CN101445552B - 水稻蛋白OsOEE3-1及其编码基因与应用 - Google Patents
水稻蛋白OsOEE3-1及其编码基因与应用 Download PDFInfo
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Abstract
本发明提供公开了一种水稻耐盐蛋白OsOEE3-1，具有序列表中SEQID NO.1所述的氨基酸序列。本发明蛋白在水稻幼苗中得到过量表达，可以提高水稻幼苗耐高盐和高温能力。若将编码该蛋白的基因转化拟南芥、辣椒、茄子、草莓、一串红、非洲菊等蔬菜、花卉等植物，有可能提高其耐高盐和高温性能，有助于增加水稻或露地蔬菜、花卉在盐碱地上的产量，提高我国滨海地区的盐碱地的利用；克服设施条件下因土壤返盐或高温引起的连作障碍，提高蔬菜、花卉等植物的产量和品质，促进农业增效和农民增收。
Description
技术领域
本发明涉及功能基因组学领域，尤其涉及一种水稻蛋白OsOEE3-1及其编码基因与应用。
背景技术
土壤盐渍化对农业的威胁是一个全球性的问题。全世界共有10亿公顷的盐碱地，约占世界陆地面积7.6％，我国盐碱地近1亿多公顷，农业耕地因盐渍化引起的减产、弃耕地就近333.5万公顷。近年来，我国设施农业的快速发展，特别是蔬菜和花卉大棚生产面积不断扩大，据统计，2005年全国蔬菜、花卉、瓜果等作物设施栽培面积达210万公顷。设施农业的发展为农业生产结构调整和提高农业生产效益发挥了重要作用，但是随着设施栽培时间延长，土壤次生盐渍化的问题日益加剧，严重影响了设施栽培作物的产量和品质，效益也随之下降，从而影响设施农业的健康发展。解决设施栽培土壤盐渍化一般采取以下两种措施，一是用石膏和硫磺等化学方法或用排水和灌溉洗盐等物理方法改良土壤；二是通过常规育种或生物技术手段培育耐盐作物品种，而前者投入成本高。通过培育适宜在盐碱地区栽培和设施栽培的农作物抗盐新品种将不仅能有效解决设施栽培土壤盐渍化问题，而且还能通过有效利用部分盐渍化土地而大大地缓解我国土地资源匮乏的问题。
近年来，随着模式植物拟南芥和水稻基因组测序完成，植物基因组学研究已转入到功能基因组学。目前一些研究功能基因组学的新方法和实验技术体系如cDNA微阵列、基因芯片、基因表达系统分析(serial analysis ofgene expression，SAGE)、基因敲除(gene knockout)和RNAi分析均能有效分析大量基因的表达和功能模式，并在耐盐性相关功能基因资源发掘上取得了一定进展。一些与渗透调节相关基因已从不同植物种类中被成功克隆并转化应用，如脯氨酸合成相关基因P5CS(Kishor PBK，Hong Z，MiaoG H，Hu CAA，Verma DPS.Overexpression of P5CS increases prolineproduction and confers osmotolerance in transgenic plants.Plant Physiol，1995，108：1387-1394)和甜菜碱脱氢酶BADH基因(肖岗，张耕耘，刘凤华等，山菠菜甜菜碱醛脱氢酶基因研究，科学通报，1995，40(8)：741-745)。
植物体内Na+离子平衡是植物自身耐盐调节的重要机制。朱健康研究小组发现拟南芥SOS基因系列的调控信号是植物自身调节Na+离子平衡的重要途径之一。2005年，林鸿宣研究小组与美国栾升教授合作，成功克隆了水稻耐盐相关的数量性状基因SKC1。该基因能控制水稻植株地上部钠离子和钾离子的含量，维持钠和钾离子平衡，使过量钠离子不在茎叶等部位积累，并使钠离子回流到根部，减轻钠离子毒害，同时增加营养元素钾离子，从而增加水稻耐盐性。
众所周知，水稻的基因图谱已经绘制完成，也就是所大部分水稻基因序列是公开的，但是具体涉及到某个基因的功能是未知的，特别是某个基因编码的蛋白以及该蛋白的对水稻产生的功能影响也是未知的。直接鉴定某个基因的功能是非常困难的，现有手段往往是先分离出植物蛋白，检测该蛋白的氨基酸序列，然后再通过氨基酸序列来比对公用数据库中的某个基因序列，从而确定该基因的功能以及该基因的应用。
发明内容
本发明提供了一种水稻蛋白，名称为OsOEE3-1，它是一个受盐诱导表达方式的蛋白，有助于提高水稻耐高盐和耐高温性能。
一种水稻蛋白，具有序列表中SEQ ID NO.1所述的氨基酸序列。
该蛋白能够上调超氧物歧化酶、过氧化氢酶、抗坏血酸过氧化物酶等抗氧化酶活性，清除因高盐或低温胁迫产生的过多的活性氧(超氧离子或过氧化氢)，维持细胞体内活性氧水平在正常范围内，保护幼苗、植株免遭因高盐或高温引发的氧化损伤，提高植物抗逆能力。
根据上述水稻蛋白OsOEE3-1的氨基酸序列，通过NCBI和TIGER数据库搜索，比对到编码水稻冷激蛋白的基因OsOEE3-1(Oryza sativaOxygen Evolving Ehancer protein 3-1)，基因OsOEE3-1的基因号为Os07g0544800(ID 4343515)，该基因的开放阅读框(ORF)为654bp，具有序列表中SEQ ID NO.2所述的核苷酸序列；mRNA长度为961bp，具有序列表中SEQ ID NO.3所述的核苷酸序列。
上述基因可以应用于提高植物耐高盐和耐高温的性能，具体操作如下：
(1)将上述基因连接Super1300载体中；
(2)将上述重组载体通过农杆菌介导转化到目标植物；
(3)以潮霉素B为抗性标记，结合高盐(200mM NaCl以上)和高温(46℃)处理为选择压进行筛选，得到具有耐高盐和耐高温性能的植物。
本发明水稻蛋白可以提高水稻耐高盐和耐高温性能，将编码该蛋白的基因导入其它植物当中，如草莓、辣椒、茄子、一串红、非洲菊等，也有可能提高这些植物的耐高盐和耐高温性能。通过上述手段，一方面可以增加作物在盐碱地上的产量，提高我国滨海地区的盐碱地的利用；另一方面可以克服设施条件下蔬菜、花卉等植物因土壤返盐和夏季高温引起的生育障碍，提高其产量和品质，增加农民收入。
具体实施方式
基因的获得
(1)以杂交水稻耐盐组合汕优10号和盐敏感组合两优培九为材料，将它们的种子播在含100mM NaCl溶液浸湿滤纸培养皿中，置于30℃培养箱中发芽，每天更换盐溶液，以保持盐浓度基本一致。待幼苗生长至10d，分别收集汕优10号和两优培九幼苗的叶片。
(2)用冷丙酮/三氯乙酸沉淀法(Salekdeh G H，Siopongco J，Wade L J，Ghareyazie B，Bennett J.A proteomic approach to analyzing drought-andsalt-responsiveness in rice.Field Crop Res，2002，76(2-3)：199-219)快速提取叶总蛋白，具体操作如下：
1)水稻叶片用液氮研磨成细粉，分装入1.5ml离心管中，加入1ml蛋白提取液I(含10％三氯乙酸和0.07％β-巯基乙醇的丙酮溶液)在-20℃沉淀粗蛋白1h，在4℃、13000rpm下离心20min，取沉淀，弃上清；
2)然后往沉淀中加入1ml蛋白提取液II(含0.07％β-巯基乙醇的丙酮溶液)，在-20℃悬浮粗蛋白丸1h，在4℃、13000rpm下离心20min，取沉淀，弃上清，再重复用蛋白提取液II，在相同条件下悬浮提取3次后，真空抽干沉淀；
3)用裂解液(含7mol/L尿素、2mol/L硫脲、4％Chaps(Ameresco公司，美国)、50mmol/L DTT(Promega公司，美国)和0.5％pH3-10的40％两性电解质)溶解沉淀，裂解液用量为25μl裂解液/mg沉淀，然后在室温下放置1h，裂解期间不断涡旋5-6次。
(2)根据Bradford法(Bradford M M.A rapid and sensitive method forthe quantitation of microgram quantities of protein utilizing the principle ofprotein-dye binding.Anal Biochem，1976，72：248-54)用考马斯亮兰G-250(Sigma公司)测定上述裂解液中的蛋白含量，上述裂解液中的蛋白用双向凝胶电泳(第一向采用17cm pH7-10的IPGs干胶条(Bio-Rad公司)聚焦，第二向采用变性/SDS-2D-PAGE分离)分离。
第一向等电聚焦分四步进行，第一步，电压250V 15min；第二步，电压10000V 5h；第三步，电压10000V，60000Vh；第四步，电压500V直到结束。
在第一向等电聚焦向第二向转换时，需要平衡胶条，分两步进行，第一步，在平衡液I(含6.0mol/L尿素、2％SDS(Promega公司，美国)、0.375mol/L pH 8.8 Tris-HCl(Promega公司，美国)、20％甘油和130mmol/L DTT(Promega公司，美国))中平衡10min；第二步，在平衡液II(含6.0mol/L尿素、2％SDS(Promega公司，美国)、0.375mol/L pH 8.8Tris-HCl(Promega公司，美国)、20％甘油和135mmol/L碘乙酰胺)中平衡10min，然后转移到第二向SDS-2D-PAGE胶，跑胶采用恒流，每块胶的电流24mA，运行5-6h。
用500ml固定液(40％甲醇和10％乙酸)固定30min，然后用500ml银氨染色液(含有3.6％氢氧化钠10.5ml、20％硝酸银9ml和5ml氨水)染色32-33min，用双蒸水冲洗4次，然后用500ml显色液(含有1％柠檬酸2.5ml和甲醛250μl)显色5-12min，最后用500ml 5％醋酸终止反应5min。
扫描后用PDQUEST(Bio-Rad公司)软件分析胶图匹配情况，结果发现在汕优10号幼苗叶片中一个高表达蛋白点，估测其等电点和分子量分别为pI 9.8和22KD左右。
(3)在胶上切下该高表达蛋白点，加入8μl 10ng/μl胰蛋白酶(Trypsin，Roche，美国)进行胶内消化，然后置于4℃冰箱放置40min使胶片完全吸收酶液，再补加10μl 25mM碳酸氢铵缓冲液(pH 8.0)，于37℃温育12h，胶内蛋白质被酶解成肽段混合物。
(4)在上述肽段混合物中加入30-50μl 5％TFA(Merk公司，德国)于40℃提取上述酶切肽段1小时一次，再用相同体积的50％CAN和2.5％TFA(Merk公司，德国)溶液于30℃提取1小时一次，最后用25μlCAN(Fischer公司，美国)超声提取一次，合并3次提取液。真空干燥，然后用4μl 0.5％三氟乙酸溶解，将0.6μl溶解物用基质辅助激光解吸离子化飞行质谱(MALDI-TOF-MS)分析，获得肽质量指纹(Peptide MassFingerprint，PMF)图谱，查询Mascot数据库，以较高分值(117)显著(比对分高于65)比对到水稻OsOEE3-1蛋白，匹配序列占总氨基酸序列37％。
根据已有的水稻OsOEE3-1的氨基酸序列，通过NCBI和TIGER数据库搜索，比对到编码水稻放氧增强子蛋白基因，基因号为Os07g0544800(ID 4343515)。该基因的开放阅读框(ORF)为654bp，mRNA长度为961bp。
基因克隆与转化
以汕优10号幼苗(播后10天)总mRNA为模板，利用RT-PCR方法扩增到OsOEE3-1基因的编码序列。
具体操作如下：首先，将mRNA反转录成第一链cDNA，所用反转录试剂盒为TaKaRa公司的High Fidelity PrimerScriptTM RT-PCR Kit，反应体系20μl，依次加入1μl 20M随机引物、1μl 10mM dNTP、2μl总RNA和DEPC水至10μl，在65℃变性5分钟，迅速在冰上冷却2分钟，稍微离心，然后依次加入4μl 5×PrimerScript RT buffer、0.5μl RNase inhibitor、0.5μl PrimerScript RTase和5μl DEPC水。轻微混合均匀，30℃反应10分钟，42℃反应30分钟，95℃5分钟使酶失活。为了去掉与cDNA互补的RNA链，加入1μl RNase H在37℃温育20min，-20℃保存。然后以第一链cDNA为摸板扩增目的基因OsOEE3-1，所用扩增配对引物：
OsOEE3-1-F，5’-TCTAGAATGGCACAGGCAATGGCGTC-3’，
OsOEE3-1-R，5’-GGTACCCTAGCCTAGCTTGGCGAGGA-3’，
PCR反应体系为50μl，依次加入2×PCR buffer 25μl、2.5mM dNTPs4μl、反转录产物2μl、20μM正向引物(OsOEE3-1-F)1μl、20μM反向引物(OsOEE3-1-R)1μl、2.5U/μl Tag DNA聚合酶0.5μl，最后加水至50μl。PCR反应条件：预变性94℃3min，变性98℃10s，退火55℃15s，延伸72℃50s，30个循环，最后延伸72℃10min，4℃保存。
将回收纯化的OsOEE3-1基因的DNA与pMD19-T载体进行连接反应，连接体系10μl，各组分分别为0.5μl pMD19-T载体、4.5μl纯化的OsOEE3-1基因的DNA、5μl Solution I。在14℃-16℃下连接8-12小时，然后将连接产物转化到大肠杆菌DH5α感受态细胞中，将OsOEE3-1基因装入pMD19-T载体后经测序正确，用TakaRa公司生产的EcoRI和HindIII酶切，酶切体系40μl，包括4μl 10×buffer、8μl已插入OsOEE3-1基因的pMD19-T载体、1μl XbaI、1μl KpnI和26μl水，在37℃水浴中温育6h。
用北京博大泰克生物技术公司生产的Glassmilk kit回收基因片段，操作如下：上述混合基因经凝胶电泳以后，从凝胶上切下所需DNA片段，放在1.5ml的Eppendorf管中。加入3倍体积的溶胶液，室温下放置5min，期间轻摇Eppendorf管几次使胶完全溶化。加入10μl玻璃奶，颠倒混匀，冰浴下放置10min。每隔2-3min混匀1次，12000rpm离心30s，吸弃上清。加入250μl漂洗液，用移液器吹打漂洗液，轻柔地将玻璃奶悬浮混匀，12000rpm离心30s，吸弃上清。重复漂洗1次。用枪头将剩余的漂洗液吸干净。然后，放置于37℃温箱干燥15-20min。加入20μl的无菌蒸馏水，混匀，60℃水浴5min，12000rpm离心1min，回收上清液，即为纯化的基因OsOEE3-1。
将回收的基因片段连入Super1300载体中，操作如下：连接体系10μl，包括2μl Super1300载体、6μl纯化的OsOEE3-1基因的DNA、1μl 10×T4连接酶buffer和1μl T4连接酶，在4-10℃下连接12h，然后将连接产物转化到大肠杆菌DH5α感受态细胞中，提取质粒进行鉴定。
基因片段连入Super1300载体后再转入EHA105农杆菌中，操作如下：取200μl农杆菌感受态细胞，加入5-10μl构建好的质粒DNA，30℃冰浴30min，液氮中速冻1min，37℃水浴5min，然后加入1ml YEB培养基(1升YEB培养基含1g酵母提取物、5g牛肉浸膏、5g蛋白胨、5g蔗糖和0.5g MgSO4·7H2O，pH 7.0)，28℃恢复培养4h；10000g离心30s，弃上清，加入0.1ml YEB培养基重新悬浮细胞，涂布于含有100μg/ml卡那霉素和125μg/ml利福平的YEB平板(1升YEB培养基含1g酵母提取物、5g牛肉浸膏、5g蛋白胨、5g蔗糖、0.5g MgSO4·7H2O和12g琼脂，pH 7.0)上，28℃培养约48h。
经鉴定正确后(挑取阳性克隆作为模板，用菌落PCR方法进行鉴定)，通过农杆菌介导转化模式植物水稻，操作如下：接种含有目的质粒的农杆菌菌落于10ml YEB培养基(含0.1％酵母提取物、0.5％牛肉浸膏、0.5％蛋白胨、0.5％蔗糖、0.05％MgSO4·7H2O、1.2％琼脂、100μg/ml卡那霉素和125μg/ml利福平)中28℃、200rpm震荡培养过夜，转化前一天按1∶50接种于200ml含相同抗生素的YEB培养液中扩大培养至OD600为0.6-0.8。取菌液，按1％～2％的比例，转入新配制的无抗生素的YEB液体培养基中，6小时后，菌液OD600为0.2～0.5时即可用于转化。
取粳稻品种爱知旭(Oryza sativa L cv Aichi Asahi)幼胚，用70％酒精浸泡1min，然后用0.1％升汞溶液灭菌30min，再用无菌水冲洗3次，置无菌滤纸上吸干，接种于诱导培养基(NB培养基外加2mg.L-12，4-D)上诱导愈伤组织11-13d后继代，继代后4d用于共培养。愈伤组织在含有100mol.L-1乙酰丁香酮和含目的基因质粒的农杆菌的液体培养基中培养20min，用滤纸吸去多余的菌液后，转到含乙酰丁香酮的固体培养基上26℃暗培养2d，经共培养的愈伤组织转移到选择培养基(NB培养基外加2mg.L-12，4-D、50mg.L-1潮霉素B和头300mg.L-1头孢噻肟)上。10d后挑选成活的愈伤组织进行复筛，抗性愈伤转移到分化培养基(NB培养基外加2mg.L-12，4-D、3mg.L-1、6-BA、0.5mg.L-1NAA、4mg.L-1KT、50mg.L-1潮霉素B和头300mg.L-1头孢噻肟)上诱导出苗。培养温度26℃，每天光照15h。抗性植株转到含1/2N6大量元素(1415mg.L-1KNO3、231.5mg.L-1NH4SO4、83mg.L-1CaCl2·2H2O、92.5mg.L-1MgSO4·7H2O、200mg.L-1KH2PO4、200mg.L-1FeSO4·7H2O和2.2mg.L-1MnSO4·4H2O)的无激素培养基上使其生根。当试管苗长到约8cm高并有发达的根系时，即可移栽入土。单株收获T1代种子。繁种并鉴定至T3代，获得纯合的转基因52个株系。
通过农杆菌介导浸花法转化模式植物拟南芥，操作如下：接种含有目的基因质粒的农杆菌菌落于10ml YEB培养基(含0.1％酵母提取物、0.5％牛肉浸膏、0.5％蛋白胨、0.5％蔗糖、0.05％MgSO4·7H2O、1.2％琼脂、100μg/ml卡那霉素和125μg/ml利福平)中28℃、200rpm震荡培养过夜，转化前一天按1∶50接种于200ml含相同抗生素的YEB培养液中扩大培养至OD600为1.2～1.6，约6h，5000g离心15min集菌，重悬于渗透缓冲液，使OD600为0.8，200ml重悬液可使用3次。转化所用浸泡液含有0.5×MS大量元素、0.5×MS微量元素、0.5mg/L VB5、5％蔗糖、44nM 6-BA(Sigma公司，美国)和0.03％Silwet L-77(LEHLE SEEDS公司，美国)。将200ml含目的农杆菌的渗透转化液置于一容器中，翻转种有拟南芥的花盆，使植株浸入含有待转化农杆菌的渗透缓冲液中，浸5分钟，缓慢取出花盆，侧放于托盘中，盖上黑塑料布避光24小时，第二天取下塑料布，直立放置花盆。
制备MS筛选平板(MS培养基外加80g/ml潮霉素和50g/ml氨苄青霉素)，转化收获的T1代种子经消毒后播种于筛选平板，每15cm的平板上可以筛选100μg左右的拟南芥种子。4℃春化3天，平放在生长箱中培养(22℃恒温，24h光照)，7-10天后挑选在筛选培养基上根系和地上部生长正常的阳性植株，移入正常MS培养基缓苗3-5天后移植入土壤，单株收获T2代种子。繁种并鉴定至T3代，获得纯合的转基因60个株系。基因功能鉴定
(1)耐高盐性能鉴定
取T3代纯合转基因株系和野生型(粳稻品种爱知旭)种子，均匀放在两层用蒸馏水润湿的发芽纸上，在25℃光照条件下培养10d，观察幼苗生长情况。然后将转基因株系和野生型的水稻幼苗分别移入含200mMNaCl或正常清水的发芽盒内，置于相同光温条件的培养箱中培养。培养5d后，观察转基因株系与野生型幼苗在高盐胁迫下的表型。结果发现在200mM NaCl处理下，野生型水稻幼苗叶片出现白化表型，严重时幼苗白化死亡，而转OsOEE3-1基因株系叶片仍保持绿色。叶片最大光化学效率测定(Fv/Fm)结果表明，在200mM NaCl下，野生型幼苗地上部最大光化学效率显著降低，而转OsOEE3-1基因株系幼苗地上部最大光化学效率未出现明显变化，说明OsOEE3-1基因在水稻中超量表达可以提高水稻幼苗耐盐性。
T3代纯合转基因株系和野生型(ecotype Columbia))拟南芥种子用1％次氯酸钠消毒，在4℃冰箱中春化3天，然后置于温度22℃、湿度50％、连续24h光照的培养箱中培养。培养7d后，观察幼苗生长情况。待幼苗子叶完全展开后，将转基因株系和野生型幼苗移入含200mM NaCl和正常的MS固体培养基(含1×大量元素、1×微量元素、1×铁盐、3％蔗糖和0.8％琼脂)上，然后置于相同光温条件(22℃、湿度50％、连续24h光照)的培养箱中培养。培养12-15d后，观察转基因株系与野生型幼苗在高盐胁迫下的表型。结果发现在200mM NaCl下，野生型幼苗全部白化死亡，转OsOEE3-1基因株系幼苗叶片仍保持绿色，说明OsOEE3-1基因超量表达可以缓解拟南芥盐害。
(2)耐高温性能鉴定
取T3代纯合转基因株系和野生型(粳稻品种爱知旭)种子，均匀放在两层用蒸馏水润湿的发芽纸上，在25℃光照条件下培养10d，观察幼苗生长情况。然后将转基因株系和野生型的水稻幼苗移入46℃高温培养箱，处理12h后，再放回到25℃光照条件下培养，4-5d后野生型幼苗叶片出现黄化，严重时幼苗叶片局部开始白化死亡，而转OsOEE3-1基因株系叶片仍保持正常绿色。叶片最大光化学效率测定(Fv/Fm)结果表明，野生型幼苗地上部最大光化学效率显著降低，而转OsOEE3-1基因株系幼苗地上部最大光化学效率未出现明显变化，说明OsOEE3-1基因在水稻中超量表达可以提高水稻幼苗耐高温性能。
取T3代纯合转基因株系和野生型拟南芥种子，播在塑料土盆中，在22℃温室内培养，取生长到4周的转基因株系和野生型幼苗，在45℃低温培养箱中处理8h，再放回到22℃温室内培养，3-4d后野生型幼苗叶片出现失水卷曲，严重时幼苗叶片开始白化，而转OsOEE3-1基因株系叶片仍保持正常绿色说明OsOEE3-1基因在拟南芥中超量表达可以提高拟南芥幼苗耐高温性能。
SEQUENCE LISTING
<110>杭州市农业科学研究院
<120>水稻蛋白OsOEE3-1及其编码基因与应用
<130>
<160>3
<170>PatentIn version 3.3
<210>1
<211>217
<212>PRT
<213>水稻
<400>1
Met Ala Gln Ala Met Ala Ser Met Thr Gly Leu Ser Gln Gly Val Gln
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Val Arg Ala Asp Ala Ala Ala Ala Asp Val Gln Thr Gly Arg Arg Ala
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Val Leu Gly Leu Val Ala Thr Gly Ile Ala Gly Gly Ala Leu Ala Gln
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Ala Ala Leu Ala Glu Ala Ala Lys Pro Ile Lys Leu Gly Pro Pro Pro
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Pro Pro Ser Gly Gly Leu Pro Gly Thr Leu Asn Ser Asp Gln Ala Arg
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Asp Thr Asp Leu Pro Leu Arg Glu Arg Phe Tyr Leu Gln Pro Leu Pro
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Pro Ala Glu Ala Ala Ala Ala Ala Lys Glu Ser Ala Gln Asp Ile Ile
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Asn Leu Lys Pro Leu Ile Glu Lys Lys Gln Trp Pro Phe Val Arg Asp
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Asp Leu Arg Leu Arg Ala Ser Tyr Leu Arg Tyr Asp Leu Lys Thr Val
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Ile Asn Ser Lys Pro Lys Asp Glu Lys Lys Gly Leu Lys Asp Leu Thr
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Gly Lys Leu Phe Ala Thr Ile Asp Gly Leu Asp His Ala Ala Lys Ile
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Lys Ser Pro Glu Glu Ala Glu Lys Tyr Tyr Thr Leu Thr Lys Ser Ala
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Leu Gly Asp Val Leu Ala Lys Leu Gly
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<210>2
<211>654
<212>DNA
<213>水稻
<400>2
atggcacagg caatggcgtc catgaccggg ctgtcgcagg gcgtgcagct gccggccggg 60
cccaggcgcg ccggcggcag gtccaggctc gccgtcgtca gggccgacgc cgccgccgcc 120
gacgtccaga ccggccgccg cgccgtgctc ggcctcgtcg ccaccgggat cgccggcggc 180
gccctcgcgc aggcggcgct cgccgaggcc gccaagccca tcaagctcgg ccccccgcca 240
ccgccctccg gtggactccc tgggacgctg aactcggacc aggcgaggga cacggacctg 300
ccgctgaggg aaaggttcta cctgcagccg ctgccgccgg cggaggcggc ggcgagggcg 360
aaggagtcgg cccaggacat catcaacctc aagccgctca tcgagaagaa gcagtggccg 420
ttcgtcaggg acgacctccg cctcagggcc tcctacctgc gctacgacct caaaaccgtc 480
atcaactcca agcccaagga cgagaagaag ggcctcaagg acctcaccgg caagctcttc 540
gccaccattg acgggcttga ccatgcagcc aagatcaaga gccccgaaga ggcggagaag 600
tactacacgt tgaccaaatc tgctcttggc gatgtcctcg ccaagctagg ctag 654
<210>3
<211>960
<212>RNA
<213>水稻
<400>3
aucugaagag agaaauucuc agcuuauagu caggugaggu cugagcugag guugagagau 60
ggcacaggca auggcgucca ugaccgggcu gucgcagggc gugcagcugc cggccgggcc 120
caggcgcgcc ggcggcaggu ccaggcucgc cgucgucagg gccgacgccg ccgccgccga 180
cguccagacc ggccgccgcg ccgugcucgg ccucgucgcc accgggaucg ccggcggcgc 240
ccucgcgcag gcggcgcucg ccgaggccgc caagcccauc aagcucggcc ccccgccacc 300
gcccuccggu ggacucccug ggacgcugaa cucggaccag gcgagggaca cggaccugcc 360
gcugagggaa agguucuacc ugcagccgcu gccgccggcg gaggcggcgg cgagggcgaa 420
ggagucggcc caggacauca ucaaccucaa gccgcucauc gagaagaagc aguggccguu 480
cgucagggac gaccuccgcc ucagggccuc cuaccugcgc uacgaccuca aaaccgucau 540
caacuccaag cccaaggacg agaagaaggg ccucaaggac cucaccggca agcucuucgc 600
caccauugac gggcuugacc augcagccaa gaucaagagc cccgaagagg cggagaagua 660
cuacacguug accaaaucug cucuuggcga uguccucgcc aagcuaggcu aggaucggca 720
uaauggccau augggguuuc gguguuuuua uguuuguuca uauggaaccg gcaauguacc 780
cuccauguug auauuguauc agcaagcacu uacguaugau ucaaucuuga guuguuguug 840
acggcuaaau cuccaagcag gcgcgauuau cagaaaacaa gggagaaauc caaaaguucu 900
gaacaaaauc aucgcauaau ucauucauau ucagaagcau cauuucacau cacaaguuua 960
Claims (3)
1.一种水稻蛋白基因在提高植物耐高盐和耐高温性能中的应用，所述的水稻蛋白基因的碱基序列如SEQ ID NO.2所示，所述的植物为水稻或拟南芥。
2.根据权利要求1所述的应用，包括以下步骤：
(1)将所述的水稻蛋白基因连接到载体中，得到重组载体；
(2)将重组载体通过农杆菌介导转化到植物中；
(3)筛选得到具有耐高盐和耐高温性能的植株。
3.根据权利要求2所述的应用，其特征在于：所述的载体为Super1300载体。
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1429269A (zh) * | 2000-03-23 | 2003-07-09 | 伊登生物科学有限公司 | 超敏反应激卫素受体及其应用 |
| CN1431309A (zh) * | 2001-11-22 | 2003-07-23 | 独立行政法人国际农林水产业研究中心 | 编码植物转录因子的基因 |
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| CN1429269A (zh) * | 2000-03-23 | 2003-07-09 | 伊登生物科学有限公司 | 超敏反应激卫素受体及其应用 |
| CN1431309A (zh) * | 2001-11-22 | 2003-07-23 | 独立行政法人国际农林水产业研究中心 | 编码植物转录因子的基因 |
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