油葵种子特异启动子Ha ds10 G1的克隆及功能鉴定
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摘要
从油葵中克隆得到LEA蛋白基因家族Ha ds10 G1基因的启动子序列,并对其进行功能分析。利用PCR技术从油葵品种"矮大头"基因组DNA中分离Ha ds10 G1基因上游的调控序列,将其与GUS基因融合,构建种子特异性表达载体p BI121-PHa ds10,通过根癌农杆菌介导法转化烟草(Nicotiana tabacum)NC89,对再生植株进行PCR、RT-PCR和GUS组织化学分析,以检测GUS基因在转基因烟草中的表达情况。结果表明,油葵Ha ds10 G1基因启动子长度为1 417 bp,与已报道的向日葵Ha ds10G1基因启动子序列同源性为89.42%。作用元件分析发现该区域除了具有启动子核心调控序列外,还含有多个与组织特异性、激素、逆境等表达相关的顺式作用元件,如RY重复元件、ABRE元件、TC-rich元件等。转基因植株的PCR结果显示,成功地获得了转基因阳性植株;GUS活性检测表明,该启动子序列仅能够驱动GUS基因在烟草种子表达,而在根、茎、叶等组织中均未检测到GUS基因表达。因此,油葵LEA蛋白基因家族Ha ds10 G1基因上游1 417 bp片段具有种子特异性启动子功能。研究结果为油葵等油料作物的油脂遗传改良提供组织特异性启动子。
The objective of this work is to clone and identify the seed-specific promoter of Ha ds10 G1 in a LEA(late embryogenesisabundant)protein gene family from oil sunflower. The upstream regulatory region of Ha ds10 G1 gene was isolated from the genomic DNA ofsunflower "Aidatou" by PCR method. The cloned region was fused to the GUS reporter gene,and plant expression vector p BI121-PHa ds10 was constructed,which was introduced into Nictiana tabacum NC89 by medicating Agrobacterium tumefaciens. The expression of GUS genein the transgenic tobacco plants was detected by PCR,RT-PCR and GUS. As results,the gene promoter of Ha ds10 G1 was 1 417 bp,andit had homology with the reported sequence at 89.42%. Cis-acting elements search in the segment revealed that in addition to kernel regulatorysequences of promoter,there was the presence of a number of motifs related to tissue specificity,hormone,and adverse environment,suchas RY-repeat elements,ABRE motif and TC-rich element,etc. PCR results showed that the transgenic plants were obtained successfully. GUSactivity assays indicated that this promoter drove the expression of GUS gene only in tobacco seed embryo,and not in the other tissues such asroots,stems and leaves. Conclusively,the cloned 1 417 bp upstream regulatory region of Ha ds10 G1 gene from the LEA protein gene family ofoil sunflower is seed-specific promoter. The results may provide the tissue-specific promoter for genetic improvement of oilseed crops such as oilsunflower.
The objective of this work is to clone and identify the seed-specific promoter of Ha ds10 G1 in a LEA(late embryogenesisabundant)protein gene family from oil sunflower. The upstream regulatory region of Ha ds10 G1 gene was isolated from the genomic DNA ofsunflower "Aidatou" by PCR method. The cloned region was fused to the GUS reporter gene,and plant expression vector p BI121-PHa ds10 was constructed,which was introduced into Nictiana tabacum NC89 by medicating Agrobacterium tumefaciens. The expression of GUS genein the transgenic tobacco plants was detected by PCR,RT-PCR and GUS. As results,the gene promoter of Ha ds10 G1 was 1 417 bp,andit had homology with the reported sequence at 89.42%. Cis-acting elements search in the segment revealed that in addition to kernel regulatorysequences of promoter,there was the presence of a number of motifs related to tissue specificity,hormone,and adverse environment,suchas RY-repeat elements,ABRE motif and TC-rich element,etc. PCR results showed that the transgenic plants were obtained successfully. GUSactivity assays indicated that this promoter drove the expression of GUS gene only in tobacco seed embryo,and not in the other tissues such asroots,stems and leaves. Conclusively,the cloned 1 417 bp upstream regulatory region of Ha ds10 G1 gene from the LEA protein gene family ofoil sunflower is seed-specific promoter. The results may provide the tissue-specific promoter for genetic improvement of oilseed crops such as oilsunflower.
引文
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[17]Bhunia RK,Chakraborty A,Kaur R,et al.Seed-specific increasedexpression of 2S albumin promoter of sesame qualifies it as auseful genetic tool for fatty acid metabolic engineering and relatedtransgenic intervention in sesame and other oil seed crops[J].Plant Mol Biol,2014,86:351-365.
[18]Arun M,Subramanyam K,Theboral J,et al.Transfer and targetedoverexpression ofγ-tocopherol methyltransferase(γ-TMT)geneusing seed-specific promoter improves tocopherol composition inIndian soybean cultivars[J].Appl Biochem Biotechnol,2014,172(4):1763-1776.
[19]Sohrabi M,Zebarjadi A,Najaphy A,et al.Isolation and sequenceanalysis of napin seed specific promoter from Iranian Rapeseed(Brassica napus L.)[J].Gene,2015,563(2):160-164.
[20]Yoshino M,Tsutsumi K,Kanazawa A.Profiles of embryonic nuclearprotein binding to the proximal promoter region of the soybeanβ-conglycininαsubunit gene[J].Plant Biol(Stuttg),2015,17(1):147-152.
[21]Reidt W,Wohlfarth T,Ellerstrom M,et al.Gene regulation duringlate embryogenesis:the RY motif of maturation-specific genepromoters is a direct target of the FUS3 gene product[J].Plant J,2000,21:401-408.
[22]Nie DM,Ouyang YD,Wang X,et al.Genome-wide analysis ofendosperm-specific genes in rice[J].Gene,2013,530(2):236-247.
[2]Senkoylu N,Dale N.Nutritional evaluation of a high-oil sunflowermeal in broiler starter diets[J].J Appl Poult Res,2006,15:40-47.
[3]Sujatha A,Vijay S,Vasavi S,et at.Agrobacterium-mediated transformation of cotyledons of mature seeds of multiple genotypes of sunflower(Helianthus annuus L.)[J].Plant Cell,Tissue and OrganCulture(PCTOC),2012,110(2):275-287.
[4]Lewi DM,Hopp HE,Escandon AS.Sunflower(Helianthus annuusL.)[J].Methods Mol Biol,2006,343:291-297.
[5]Skori?D,Joci?S,Sakac Z,et al.Genetic possibilities for alteringsunflower oil quality to obtain novel oils[J].Can J PhysiolPharmacol,2008,86(4):215-221.
[6]Tishchenko EN,Komisarenko AG,Mikhal’skaia SI,et al.Agrobacterium-mediated sunflower transformation(Helianthus annuus L.)in vitro and in Planta using strain of LBA4404 harboringbinary vector p Bi2E with ds RNA-suppressor proline dehydrogenasegene[J].Tsitol Genet,2014,48(4):19-30.
[7]Covey SN,Lomonossoff GP,Hull R.Characterisation of cauliflowermosaic virus DNA sequences which encode major polyadenylatedtranscripts[J].Nucleic Acids Res,1981,9:6735-6748.
[8]李田,孙景宽,刘京涛.植物启动子研究进展[J].生物技术通报,2015,31(2):18-25.
[9]Zhao Y,Sha W,Wang QY,et al.Molecular cloning and activityanalysis of a seed-specific FAD2-1B gene promoter from Glycine max[J].Cell Mol Biol,2015,61(4):85-89.
[10]Hincha DK,Thalhammer A.LEA proteins:IDPs with versatilefunctions in cellular dehydration tolerance[J].Biochem SocTrans,2012,40(5):1000-1003.
[11]Dure L.A repeating 11-mer amino acid motif and plantdesiccation[J].Plant J,1993,3(3):363-369.
[12]Almoguera C,Jordano J.Developmental and environmentalconcurrent expression of sunflower dry-seed-stored low-molecularweight heat-shock protein and Lea m RNAs[J].Plant Mol Biol,1992,19(5):781-792.
[13]Prieto-Dapena P,Almoguera C,Rojas A,et al.Seed-specificexpression patterns and regulation by ABI3 of an unusual lateembryogenesis-abundant gene in sunflower[J].Plant Mol Biol,1999,39:615-627.
[14]Doyle JJ,Doyle JL.A rapid DNA isolation procedure for smallquantities of fresh leaf tissue[J].Phytochem Bull,1987,19:11-15.
[15]Horsch RB,Fry JE,Hoffman NL.A simple and general method fortransferring gene into plant[J].Science,1985,227(4691):1229-1231.
[16]Jefferson RA,Kavanagh TA,Bevan MW.GUS fusions:β-glucuronidase as a sensitive and versatile gene fusion marker inhigher plants[J].EMBO J,1987,6(13):3901-3907.
[17]Bhunia RK,Chakraborty A,Kaur R,et al.Seed-specific increasedexpression of 2S albumin promoter of sesame qualifies it as auseful genetic tool for fatty acid metabolic engineering and relatedtransgenic intervention in sesame and other oil seed crops[J].Plant Mol Biol,2014,86:351-365.
[18]Arun M,Subramanyam K,Theboral J,et al.Transfer and targetedoverexpression ofγ-tocopherol methyltransferase(γ-TMT)geneusing seed-specific promoter improves tocopherol composition inIndian soybean cultivars[J].Appl Biochem Biotechnol,2014,172(4):1763-1776.
[19]Sohrabi M,Zebarjadi A,Najaphy A,et al.Isolation and sequenceanalysis of napin seed specific promoter from Iranian Rapeseed(Brassica napus L.)[J].Gene,2015,563(2):160-164.
[20]Yoshino M,Tsutsumi K,Kanazawa A.Profiles of embryonic nuclearprotein binding to the proximal promoter region of the soybeanβ-conglycininαsubunit gene[J].Plant Biol(Stuttg),2015,17(1):147-152.
[21]Reidt W,Wohlfarth T,Ellerstrom M,et al.Gene regulation duringlate embryogenesis:the RY motif of maturation-specific genepromoters is a direct target of the FUS3 gene product[J].Plant J,2000,21:401-408.
[22]Nie DM,Ouyang YD,Wang X,et al.Genome-wide analysis ofendosperm-specific genes in rice[J].Gene,2013,530(2):236-247.