一个水稻胚乳特异表达基因的筛选及初步分析
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摘要
在已有的启动子捕获体系的基础上,我们构建了无启动子的T-DNA插入捕获载体pGUSA-1300。将该载体通过农杆菌介导的方法转化籼稻品种明恢86(Oryzasativa.Indica.Minghui86),构建籼稻启动子捕获系突变体库,对其中2653个T0代独立启动子捕获系的根、茎、叶、花、胚和胚乳等组织进行β-葡糖醛酸糖苷酶(glucuronidase,gus)染色检测,获得了61个GUS活性表现的启动子捕获系,其中胚乳中特异表达的有20个。
w9101是一个T_0及T_1代GUS组织化学检测均表现胚乳特异表达的启动子捕获系,其T_0、T_1代自交后代标记基因分离分析及Southern blot分析表明它是一个单T-DNA插入的启动子捕获系;通过TAIL-PCR获得其T-DNA插入的侧翼序列,并在NCBI上对其侧翼序列进行比对,发现该启动子捕获系T-DNA插入位点位于一个推测的寡肽转运子基因(暂命名为Peptide Transorter 9101,PTR9101)启动子内;w9101不同发育时期不同组织RT-PCR检测表明,PTR9101为胚乳特异性表达基因:生物信息学分析表明PTR9101蛋白整条肽链表现疏水性,其跨膜区域和CHL1(The Nitrate TransporterAtNRT1.1)相似有11个α—螺旋跨膜区,因此推测PTR9101是一个寡肽转运子基因。水稻胚乳由一群高度特化的细胞组成,结合PTR9101基因家族已报道的功能,由此推测PTR9101基因可能与寡肽运输有关。
基于候选基因信息和上游调控序列,结合PLANTCARE软件分析,我们分别构建了PTR9101的cDNA正义表达载体Gt1 PTR9101-1300、PTR9101上游启动子区与GUS报告基因融合的表达载体PTR9101P-GUS,以期进一步了解它们的表达特征及其生物学功能。
We utilized gene trap method to isolate rice target gene, plasmid pGUS1300 was constructed to develop a gene trap system which contained a promoterlessβ-glucuronidase (GUS) reporter gene. We transformed the plasmid pGUS1300 into rice Oryza sativa. Indica Minghui86 which was mediated by Agrobacterium tumefaciens, and got 2653 independent transgenic lines. We detected 61 GUS-positive transgenic plants in different tissues and organs in 2653 independent transgenic lines, and 20 were endosperm-specific.
Using the promoter trap method, we identified a rice mutant plant, named w9101, in which theβ-glucuronidase (GUS) reporter gene was expressed endosperm-specificly in both T_0 and T_1 generations. A single copy of T-DNA was inserted into the plant; and the flanking sequences contiguous to the T-DNA were isolated by TAIL-PCR, and the BLAST results showed that the T-DNA of w9101 was inserted into the promoter motifs of a putative Peptide Transorter gene, temporarily named PTR9101; different tissue and developmental period RT-PCR analysis revealed that PTR9101 protein specifically expressed in rice endosperm; Further analysis by bioinformatics method indicated that PTR9101 protein was a hydrophobic protein with 11α- helix membrane-spanning domain, which was similar with that of CHL1 (The Nitrate Transporter AtNRT1.1) in Arabidopsis. These results suggested the PTR9101 was a hydrophobic peptide transporter gene, might regulate the cell development in endosperm.
Based on the bioinformatics analysis of PTR9101 gene and its upper regulation sequence, we constructed a PTR9101 gene c-DNA sense expression vector Gt1 PTR9101-1300 and a fusing expression vector PTR9101P-GUS with PTR9101 promoter sequences and GUS respectively to research more.
w9101是一个T_0及T_1代GUS组织化学检测均表现胚乳特异表达的启动子捕获系,其T_0、T_1代自交后代标记基因分离分析及Southern blot分析表明它是一个单T-DNA插入的启动子捕获系;通过TAIL-PCR获得其T-DNA插入的侧翼序列,并在NCBI上对其侧翼序列进行比对,发现该启动子捕获系T-DNA插入位点位于一个推测的寡肽转运子基因(暂命名为Peptide Transorter 9101,PTR9101)启动子内;w9101不同发育时期不同组织RT-PCR检测表明,PTR9101为胚乳特异性表达基因:生物信息学分析表明PTR9101蛋白整条肽链表现疏水性,其跨膜区域和CHL1(The Nitrate TransporterAtNRT1.1)相似有11个α—螺旋跨膜区,因此推测PTR9101是一个寡肽转运子基因。水稻胚乳由一群高度特化的细胞组成,结合PTR9101基因家族已报道的功能,由此推测PTR9101基因可能与寡肽运输有关。
基于候选基因信息和上游调控序列,结合PLANTCARE软件分析,我们分别构建了PTR9101的cDNA正义表达载体Gt1 PTR9101-1300、PTR9101上游启动子区与GUS报告基因融合的表达载体PTR9101P-GUS,以期进一步了解它们的表达特征及其生物学功能。
We utilized gene trap method to isolate rice target gene, plasmid pGUS1300 was constructed to develop a gene trap system which contained a promoterlessβ-glucuronidase (GUS) reporter gene. We transformed the plasmid pGUS1300 into rice Oryza sativa. Indica Minghui86 which was mediated by Agrobacterium tumefaciens, and got 2653 independent transgenic lines. We detected 61 GUS-positive transgenic plants in different tissues and organs in 2653 independent transgenic lines, and 20 were endosperm-specific.
Using the promoter trap method, we identified a rice mutant plant, named w9101, in which theβ-glucuronidase (GUS) reporter gene was expressed endosperm-specificly in both T_0 and T_1 generations. A single copy of T-DNA was inserted into the plant; and the flanking sequences contiguous to the T-DNA were isolated by TAIL-PCR, and the BLAST results showed that the T-DNA of w9101 was inserted into the promoter motifs of a putative Peptide Transorter gene, temporarily named PTR9101; different tissue and developmental period RT-PCR analysis revealed that PTR9101 protein specifically expressed in rice endosperm; Further analysis by bioinformatics method indicated that PTR9101 protein was a hydrophobic protein with 11α- helix membrane-spanning domain, which was similar with that of CHL1 (The Nitrate Transporter AtNRT1.1) in Arabidopsis. These results suggested the PTR9101 was a hydrophobic peptide transporter gene, might regulate the cell development in endosperm.
Based on the bioinformatics analysis of PTR9101 gene and its upper regulation sequence, we constructed a PTR9101 gene c-DNA sense expression vector Gt1 PTR9101-1300 and a fusing expression vector PTR9101P-GUS with PTR9101 promoter sequences and GUS respectively to research more.
引文
[1]张晖.水稻胚乳研究进展.现代农业科技[J].2008.22:160-162.
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[28]Paulsen IT.and Skurray RA.The POT family of transport proteins.Trends Biochem.Sci.1994,19,404.
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[31]Guo F Q,Young J,Crawford N M.The nitrate transporter AtNRT1.1 CHL1functions in stomatal opening and contributes to drought susceptibility in A rabidopsis.Plant Cell[J],2003,15(1):107-117.
[32]Alboresi A,Gestin C,Leydecker M T,et al.Nitrate,a signal relieving seed dormancy in A rabidopsis Plant Cell & Environment[J],2005,28(4):500-512.
[33]Little D Y,Rao H,Oliva S,et al.The putative high 2 affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues Proceedings of the National Academy of Sciences,USA[J],2005,102(38):13693-13698.
[34]Remans T,Nacry P,Pervent M,et al.A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to N limitation in A rabidopsis thaliana[J].Plant Physiology,2006,140(3):909-921.
[35]Chiu C C,Lin C S,Hsia A P,et al.Mutation of a nitrate transporter,AtNRT1:4,results in a reduced petiole nitrate content and altered leaf development[J].Plant Cell & Physiology,2004,45(9):1139-1148.
[36]徐海荣,邓若磊,曹云飞等.植物NO_3-转运蛋白的结构、功能及基因表达调控.草叶学报[J].2008.17(4):159-165.
[37]徐海荣,谷俊涛,路文静等。水稻新型硝酸盐转运蛋白基因.OsTNrt2.1的克隆、表达和遗传转化。河北农业大学硕士学位论文[D]。2007。
[38]Lubkowitz MA,Barnes D,Breslav M.et.al.Schizosaccharomyces pombe is p4encodes a transporter representing a novel family of oligopeptide transporters.Mol.Microbiol.1998,28,729-741.
[39]Hauser,M.,Narita,V.,Donhardt,A M.et al.Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae.Mot.Membr.Biol.2001,18,105-112.
[40]Lubkowitz,M.A.Hauser,L.,Breslav,M.et al.An oligopeptide transport gene from Candida albicans.Microbiology,1997.143,387-396.
[41]Bourbouloux A,Shahi P,Chakladar A,et al.Hgtlp,a high affinity glutathione transporter from the yeast Saccharomyces cerevisiae.J.Biol.Chem.2000,275:13259-13265.
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[2]Brink RA,Cooper D C.The endosperm in seed development.Bot Rev.1947,13:423-541.
[3]John S Greenwood,Michael Helm,Christine Gietl.et al.Ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during Ricinus seed development.PNAS,2005,2(102):2238-2243
[4]Kranz E,yon Wiegen P,Quader H.et al.Endosperm development after fusion of isolated single maize sperm and central cells in vitro.Plant Cell,1998,10:511-524.
[5]Kohler C,Hennig L,Grossniklaus U,et al.The Polycomb-group protein MEDEA regulates seed development by controlling expression of the MADS box.gene PHERES1.Genes Dev,2003,17:1540-1553.
[6]Jose F.Gutierrez-Marcos,Liliana M.Costa,Matthew M.S.Evans.et al.Maternal Gametophytic baselessl Is Required for Development of the Central Cell and Early Endosperm Patterning in Maize(Zea mays).Genetics,2006,174:317-329.
[7]Kluth A,Sp runck S,Becker D.5 'deletion of a gbssI promoter region leads to changes in tissue and developmental specificities.Plant Molecular Biology,2002,49(6):669-682.
[8]Gerhard P,Schwall Richard Safford,Roger J Wescott,et al.Nature Biotechnology,2000,18:551-556.
[9]Fujita N,Yoshida M,Kondo T,et al.Characterization of SSⅢa-Deficient Mutants of Rice:The Function of SSⅢa and Pleiotropic Effects by SSⅢa Deficiency in the Rice Endosperm.Plant Physiology,2007,144:2009-2023.
[10]Douglas A.Russell,Michael E.Fromm.Tissue specific expression in transgenic maize of four endosperm promoters from maize and rice.Transgenic Research,1997,6(2):157-168.
[11]RossakM,Smith M,Kunst L.Expression of the FAE1 gene and FAE1 promoter activity in developing seeds of Arabidopsis thaliana.Plant Molecular Biology,2001,46(6):717-725.
[12]王颖,麦维军,梁承邺等.高等植物启动子的研究进展.西北植物学报[J],2003,23(11):2040-2048.
[13]张春晓,王文棋,陈雪梅等.植物基因启动子研究进展.遗传学报[J],2004,31(12):1 455-464.
[14]Visser RGF,StolteA,Jacobsen E.Expression of a chimaeric granul-bound starch synthase-GUS gene in transgenic potato plants.Plant Mol Biol,1991,17:691-699
[15]Zhangying Wang,Xiaoping Chen,Jianhua Wang,et al.Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic.Plant Cell Tiss Organ Cult.2007,88:83-92.
[16]Ye XD,AI-Babili S,Kl(o)ti A,etal..Engineering the provitamin A(β-carotene)biosynthetic pathway into(carotenoid-free) rice endosperm.Science,2000,287(2):303-305.
[17]Takaiwa F,Takagi H,Hirose S,etal.Endosperm tissue is good production platform for artificial recombinant proteins in transgenic rice.Plant Biotechnol J,2007:5(1):84-89.
[18]An,S.et al.Generation and analysis of end-sequence database for T-DNA tagging lines in rice.Plant Physiol.2003,133:2040-2047.
[19]Yi-Fang Tsay,Julian I.Schroeder,Kenneth A.Feldmann,etal.The Herbicide Sensitivity.Gene CHLI of Arabidopsis Encodes a Nitrate-Inducible Nitrate Transporter.Cell[J],1993,72,705-713.
[20]ChiangC S,StaceyG,TsayY F.Mechanisms and functional properties of two peptide transporters,AtPTR2 and fPTR2.J Biol Chem[J].2004,279.30150-30157.
[21]Delrot.S,Atanassova R,Maurousset L.Regulation of sugar,amino acid and peptide plant membrane transporters.Biochim.Biophys.Acta.2000,1465,281-306.
[22]Gary Stacey,Serry Koh,Cheryl Granger et al.Peptide transport in plants.TREND Science.2002,7,257-263.
[23]Perry,J.R.Basrai,M.A.Steiner,H.Y.et al.Isolation and characterization of a Saccharomyces cerevisiae peptide transport gene.Mol.Cell.Biol.1994,14,104-115.
[24]Becker,J.M.and Naider,F.Transport and utilization of peptides by yeast.In Microorganisms and Nitrogen Sources(Payne J.W.,ed.),1980,257-279.
[25]Ganapathy,V.and Leibach,F.H.Proton-coupled solute transport in the animal cell plasma membrane.Curr.Opin.Cell Biol.1991,3,695-701.
[26]Payne JW.and Smith MW.Peptide transport by microorganisms.Adv.Microb.Physiol.1994,36,1-80.
[27]王华丙,张振义,包锐等.ABC转运蛋白的结构与转运机制.生命的化学[J].2007:27(3):208-210.
[28]Paulsen IT.and Skurray RA.The POT family of transport proteins.Trends Biochem.Sci.1994,19,404.
[29]Steiner,H.Y.Naider F,Becker JM.et.al.The PTR family:a new group of peptide transporters.Mol.Microbiol.1995.16,825-834.
[30]Yeung AK,Basu SK,Wu SK,et.al.Molecular identification of a role for tyrosine 167 in the function of the human intestinal proton-coupled dipeptides transporter (hPEPT1).Biochem.Biophys.Res.Commun.1998,250,103-107.
[31]Guo F Q,Young J,Crawford N M.The nitrate transporter AtNRT1.1 CHL1functions in stomatal opening and contributes to drought susceptibility in A rabidopsis.Plant Cell[J],2003,15(1):107-117.
[32]Alboresi A,Gestin C,Leydecker M T,et al.Nitrate,a signal relieving seed dormancy in A rabidopsis Plant Cell & Environment[J],2005,28(4):500-512.
[33]Little D Y,Rao H,Oliva S,et al.The putative high 2 affinity nitrate transporter NRT2.1 represses lateral root initiation in response to nutritional cues Proceedings of the National Academy of Sciences,USA[J],2005,102(38):13693-13698.
[34]Remans T,Nacry P,Pervent M,et al.A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to N limitation in A rabidopsis thaliana[J].Plant Physiology,2006,140(3):909-921.
[35]Chiu C C,Lin C S,Hsia A P,et al.Mutation of a nitrate transporter,AtNRT1:4,results in a reduced petiole nitrate content and altered leaf development[J].Plant Cell & Physiology,2004,45(9):1139-1148.
[36]徐海荣,邓若磊,曹云飞等.植物NO_3-转运蛋白的结构、功能及基因表达调控.草叶学报[J].2008.17(4):159-165.
[37]徐海荣,谷俊涛,路文静等。水稻新型硝酸盐转运蛋白基因.OsTNrt2.1的克隆、表达和遗传转化。河北农业大学硕士学位论文[D]。2007。
[38]Lubkowitz MA,Barnes D,Breslav M.et.al.Schizosaccharomyces pombe is p4encodes a transporter representing a novel family of oligopeptide transporters.Mol.Microbiol.1998,28,729-741.
[39]Hauser,M.,Narita,V.,Donhardt,A M.et al.Multiplicity and regulation of genes encoding peptide transporters in Saccharomyces cerevisiae.Mot.Membr.Biol.2001,18,105-112.
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