad2-1 fatty acid desaturase 5'utr intron and the molecular systematics of gossypium(malvaceae).American Journal of Botany,88(1):92-102.
[3]牛俊奇,吴朝兴,杨丽涛,等.甘蔗So NIN1基因结构及其内含子信息分析[J].生物技术通报,2014,(12):147-152.Niu Jun-qi,Wu Chao-xing,Yang Li-tao,et al.(2014).Analysis of Genomic Structure and Introns of So NIN1 Gene from Sugarcane[J].Biotechnology Bulletin,(12):147-152.(in Chinese)
[4]李秀兰,姜曰水.高等植物细胞器Ⅱ类内含子剪接的研究进展[J].植物生理学报,2017,53(8):1 365-1 371.LI Xiu-Lan,JIANG Yue-Shui.(2017).Research progress of group II intron splicing in higher plant organelles[J].Plant Physiology Journal,53(8):1,365-1,371.(in Chinese)
[5]Zhang,J.,Liu,H.,Sun,J.,Li,B.,Zhu,Q.,&Chen,S.,et al.(2012).Arabidopsis fatty acid desaturase fad2 is required for salt tolerance during seed germination and early seedling growth.Plos One,7(1):e30355.
[6]Matteucci,M.,D'Angeli,S.,Errico,S.,Lamanna,R.,Perrotta,G.,&Altamura,M.M.(2011).Cold affects the transcription of fatty acid desaturases and oil quality in the fruit of olea europaea l.genotypes with different cold hardiness.Journal of Experimental Botany,62(10):3,403-3,402.
[7]Kargiotidou,A.,Deli,D.,Galanopoulou,D.,Tsaftaris,A.,&Farmaki,T.(2008).Low temperature and light regulate delta 12 fatty acid desaturases(fad2)at a transcriptional level in cotton(gossypium hirsutum).Journal of Experimental Botany,59(8):2,043-2,056.
[8]Donath,M.,Mendel,R.,Cerff,R.,&Martin,W.(1995).Intron-dependent transient expression of the maize gapa1,gene.Plant Molecular Biology,28(4):667-676.
[9]Callis,J.,Fromm,M.,&Walbot,V.(1987).Introns increase gene expression in cultured maize cells.Genes&Development,1(10):1,183-1,200.
[10]Maas,C.,Laufs,J.,Grant,S.,Korfhage,C.,&Werr,W.(1991).The combination of a novel stimulatory element in the first exon of the maize shrunken-1 gene with the following intron1 enhances reporter gene expression up to 1,000-fold.Plant Molecular Biology,16(2):199-207.
[11]Morello,L.,Bardini,M.,Cricrì,M.,Sala,F.,&Breviario,D.(2006).Functional analysis of dna sequences controlling the expression of the rice oscdpk2,gene.Planta,223(3):479-491.
[12]Jeon Jong Seong,Lee Si Chul,Jung Ki Hong,Jun Sung Hoon,Kim Chan Hong,&An Gyn Heung.(2000).Tissue-preferential expression of a riceα-tubulin gene,ostuba1,mediated by the first intron.Plant Physiology,123(3):1,005-1,014.
[13]Dean,C.,Favreau,M.,Bond-Nutter,D.,Bedbrook,J.,&Dunsmuir,P.(1989).Sequences downstream of translation start regulate quantitative expression of two petunia rbcs genes.Plant Cell,1(2):201-208.
[14]Leon,P.,&Walbot,V.(1991).Transient gene expression in protoplasts of phaseolus vulgaris isolated from a cell suspension culture.Plant Physiology,95(3):968-972.
[15]Bourdon,V.,Harvey,A.,&Lonsdale,D.M.(2001).Introns and their positions affect the translational activity of mrna in plant cells.Embo Reports,2(5):394-398.
[16]Vasil,V.,Clancy,M.,Ferl,R.J.,Vasil,I.K.,&Hannah,L.C.(1989).Increased gene expression by the first intron of maize shrunken-1 locus in grass species.Plant Physiology,91(4):1,575-1,579.