垫状卷柏海藻糖-6-磷酸合成酶基因（SpTPS1）的克隆及其功能分析
|
摘要
水是植物生命活动的物质基础,干旱对植物有广泛的影响。在干旱胁迫条件下,植物新陈代谢发生变化,生理活动受到阻碍,生长发育各阶段受到影响,植物出现萎蔫现象。
垫状卷柏(Selaginella pulvinata Maxim),俗称“九死还魂草”,极耐干旱。它能经受脱水与再水化的循环,一旦获得水分,植物枯黄卷缩的部分可以迅速张开,重新复活。有研究表明垫状卷柏这类复苏植物体内含有大量的应激代谢物—海藻糖。海藻糖对生物体有特殊保护作用,它能使许多生物在高温、脱水和冷冻等异常条件下仍保持原活性。以UDP-葡萄糖和6-磷酸葡萄糖为底物的海藻糖合成途径广泛存在于细菌、真菌和植物体内,其中由TPS1基因编码的海藻糖-6-磷酸合成酶是合成途径的关键酶。研究人员已在植物体内克隆出了TPS1基因,但除了在复苏植物体内检测到大量海藻糖积累外,在其他植物体内没有检测到海藻糖的存在。国内外已有将酵母和大肠杆菌的TPS1基因对植物体进行遗传转化研究,转化植株体内海藻糖含量有所增加,抗旱性得到增强,但植株在生长发育和形态上有不同程度的改变。
探索利用耐旱能力高,适宜玉米生理代谢机制与生产要求的耐旱基因,是转基因耐旱玉米培育的技术关键。本研究选择了极耐旱的垫状卷柏做为试验材料,采用RACE技术克隆海藻糖-6-磷酸合成酶基因(SpTPS1),全长3223 bp,包含一个2790bp的完整开放阅读框(ORF),并对SpTPS1基因进行核酸序列和成熟蛋白生物信息学的初步分析。
为了研究垫状卷柏海藻糖-6-磷酸合成酶基因(SpTPS1)的功能,将SpTPS1基因的开放阅读框ORF亚克隆到载体pUC119中,构建载体OpUC119。然后选择适当的酶切位点分别酶切载体OpUC119和质粒载体pRS6/AtTPS1,将ORF片段与质粒载体骨架pRS6相连,最终得到真核表达载体OpRS6。用LiAc转化法将真核表达载体OpRS6和阳性对照质粒pRS6/AtTPS1转入缺失酵母菌株tps1△,YSH290中,转化混合液涂在缺失组氨酸的半乳糖培养基上培养。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株能在缺失组氨酸的半乳糖培养基上生长。将培养基中的2%半乳糖换为2%葡萄糖,挑选阳性转化子划线于该培养基中。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在缺失组氨酸的葡萄糖培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株tps1△,由于恢复了功能,故能在含有葡萄糖的培养基上生长。提取阳性菌株质粒DNA,经PCR检测正确。通过酵母功能互补试验证明了SpTPS1基因具有编码海藻糖-6-磷酸合成酶的功能。
本研究从垫状卷柏中克隆了具有抗旱耐盐的SpTPS1基因,它作为培育抗旱耐盐新品种的抗旱基因,具有广泛的应用价值。并为后续的抗旱耐盐转基因新品种的培育提供了耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因。
Water is the fundamental material in plants, drought have a wide range of impact on plants. In drought stress conditions, plant metabolism has changed, physical activities blocked, the various stages of growth impacted, and appears wilt phenomenon.
Selaginella pulvinata, commonly known as the "resurrection grass", displays extremely tolerance to desiccation. It can survive in the dehydration and re-hydration cycle, the curling yellow parts can be quickly extended and resurvived once got water. Studies have shown that the "resurrection plant", such as Selaginella pulvinata, contains a lot of stress metabolites-trehalose. Trehalose plays a special protective role in organisms, it can maintain the biological activities, even in the abnormal conditions, such as high temperature, dehydration and freeze. The trehalose synthesis pathway, which uses UDP-glucose and 6-phosphate glucose as substrate, is the most widely distributed in eubacteria, fungi and plants, the Trehalose-6-phosphate synthase, encoded by TPS1 gene, is the key enzyme in the pathway. The researchers have been cloned TPS1 in plants, but they found there is no accumulation of trehalose in vivo, except for the resurrection plants. They also have been transformed the S. cerevisiae TPS1 and E.coli otsA into plants, the positive plants increased the trehalose content in vivo and tolerance to desiccation, but had varying degrees of changes in development and morphology.
It is a transgenic techno-key to gain the higher drought-resistant gene that is fit for corn physiological mechanisms and production. Our research chose the extremely drought-tolerant Selaginella pulvinata to clone trehalose-6-phosphate synthase gene (SpTPS1) by RACE, the gene whole length 3223 bp, containing 2790 bp open reading frame, and analyzed the nucleotide and mature protein through bioinformatics.
To research the SpTPS1 gene function, we subcloned the ORF into vector pUC119, and then select the appropriate restriction site to respectively digest the vector OpUC119 and pRS6/AtTPS1, the ORF fragment and the pRS6 framework from pRS6/AtTPS1 were linked, and finally we got the eukaryotic expression vector OpRS6. Transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 into the host yeast strain tps1△, YSH290 by lithium acetate, and then spread the dilution onto a selection medium which contains the appropriate galactose and lacks Histidine. The result shows that wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 can grow in the medium. We substituted 2% galactose for 2% glucose in the selection medium, and selected the positive transformants to grow on the medium. The result shows that the wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium which contains glucose and lacks Histidine; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 recoveried the function of deletion yeast strain tps1△, and can grow in the medium containing glucose. Extracted the plasmid DNA of the positive strains which is testified correct by PCR detection. Consequen -tly the SpTPS1 gene can be testified to encode the active trehalose-6-phosphate synthase by the yeast fuctional complementary test.
In this study, we cloned drought-tolerant gene SpTPS1, it is valuable for a wide range of applications to cultivate new drought-resistant varieties. It provide a higher drought-tolerant gene that is fit for corn physiological mechanisms and production to cultivate new drought-tolerant transgenic cores.
垫状卷柏(Selaginella pulvinata Maxim),俗称“九死还魂草”,极耐干旱。它能经受脱水与再水化的循环,一旦获得水分,植物枯黄卷缩的部分可以迅速张开,重新复活。有研究表明垫状卷柏这类复苏植物体内含有大量的应激代谢物—海藻糖。海藻糖对生物体有特殊保护作用,它能使许多生物在高温、脱水和冷冻等异常条件下仍保持原活性。以UDP-葡萄糖和6-磷酸葡萄糖为底物的海藻糖合成途径广泛存在于细菌、真菌和植物体内,其中由TPS1基因编码的海藻糖-6-磷酸合成酶是合成途径的关键酶。研究人员已在植物体内克隆出了TPS1基因,但除了在复苏植物体内检测到大量海藻糖积累外,在其他植物体内没有检测到海藻糖的存在。国内外已有将酵母和大肠杆菌的TPS1基因对植物体进行遗传转化研究,转化植株体内海藻糖含量有所增加,抗旱性得到增强,但植株在生长发育和形态上有不同程度的改变。
探索利用耐旱能力高,适宜玉米生理代谢机制与生产要求的耐旱基因,是转基因耐旱玉米培育的技术关键。本研究选择了极耐旱的垫状卷柏做为试验材料,采用RACE技术克隆海藻糖-6-磷酸合成酶基因(SpTPS1),全长3223 bp,包含一个2790bp的完整开放阅读框(ORF),并对SpTPS1基因进行核酸序列和成熟蛋白生物信息学的初步分析。
为了研究垫状卷柏海藻糖-6-磷酸合成酶基因(SpTPS1)的功能,将SpTPS1基因的开放阅读框ORF亚克隆到载体pUC119中,构建载体OpUC119。然后选择适当的酶切位点分别酶切载体OpUC119和质粒载体pRS6/AtTPS1,将ORF片段与质粒载体骨架pRS6相连,最终得到真核表达载体OpRS6。用LiAc转化法将真核表达载体OpRS6和阳性对照质粒pRS6/AtTPS1转入缺失酵母菌株tps1△,YSH290中,转化混合液涂在缺失组氨酸的半乳糖培养基上培养。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株能在缺失组氨酸的半乳糖培养基上生长。将培养基中的2%半乳糖换为2%葡萄糖,挑选阳性转化子划线于该培养基中。结果显示野生型菌株W303-1A和缺失酵母菌株tps1△不能在缺失组氨酸的葡萄糖培养基上生长;转化了载体OpRS6和阳性对照质粒pRS6/AtTPS1的菌株tps1△,由于恢复了功能,故能在含有葡萄糖的培养基上生长。提取阳性菌株质粒DNA,经PCR检测正确。通过酵母功能互补试验证明了SpTPS1基因具有编码海藻糖-6-磷酸合成酶的功能。
本研究从垫状卷柏中克隆了具有抗旱耐盐的SpTPS1基因,它作为培育抗旱耐盐新品种的抗旱基因,具有广泛的应用价值。并为后续的抗旱耐盐转基因新品种的培育提供了耐旱能力更高,适宜玉米生理代谢机制与生产要求的耐旱基因。
Water is the fundamental material in plants, drought have a wide range of impact on plants. In drought stress conditions, plant metabolism has changed, physical activities blocked, the various stages of growth impacted, and appears wilt phenomenon.
Selaginella pulvinata, commonly known as the "resurrection grass", displays extremely tolerance to desiccation. It can survive in the dehydration and re-hydration cycle, the curling yellow parts can be quickly extended and resurvived once got water. Studies have shown that the "resurrection plant", such as Selaginella pulvinata, contains a lot of stress metabolites-trehalose. Trehalose plays a special protective role in organisms, it can maintain the biological activities, even in the abnormal conditions, such as high temperature, dehydration and freeze. The trehalose synthesis pathway, which uses UDP-glucose and 6-phosphate glucose as substrate, is the most widely distributed in eubacteria, fungi and plants, the Trehalose-6-phosphate synthase, encoded by TPS1 gene, is the key enzyme in the pathway. The researchers have been cloned TPS1 in plants, but they found there is no accumulation of trehalose in vivo, except for the resurrection plants. They also have been transformed the S. cerevisiae TPS1 and E.coli otsA into plants, the positive plants increased the trehalose content in vivo and tolerance to desiccation, but had varying degrees of changes in development and morphology.
It is a transgenic techno-key to gain the higher drought-resistant gene that is fit for corn physiological mechanisms and production. Our research chose the extremely drought-tolerant Selaginella pulvinata to clone trehalose-6-phosphate synthase gene (SpTPS1) by RACE, the gene whole length 3223 bp, containing 2790 bp open reading frame, and analyzed the nucleotide and mature protein through bioinformatics.
To research the SpTPS1 gene function, we subcloned the ORF into vector pUC119, and then select the appropriate restriction site to respectively digest the vector OpUC119 and pRS6/AtTPS1, the ORF fragment and the pRS6 framework from pRS6/AtTPS1 were linked, and finally we got the eukaryotic expression vector OpRS6. Transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 into the host yeast strain tps1△, YSH290 by lithium acetate, and then spread the dilution onto a selection medium which contains the appropriate galactose and lacks Histidine. The result shows that wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 can grow in the medium. We substituted 2% galactose for 2% glucose in the selection medium, and selected the positive transformants to grow on the medium. The result shows that the wild-type strain W303-1A and deletion yeast strain tps1△can not grow in the medium which contains glucose and lacks Histidine; but the yeast strain which transformed the vector OpRS6 and positive control plasmid pRS6/AtTPS1 recoveried the function of deletion yeast strain tps1△, and can grow in the medium containing glucose. Extracted the plasmid DNA of the positive strains which is testified correct by PCR detection. Consequen -tly the SpTPS1 gene can be testified to encode the active trehalose-6-phosphate synthase by the yeast fuctional complementary test.
In this study, we cloned drought-tolerant gene SpTPS1, it is valuable for a wide range of applications to cultivate new drought-resistant varieties. It provide a higher drought-tolerant gene that is fit for corn physiological mechanisms and production to cultivate new drought-tolerant transgenic cores.
引文
[1]孙梅霞,祖朝龙,徐经年.干旱对植物影响的研究进展[J].安徽农业科学,2004,32(2):365-367.
[2]张正斌.作物抗旱节水的生理遗传育种基础[M].北京:科学出版社,2003.
[3]徐云刚,詹亚光.植物抗旱机理及相关基因研究进展[J]_生物技术通报,2009,2:11-17.
[4]衫本利行.糖类新资源的开发现状和应用[J].食品工业,1994:34-38.
[5]涂国云.海藻糖的性质、生产及应用[J].山西食品工业,2003,3:33-35.
[6]Krag K.T,Koeler I.M,Wright R.W.Trehalose:A non-permeable cryoprotectant for direct freezing of early stage murine embryos[J].Theriogenology,1995,23:199-206.
[7]Borelli M.I,Semino M C,Gagliardino J et al.Changes in insulin secretion and calcium distribution within B cells induced by gliclazide[J].Methods and findings in experimental and clinical pharmacology,1987,9(12):787-791.
[8]Crowe JH,Crowe LM,Corpenter J F et al.Interaction of sugers with membranes[J].Biochem Biaphys Acta,1988,947:367-384.
[9]Leslie S B.Trehalose lowers membrane phase transitions in dry yeast cells[J].Blochimicaet Biophysca Acia,1987,1192:7-13.
[10]Koichi Yoshinaga,Hiroe Yoshioka al el.Protection by trehalose of DNA from Radiation Damage [J].Biochem,1997,84:157-164.
[11]黄成根,安国瑞,王庆敏等.海藻糖对医用诊断工具酶活性保护研究[J].微生物学通报,1997,24(6):341-343.
[12]Mozhaev VV.Structure stability relationships in proteins:new approaches to stabilizing Enzyme[J].Microb TFchnol,1984,6(2):435-439.
[13]李浩明,高兰.海藻糖在生物制品干燥与保存中的应用研究[J].食品与发酵工业,1994,4:49-52.
[14]Lucy LA.The fusion of biological membranes[J].Nature,1970,227:815.
[15]Otting G.Protein hydration in aqueons[J].Science,1991,254:974.
[16]董志杨,张树政,方宜揭等.海藻糖的生物合成及抗塑研究机理[J].核农学研究进展,1993,4:121-123.
[17]张红缨,刘洋,张今等.海藻糖的生物合成及相关的特性[J].微生物学通讯,1998,25(4):134-139.
[18]F Sussich,R Urbani,F Princivalle.Polymorphic Amorphous and Crystalline Forms of Trehaloase [J].Amer Chem Soc,1998,31:7893-7899.
[19]杨小民,杨基础.几种糖对纤维素酶热稳定性影响的研究[J].清华大学学报,2000,40(2):51-58.
[20]Yoda H.Mol Genet Genomies,2002,267:154-161.
[21]杨平,李敏,潘克俭.海藻糖的生物合成与分解途径及其生物学功能[J].生命的化学,2006,26(3):233-235.
[22]Kassen I,P Falkenberg,O B Styrvold et al.Molecular cloning and physical mapping of otsBA genes,which encode the osmoregulatory trehalose pathway of Escherichia coli:evidence that transcription is activated by KatF[J].J Bacteriol,1992,174(3):889-898.
[23]Aronis R H,Klein W,Lange R et al.Trehalose synthesis genes are controlled by the putative sigma faltor encoded by tpos and are involved in stationary phase thermotolerance in Escherichia coli[J].Bacter-al,1991,173:7918-7924.
[24]Kassen I,Mcdougall J,Strom A R.Analysis of the otsBA operon for osmotegulatory trehalose synthesis in Escherichia coli and homology of the otsA and otsB proteins to the yeast trehalose-6-phosphate synthase/phosphatase complex[J].Gene,1994,145:9-15.
[25]De Vilgilio C,Burckert N,Bell W.Disruption of TPS2,the gene encoding the 100-K da subunit of trehaolose-6-phosphate synthase/phsophatase complex in saccharomyces cerevisiae,causes accumulation of trehalose-6-phsophatase and loss of trehalose-6-phosphate phsophatase activity[J].Eur J Biochem.1993,216:849-861.
[26]Londesborough J,Vuorio O E,Kalkkinen N.Cloning of two related genes encoding the 56 kDa and 123 kDa subunits of trehalose synthase from the yeast saccharomyces cerevisiae[J].Eur J Biochem,1993,216:848-861.
[27]Bell W,P.Klaasen,M.Ohnacker,T.Boller,M.Herweijer et al.Characterization of the 56Kda subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIFI,a regulator of carbon catabolite inactivation[J].Eur.J.Biochem,1992,209:951-959.
[28]De Virgilio C,Hottinger T,Dominguez,J.Boller.The role of trehalose synthesis for the acquisition of thermatollerance in yeast,genetic evidence that trehalose is a thermoprotectant[J].Eur J Biochem,1994,219:179-186.
[29]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of trehalose biosynthesis genes from Arthrobacter sp Q36[J].Biochim Biophys Acta,1996,1289:10-13.
[30]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp.M11[J].Biosci Biotechnol Biochem,1996,60:717-720.
[31]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of a cluster of genes encoding novel enzymes of trehalose biosynthesis from thermophilic Archacbacterium Sulfolobus acidocaldarius [J].Biochim Biophys Acta,1996,1291:177-188.
[32]Tsusaki K,Nishimoto T,Nakada T et al.Cloning and sequencing of trehalose synthase gene from Pimelobacter sp R48[J].Biochim Biophys Acta,1996,1290:1-3.
[33]Tsusaki K,Nishimoto T,Nakada T et al.Cloning and sequencing of trehalose synthase gene from Thermus aquaticus ATCC33923[J].Biochim Biophys Acta,1997,1334:28-32.
[34]Blazquez M.A,Santos E,Flores C L et al.Isolation and molecular characterization of the Arabidopsis TPS1 gene encoding trehalose-6-phosphate synthase[J].Plant J,1998,13:685-689.
[35]Vogel G,Aeschbacher R A,Muller J et al.Trehatose-6-phosphate phosphataes from Arabidopsis thaliana identification by functional complementation of the yeast tps2 mutant[J].Plant J,13:673-683.
[36]Pilon Smits E A H.Trehalose producing transgenic under drought stress[J].Plant Physiol,1998,152:225-233.
[37]Holmstrom K O,Mantyla E,Welin Bet al.Drought tolerance in tobacco[J].Nature,1996,379:683-684.
[38]Holmstrom K O.Engineering plant adaptation to water stress[J],Acta-Universitatis-Agricult-ure -Sueciae-Agraia,1998,84:49.
[39]Goddijn O J M,Verwoerd TC,Voogd E et al.Inhibition of trehalose activity enhances trehalose accumulation in transgenic[J].Plant Physiology,1997,113:181-190.
[40]Romero C,Belles JM,Vaya JL et al.Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco[J].Plants,1997,201:293-297.
[41]赵恢武,陈杨坚,胡莺雷等.干旱诱导性启动子驱动的海藻糖-磷酸合酶荃因载体的构建及转基因烟草的耐早性.植物学报,2000,42(6):616-619.
[42]王忆琴,戴秀玉,王锡拘,周坚.大肠杆菌otsA基因的克隆和表达[J].微生物学报,2000(3):270-274.
[43]Yeo ET,Kwn HB,Han SE et al.Genetic engineering of drought resistant introduction of the trehalose-6-phosphonte synthase(TPS1) gene from saccharamyces cerevisiae[J].Mol Cells,2000,10(3): 263-268.
[44]Ajay K Garg,Ju-kon Kim,Thomas G.Owens et al.Trehalose accumulation in rice plants confers high tolerance levels to diferent abiotic stresses[J].Proc.Natl.Acad Sci.USA,2002,99(25):15898-15903.
[45]Gill RW,Sanseau P.Rapid in silicon cloning of genes using expressed sequence tags(ESTs)[J].Biotech Ann Rev,2000,5:25-44
[46]Liu J,Ishitani M,Halfter U,Kim CS,Zhu JK.The Arabidopsis thalisns SOS2 gene encodes a protein kinase that is required for salt tolerance[J].Proc.Natl.Acad Sci.USA,2000,97:3730-3734.
[47]Maruyama K,Sugano S.Oligo-capping:a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides[J].Gene,1994,138:171-174.
[48]张洁,陆海峰,李有志.电子PCR分子植物育种[J].2004,2(1):139-145.
[49]宋宇轩,曹斌云.表达序列标签(ESTs)及其应用[J].家畜生态学,2004,25(4):152-156.
[50]黄骥,张红生,曹雅君等.水稻功能基因的电子克隆策略[J].中国水稻科学,2002,16:(4):-298.
[51]黄骥,张红生,曹雅君等.一个新的水稻C2H2型锌指蛋白cDNA的克隆与序列分析[J].南京农业大学(自然科学版),2002a,25(2):110-112.
[52]黄骥,王建飞,张红生等.水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆[J].遗传学报,2002b,29(11):1012-1016.
[53]Kozak M.An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[J].Nucl AcidRes,1987,15:8125-8148.
[54]王峦,薛鹏,程睿.人类新基因ACBP5 cDNA的克隆及其同源物在小鼠/鸡胚发育过程中的表达[J].中国生物化学与分子生物学学报,2005,21(2):164-170.
[55]李慧,李亦平,慈宏亮.编码锌指蛋白的人类新基因TFL76的电子克隆[J].生物技术,2003,15(12):15-17
[56]刘庆波,防英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆[J].生物信息学,2005,2:5-9.
[57]李常银,杨谦,王晓飞等.抗稻瘟病新基因pi-hit-1的克隆与功能研究[J].高技术通讯,2004,10:20-26.
[58]林慧贤,刘莜斌,李发强等.水稻小GTP蛋白基因Osrab5B基因的克隆和鉴定[J].高技术通讯,2001.3:9-14.
[59]陈军方,任正隆,孔秀英,吴佳洁,周荣华,贾继增.小麦中雄性不育同源基因序列的分离、鉴定及表达分析.遗传学报,2005,32(6):586-570.
[60]Trilia T,Peterson MG,Kemp DJ.A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences[J].Nucleic Acids Research,1988,16(16):8186
[61]Renato Mastrangeli,Emilia Micangdi,Silvia Donini.Cloning of Murine LAG-3 by Magnetic Bead Bound Homologous Probes and PCR(GENE-CAPTURE PCR)[J].Analytical Biochemistry,1996,241(1):93-102.
[62]Maria Lagerstre Fermer,Dan Larhemmar,Elsy Johnsen,Uif Landegren.Comparative Genomics by Capture PCR[J].Genomics,2002,79(3):442-446.
[63]Victor Ravin,Tapani Alatossava.Three new insertion sequence elements ISLd12,ISLd13,and ISLd13 in Lactobacillus delbrueckii:isolation,molecular characterization and potential use for strain identification[J].Plasmid,2003,49(3):253-268.
[64]罗丽娟,施季森.一种DNA侧翼序列分离技术-TAIL-PCR[J].南京林业大学学报,2003,4:87.
[65]Woodget JR.,Gould KL,Hunter T,Eur J.Substrate specificity of protein kinase C.Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements[J].Biochem,1986,161:177-184.
[66]Hester G,Kaku H,Goldstein IJ,Wright CS.Structure ofmannose-specific snowdrop(Galanthus nivalis) lectin is representative of a new plant lectin family[J].Nature Struct Biol,1995,2:472-479.
[67]Robert P.Gibson,Johan P.Turkenburg,Simon J.Charnock et al.Insights into Thrhalose Synthesis Provided by the Structure of the Retaining Glucosyltransferase OtsA[J].Chemistry&Biology,2002,9:1337-1346.
[68]Vogel G,Fiehn O,BresselLJR,Boller T,Wiemken A,Aeschbacher RA,Wingler A.Trehalose metabolism in Arabidopsis:occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues[J].Exp Bot,2001,52:1817-1826.
[69]Bell W,Sun W,Hohmann S,Wera S,Reinders A,De Virgilio C,Wiemken A,Thevelein JM.Composition and functional analysis of the Saccaharomyces cerevisiae trehalose synthase complex[J].Biol Chem,1998,273:33311-33319.
[70]S.Hohmann,M.J.Neves,W.de Koning,R.Alijo,J.Ramos,J.M.Thevelein.The growth and signalling defects of the ggs1(fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII[J].Curr.Genet,1993,23:281-289.
[71]M.A.Blazquez,R.Lagunas,C.Gancedo,J.M.Gancedo.Trehalose-6-phosphate,a new regulator of yeast glycolysis that inhibits hexokinases[J].FEBS Lett,1993,329:51-54.
[2]张正斌.作物抗旱节水的生理遗传育种基础[M].北京:科学出版社,2003.
[3]徐云刚,詹亚光.植物抗旱机理及相关基因研究进展[J]_生物技术通报,2009,2:11-17.
[4]衫本利行.糖类新资源的开发现状和应用[J].食品工业,1994:34-38.
[5]涂国云.海藻糖的性质、生产及应用[J].山西食品工业,2003,3:33-35.
[6]Krag K.T,Koeler I.M,Wright R.W.Trehalose:A non-permeable cryoprotectant for direct freezing of early stage murine embryos[J].Theriogenology,1995,23:199-206.
[7]Borelli M.I,Semino M C,Gagliardino J et al.Changes in insulin secretion and calcium distribution within B cells induced by gliclazide[J].Methods and findings in experimental and clinical pharmacology,1987,9(12):787-791.
[8]Crowe JH,Crowe LM,Corpenter J F et al.Interaction of sugers with membranes[J].Biochem Biaphys Acta,1988,947:367-384.
[9]Leslie S B.Trehalose lowers membrane phase transitions in dry yeast cells[J].Blochimicaet Biophysca Acia,1987,1192:7-13.
[10]Koichi Yoshinaga,Hiroe Yoshioka al el.Protection by trehalose of DNA from Radiation Damage [J].Biochem,1997,84:157-164.
[11]黄成根,安国瑞,王庆敏等.海藻糖对医用诊断工具酶活性保护研究[J].微生物学通报,1997,24(6):341-343.
[12]Mozhaev VV.Structure stability relationships in proteins:new approaches to stabilizing Enzyme[J].Microb TFchnol,1984,6(2):435-439.
[13]李浩明,高兰.海藻糖在生物制品干燥与保存中的应用研究[J].食品与发酵工业,1994,4:49-52.
[14]Lucy LA.The fusion of biological membranes[J].Nature,1970,227:815.
[15]Otting G.Protein hydration in aqueons[J].Science,1991,254:974.
[16]董志杨,张树政,方宜揭等.海藻糖的生物合成及抗塑研究机理[J].核农学研究进展,1993,4:121-123.
[17]张红缨,刘洋,张今等.海藻糖的生物合成及相关的特性[J].微生物学通讯,1998,25(4):134-139.
[18]F Sussich,R Urbani,F Princivalle.Polymorphic Amorphous and Crystalline Forms of Trehaloase [J].Amer Chem Soc,1998,31:7893-7899.
[19]杨小民,杨基础.几种糖对纤维素酶热稳定性影响的研究[J].清华大学学报,2000,40(2):51-58.
[20]Yoda H.Mol Genet Genomies,2002,267:154-161.
[21]杨平,李敏,潘克俭.海藻糖的生物合成与分解途径及其生物学功能[J].生命的化学,2006,26(3):233-235.
[22]Kassen I,P Falkenberg,O B Styrvold et al.Molecular cloning and physical mapping of otsBA genes,which encode the osmoregulatory trehalose pathway of Escherichia coli:evidence that transcription is activated by KatF[J].J Bacteriol,1992,174(3):889-898.
[23]Aronis R H,Klein W,Lange R et al.Trehalose synthesis genes are controlled by the putative sigma faltor encoded by tpos and are involved in stationary phase thermotolerance in Escherichia coli[J].Bacter-al,1991,173:7918-7924.
[24]Kassen I,Mcdougall J,Strom A R.Analysis of the otsBA operon for osmotegulatory trehalose synthesis in Escherichia coli and homology of the otsA and otsB proteins to the yeast trehalose-6-phosphate synthase/phosphatase complex[J].Gene,1994,145:9-15.
[25]De Vilgilio C,Burckert N,Bell W.Disruption of TPS2,the gene encoding the 100-K da subunit of trehaolose-6-phosphate synthase/phsophatase complex in saccharomyces cerevisiae,causes accumulation of trehalose-6-phsophatase and loss of trehalose-6-phosphate phsophatase activity[J].Eur J Biochem.1993,216:849-861.
[26]Londesborough J,Vuorio O E,Kalkkinen N.Cloning of two related genes encoding the 56 kDa and 123 kDa subunits of trehalose synthase from the yeast saccharomyces cerevisiae[J].Eur J Biochem,1993,216:848-861.
[27]Bell W,P.Klaasen,M.Ohnacker,T.Boller,M.Herweijer et al.Characterization of the 56Kda subunit of yeast trehalose-6-phosphate synthase and cloning of its gene reveal its identity with the product of CIFI,a regulator of carbon catabolite inactivation[J].Eur.J.Biochem,1992,209:951-959.
[28]De Virgilio C,Hottinger T,Dominguez,J.Boller.The role of trehalose synthesis for the acquisition of thermatollerance in yeast,genetic evidence that trehalose is a thermoprotectant[J].Eur J Biochem,1994,219:179-186.
[29]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of trehalose biosynthesis genes from Arthrobacter sp Q36[J].Biochim Biophys Acta,1996,1289:10-13.
[30]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of trehalose biosynthesis genes from Rhizobium sp.M11[J].Biosci Biotechnol Biochem,1996,60:717-720.
[31]Maruta K,Hattori K,Nakada T,Kubota M.Cloning and sequencing of a cluster of genes encoding novel enzymes of trehalose biosynthesis from thermophilic Archacbacterium Sulfolobus acidocaldarius [J].Biochim Biophys Acta,1996,1291:177-188.
[32]Tsusaki K,Nishimoto T,Nakada T et al.Cloning and sequencing of trehalose synthase gene from Pimelobacter sp R48[J].Biochim Biophys Acta,1996,1290:1-3.
[33]Tsusaki K,Nishimoto T,Nakada T et al.Cloning and sequencing of trehalose synthase gene from Thermus aquaticus ATCC33923[J].Biochim Biophys Acta,1997,1334:28-32.
[34]Blazquez M.A,Santos E,Flores C L et al.Isolation and molecular characterization of the Arabidopsis TPS1 gene encoding trehalose-6-phosphate synthase[J].Plant J,1998,13:685-689.
[35]Vogel G,Aeschbacher R A,Muller J et al.Trehatose-6-phosphate phosphataes from Arabidopsis thaliana identification by functional complementation of the yeast tps2 mutant[J].Plant J,13:673-683.
[36]Pilon Smits E A H.Trehalose producing transgenic under drought stress[J].Plant Physiol,1998,152:225-233.
[37]Holmstrom K O,Mantyla E,Welin Bet al.Drought tolerance in tobacco[J].Nature,1996,379:683-684.
[38]Holmstrom K O.Engineering plant adaptation to water stress[J],Acta-Universitatis-Agricult-ure -Sueciae-Agraia,1998,84:49.
[39]Goddijn O J M,Verwoerd TC,Voogd E et al.Inhibition of trehalose activity enhances trehalose accumulation in transgenic[J].Plant Physiology,1997,113:181-190.
[40]Romero C,Belles JM,Vaya JL et al.Expression of the yeast trehalose-6-phosphate synthase gene in transgenic tobacco[J].Plants,1997,201:293-297.
[41]赵恢武,陈杨坚,胡莺雷等.干旱诱导性启动子驱动的海藻糖-磷酸合酶荃因载体的构建及转基因烟草的耐早性.植物学报,2000,42(6):616-619.
[42]王忆琴,戴秀玉,王锡拘,周坚.大肠杆菌otsA基因的克隆和表达[J].微生物学报,2000(3):270-274.
[43]Yeo ET,Kwn HB,Han SE et al.Genetic engineering of drought resistant introduction of the trehalose-6-phosphonte synthase(TPS1) gene from saccharamyces cerevisiae[J].Mol Cells,2000,10(3): 263-268.
[44]Ajay K Garg,Ju-kon Kim,Thomas G.Owens et al.Trehalose accumulation in rice plants confers high tolerance levels to diferent abiotic stresses[J].Proc.Natl.Acad Sci.USA,2002,99(25):15898-15903.
[45]Gill RW,Sanseau P.Rapid in silicon cloning of genes using expressed sequence tags(ESTs)[J].Biotech Ann Rev,2000,5:25-44
[46]Liu J,Ishitani M,Halfter U,Kim CS,Zhu JK.The Arabidopsis thalisns SOS2 gene encodes a protein kinase that is required for salt tolerance[J].Proc.Natl.Acad Sci.USA,2000,97:3730-3734.
[47]Maruyama K,Sugano S.Oligo-capping:a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides[J].Gene,1994,138:171-174.
[48]张洁,陆海峰,李有志.电子PCR分子植物育种[J].2004,2(1):139-145.
[49]宋宇轩,曹斌云.表达序列标签(ESTs)及其应用[J].家畜生态学,2004,25(4):152-156.
[50]黄骥,张红生,曹雅君等.水稻功能基因的电子克隆策略[J].中国水稻科学,2002,16:(4):-298.
[51]黄骥,张红生,曹雅君等.一个新的水稻C2H2型锌指蛋白cDNA的克隆与序列分析[J].南京农业大学(自然科学版),2002a,25(2):110-112.
[52]黄骥,王建飞,张红生等.水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆[J].遗传学报,2002b,29(11):1012-1016.
[53]Kozak M.An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs[J].Nucl AcidRes,1987,15:8125-8148.
[54]王峦,薛鹏,程睿.人类新基因ACBP5 cDNA的克隆及其同源物在小鼠/鸡胚发育过程中的表达[J].中国生物化学与分子生物学学报,2005,21(2):164-170.
[55]李慧,李亦平,慈宏亮.编码锌指蛋白的人类新基因TFL76的电子克隆[J].生物技术,2003,15(12):15-17
[56]刘庆波,防英,董辉.水稻线粒体丝氨酸羟甲基转移酶基因的电子克隆[J].生物信息学,2005,2:5-9.
[57]李常银,杨谦,王晓飞等.抗稻瘟病新基因pi-hit-1的克隆与功能研究[J].高技术通讯,2004,10:20-26.
[58]林慧贤,刘莜斌,李发强等.水稻小GTP蛋白基因Osrab5B基因的克隆和鉴定[J].高技术通讯,2001.3:9-14.
[59]陈军方,任正隆,孔秀英,吴佳洁,周荣华,贾继增.小麦中雄性不育同源基因序列的分离、鉴定及表达分析.遗传学报,2005,32(6):586-570.
[60]Trilia T,Peterson MG,Kemp DJ.A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences[J].Nucleic Acids Research,1988,16(16):8186
[61]Renato Mastrangeli,Emilia Micangdi,Silvia Donini.Cloning of Murine LAG-3 by Magnetic Bead Bound Homologous Probes and PCR(GENE-CAPTURE PCR)[J].Analytical Biochemistry,1996,241(1):93-102.
[62]Maria Lagerstre Fermer,Dan Larhemmar,Elsy Johnsen,Uif Landegren.Comparative Genomics by Capture PCR[J].Genomics,2002,79(3):442-446.
[63]Victor Ravin,Tapani Alatossava.Three new insertion sequence elements ISLd12,ISLd13,and ISLd13 in Lactobacillus delbrueckii:isolation,molecular characterization and potential use for strain identification[J].Plasmid,2003,49(3):253-268.
[64]罗丽娟,施季森.一种DNA侧翼序列分离技术-TAIL-PCR[J].南京林业大学学报,2003,4:87.
[65]Woodget JR.,Gould KL,Hunter T,Eur J.Substrate specificity of protein kinase C.Use of synthetic peptides corresponding to physiological sites as probes for substrate recognition requirements[J].Biochem,1986,161:177-184.
[66]Hester G,Kaku H,Goldstein IJ,Wright CS.Structure ofmannose-specific snowdrop(Galanthus nivalis) lectin is representative of a new plant lectin family[J].Nature Struct Biol,1995,2:472-479.
[67]Robert P.Gibson,Johan P.Turkenburg,Simon J.Charnock et al.Insights into Thrhalose Synthesis Provided by the Structure of the Retaining Glucosyltransferase OtsA[J].Chemistry&Biology,2002,9:1337-1346.
[68]Vogel G,Fiehn O,BresselLJR,Boller T,Wiemken A,Aeschbacher RA,Wingler A.Trehalose metabolism in Arabidopsis:occurrence of trehalose and molecular cloning and characterization of trehalose-6-phosphate synthase homologues[J].Exp Bot,2001,52:1817-1826.
[69]Bell W,Sun W,Hohmann S,Wera S,Reinders A,De Virgilio C,Wiemken A,Thevelein JM.Composition and functional analysis of the Saccaharomyces cerevisiae trehalose synthase complex[J].Biol Chem,1998,273:33311-33319.
[70]S.Hohmann,M.J.Neves,W.de Koning,R.Alijo,J.Ramos,J.M.Thevelein.The growth and signalling defects of the ggs1(fdp1/byp1) deletion mutant on glucose are suppressed by a deletion of the gene encoding hexokinase PII[J].Curr.Genet,1993,23:281-289.
[71]M.A.Blazquez,R.Lagunas,C.Gancedo,J.M.Gancedo.Trehalose-6-phosphate,a new regulator of yeast glycolysis that inhibits hexokinases[J].FEBS Lett,1993,329:51-54.