CN1273483C - 一个玉米bZIP类转录因子及其编码基因与应用 - Google Patents
一个玉米bZIP类转录因子及其编码基因与应用 Download PDFInfo
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- CN1273483C CN1273483C CNB021271879A CN02127187A CN1273483C CN 1273483 C CN1273483 C CN 1273483C CN B021271879 A CNB021271879 A CN B021271879A CN 02127187 A CN02127187 A CN 02127187A CN 1273483 C CN1273483 C CN 1273483C
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Abstract
本发明采用分子生物学的方法克隆了玉米bZIP类转录因子ABP9的基因，ABP9基因是一个新的bZIP类转录调控基因，其编码的蛋白是一个新的转录调控因子。ABP9基因产物是具有ABRE结合特异性和转录激活功能的转录因子。ABP9是与植物抗非生物逆境密切相关的转录因子，ABP9可以提高植物对低温、干旱和盐渍等多种非生物逆境的抵抗能力。
Description
技术领域
本发明属于植物基因工程领域，是采用分子生物学的方法克隆了一个新的转录因子ABP9的基因，ABP9是与植物抗非生物逆境密切相关的转录因子，ABP9可以提高植物对低温、干旱和盐渍等多种非生物逆境的抵抗能力。
背景技术
在干旱、高盐和低温等非生物逆境条件下，植物并不仅仅是被动的接受，而是积极地调动体内的防卫体系来对抗外来的逆境，这时在植物体内发生了很多的变化，如新蛋白质的合成，代谢的转变，抗逆境物质的积累等等(Hans J.1995，Plant cell.7：1099-1111)。其中有很多的蛋白参与了植物对非生物逆境的反应(Ashwani Pareek，1998，CurrentScience.75：1170-1174)，它们协同调整植物生理生化以及代谢的变化，提高植物对非生物逆境的抗性，研究表明植物对非生物逆境的抵抗并不是依靠某一个两个功能基因就能完成的。
在非生物逆境条件下，植物体内被诱导产生的蛋白中有许多是与植物抗逆境密切相关的基因，其中的一些基因已被克隆(Anil Grover，1998，Current Science.75：689-695)。为了提高植物的抗寒、抗旱和抗盐等非生物逆境的能力，人门也克隆了许多其它不同来源的抗逆相关基因，并转化到植物中(Shavindra Bajaj，1999，Molecular Breeding.5：493-503)。这些被克隆并进行转化基因可分为3类：1)合成渗透调节因子的酶类：如向烟草转入E.coli来源的mtlD基因，可提高植物的甘露醇含量；向烟草、水稻转入P5CS，可提高植物的脯氨酸含量；向拟南芥、水稻转入codA，提高了植物甜菜碱的含量(Sakamoto A.，1998，PMB.38：1011-1019)。2)LEA及其相关蛋白：如向拟南芥转入cor15a基因(Steponkus PL，1998，PNAS.95：14570-14575)。3)氧化逆境相关蛋白：如向苜蓿中转入Mn-SOD(Mckersic BD，1999，Plant Physiol.111：1177-1181)；向烟草中转入GST。这些功能基因转入到植物中后，在实验室条件下，植株在某一方面的抗逆性有所提高，但所取得的效果并不理想。最近，有人向拟南芥中转入抗逆相关的转录因子CBF1(C-repeatBinding Factor)，CBF1可调控一系列的抗寒相关基因的表达，与上述转单个的功能基因相比，CBF1明显提高拟南芥的抗寒性能(Kirsten R.1998，Science.280：104-106)。同样，向拟南芥转入DREB1A因子，它可调控与抗非生物逆境相关的多个基因的表达，明显提高了植株的抗盐、抗寒、抗旱的能力(Mie Kasuga，1999，Nature Biotechnology.17：287-291)。
已有的研究表明在干旱、盐渍和低温等逆境条件下，植物体内会产生大量的活性氧，形成氧化逆境(Zhu J.K.，2001，Trends Plant Sci.6：66-71)。由于活性氧具有非常活泼的化学性质，它会对细胞造成严重的伤害，如损伤细胞膜、失活重要的酶类及DNA断裂等，因此活性氧的及时清除对植物抗非生物逆境能力的提高具有重要意义。在活性氧的清除过程中，过氧化氢酶CAT1起作重要作用，但在逆境条件下，植物本身的抗氧化系统受诱导表达的能力比较弱，不能及时地清除体内的活性氧，影响了植物抗逆性的进一步提高，所以克隆调控Cat1表达的转录因子不仅有助于我们对ROS信号传导路径的理解，而且对培育具有抗旱、抗盐、抗寒等逆境能力的作物具有重要的指导意义。因为这样的反式因子它不仅仅是能够调控Cat1基因的表达，还能调控其它的一系列的抗氧化基因和抗非生物逆境基因的表达。
ABRE是存在于许多逆境响应基因启动子区的ABA响应元件(ABAResponsive elelment)，其特征序列是(C/G/T)ACGTG(G/T)(A/C)(Wenqiong Chen 2001，Plant Cell.14：559-574)。在Cat1基因的启动子区含有一个重要的与逆境相关的顺式调控元件：ABRE1和ABRE2，缺失分析表明：ABRE2(5’-GAAGTCCACGTGGAGGTGG)是Cat1基因响应ABA调控所必需的顺式元件。在玉米幼胚的发育过程中，随着ABA含量的升高，Cat1基因的表达也随之增强，事实上，胚发育的过程就是有机物质不断积累，细胞不断脱水的过程，也是细胞抗逆境能力不断提高的过程。研究表明在玉米幼胚的发育过程中，细胞中存在着能够与ABRE2相互作用的反式因子，可分为两类：一类是依赖于ABA的能够与Cat1基因ABRE2元件相互作用的反式因子(命名为Cat1 promoter Binding Factor 1，CBF1)，另一类是不依赖于ABA的能够与Cat1基因ABRE2元件相互作用的反式因子(命名为Cat1 promoter Binding Factor 2，CBF2)(LingqingM.Guan，2000，The Plant Journal.22(2)：87-95)。目前还没有关于这两类重要调控因子的报道。
发明内容
本发明总的目的是从重要农作物玉米中分离、克隆具有ABRE结合特异性的转录调控因子，该调控因子可作用于多个抗非生物逆境相关基因启动子区的ABRE顺式作用元件，调控抗非生物逆境相关基因的表达，提高植物对非生物逆境的抵抗能力。
本发明采用酵母单杂交的方法对所构建的玉米授粉后17天(17dpp)幼胚cDNA文库进行了筛选，得到1个阳性的克隆，命名为ABRE结合蛋白ABP9(ABRE Binding Protein 9)。把本发明所克隆的ABP9的蛋白序列输入到GenBank中BLAST分析，结果表明ABP9属于bZIP类转录因子，与已报道的bZIP类转录因子相比，ABP9与它们之间的同源性均很低，ABP9与来源于模式植物拟南芥的ABF3的同源性最高，但也只有18.8％。
本发明采用5’RACE的方法，获得了ABP9基因的全长cDNA序列。ABP9基因的全长cDNA序列包含1510bp(图1)，推测的读码框为1158bp，编码385个氨基酸(图2)。本发明还包括该残基序列经过一个或几个氨基酸残基的取代、缺失或添加且具有与序列2的氨基酸残基序列相同活性的序列衍生的蛋白质；以及编码该蛋白质的DNA序列，且具有与序列1同源性为90％以上的DNA序列。
本发明把所克隆的ABP9基因分别构建到酵母表达载体pPC86和原核表达载体pGEX4T-1上，实验结果表明ABP9基因产物不但在酵母体内具有ABRE结合特异性(图3)，在体外也同样具有ABRE结合特异性(图4)，ABP9基因产物能够特异作用于含有核心元件序列为(C/G/T)ACGTG(G/T)(A/C)的ABRE顺式元件。
本发明把所克隆的ABP9基因分别构建到酵母表达载体YepGAP和植物表达载体pBI121上，实验结果表明ABP9基因产物在酵母(图5)和玉米悬浮细胞(图6)中均具有ABRE结合特异性和转录激活功能，因此，ABP9基因产物是具有ABRE结合特异性和转录激活功能的转录因子。且ABP9基因受到盐、干旱、过氧化氢、ABA等逆境条件的诱导表达(图7)。
综上所述，本发明报道的ABP9基因是一个新的转录调控基因，其编码的蛋白是一个新的转录调控因子。
本发明把ABP9基因构建到植物转化载体上(图8)，并转化到农杆菌中，用农杆菌转化拟南芥并获得了转基因植株。对转基因植株在不同的逆境条件下进行处理，结果证明ABP9能够提高转基因植物的抗旱(图9)、抗盐(图10)和抗寒(图11)等非生物逆境的能力。
附图简要说明
图1、ABP9的cDNA序列
图2、ABP9的蛋白序列
图3、ABP9与ABRE的体内结合特异性
图4、ABP9与ABRE的体外结合特异性
图5、ABP9在酵母体内的ABRE结合特异性及转录激活功能
图6、ABP9在玉米细胞中的ABRE结合特异性及转录激活功能
图7、ABP9在盐、干旱、过氧化氢、ABA、低温等逆境条件的表达
图8、ABP9转基因表达载体的构建
图9、ABP9转基因拟南芥的抗旱试验
图10、ABP9转基因拟南芥的抗寒试验
图11、ABP9转基因拟南芥的抗盐试验
具体实施方式
实施例1：
玉米材料：玉米选用齐319授粉后17天(17dpp)的幼胚。菌种：大肠杆菌E.coli DH5α、JM109及酵母菌株yWAM2(Leu-，His-，Trp-)。载体：pBSK+及pRS315、pPC86。工具酶和修饰酶：各种限制性内切酶和修饰酶购自Promega公司，New England Biolab公司和Gibco公司。化学试剂：酵母培养用试剂均购自Sigma公司和Oxford公司；其它化学药品均为国产分析纯。试剂盒：质粒DNA提取用Promega公司的WizardTMMinipreps DNA Purification System和WizardTM Maxipreps DNAPurification System；回收DNA用鼎国公司的DNA片段快速纯化/回收试剂盒；RNA提取用Promega公司的RNAgents Total RNA Isolation Systemkit和PolyATtract mRNA Isolation System；文库构建用GibcoBRL公司的SuperScriptTM Plasmid System for cDNA Synthesis and PlasmidCloning kit。引物合成：由北京赛百盛生物工程公司和上海博亚生物技术有限公司合成。测序：由上海博亚生物技术有限公司完成。
RNA的提取和mRNA的分离：
RNA的提取和mRNA的分离分别按Promega公司的RNAgents Total RNAIsolation System kit和PolyATtract mRNA Isolation System进行。取玉米17dpp幼胚1g，提取总RNA 2.834mg，共分离出mRNA 43.7μg。取玉米21dpp幼胚1g，提取总RNA 2.427mg，共分离出mRNA 41.6μg。
玉米幼胚17dpp、21dpp cDNA文库的构建及扩增：
按GibcoBRL公司的SuperScriptTM Plasmid System for cDNASynthesis and Plasmid Cloning kit进行，取出5μg 17dpp的mRNA用于cDNA文库构建，反转录引物用：
Not I adapter：5’-pGACTAGTTCTAGATCGCGAGCGGCCGCCC(T)15-3’，
双链合成后加Sal I adapter：5’-TCGACCCACGCGTCCG-3’；
GGGTGCGCAGGCp-5’；
再用Not I酶切，构建到经Sal I和Not I酶切纯化后的pPC86载体上(Trp+)。转化E.coli.DH10B，得到库容量为5.2×106cfu的cDNA文库。
cDNA文库的扩增：
配2×LB培养基4L(Bacto-Trypton 20g/L，Bacto-Yeast Extract10g/L，NaCl 10g/L，SeaPrep agarose 3g/L，pH7.0)，121℃30min灭菌后，37℃温育2小时；加入氨苄青霉素至终浓度200mg/L；加入文库至106cfu/L；混匀后，分装20~30mL到50的培养管中；置于冰上1小时；30℃培养40小时；8000rpm，10min，离心收集菌体；弃上清，加入200mL 2×LB(含甘油12.5％)悬浮细胞，分装成10mL一份于-70℃保存备用。
4mer ABRE诱饵载体及4mer mutant ABRE(mABRE)挽救载体的构建
合成ABRE(+)：5’GAAGTCCACGTGGAGGTGG3’和ABRE(-)：5’TCCCACCTCCACGTGGACT 3’，各取20μL(1μg/μL)的ABRE(+)和ABRE(-)，混匀，加4μL 3M的NaOAc和100μL的无水乙醇，-20℃，30分钟后离心沉淀DNA，70％乙醇洗一次，干燥，加6.5μL无菌水，1μL 10×的T4 polynucleotide kinase buffer，退火：条件是88℃，2’；65℃，10’；37℃，10’；25℃；5’，加1.5μL 20mM的ATP和1μL T4polynucleotide kinase，37℃反应2小时，加酚氯仿和氯仿各抽提一次，用无水乙醇沉淀DNA。再加2μL 10×ligase buffer，1μLligase(5units/μL)，17μL无菌水，16℃连接过夜，用2％的琼脂糖电泳，分离大小约为80bp的DNA片段，克隆到pBSK+载体上(经Spe I酶切，补平)，测序，获得pA4质粒。合成mABRE(+)：5’GAAGTaacatgttcGGTGG3’和mABRE(-)：5’TCCCACCgaacatgttACT 3’，方法同上，获得pmA4质粒。
pRS315His(Leu+)载体及pA4和pmA4质粒均用BamH I、Xba I双切后纯化，把4连ABRE和4连mABRE克隆到pRS315His，得到诱饵载体pRSA4(Leu+)和挽救载体pRSmA4(Leu+)质粒。
玉米幼胚17dpp cDNA文库的筛选：
制备yWAM2感受态细胞，把pRSA4转化到酵母菌株yWAM2(Leu-，His-，Trp-)中，转化方法参照Two Hybrid System TRAFO Protocol进行。获得含有pRSA4的酵母菌株yA4(His-，Trp-)。用含有诱饵载体的yA4酵母对文库进行筛选，转化方法同上，将转化细胞涂到His-选择培养基上，28℃培养3~5天。待酵母长出后，提酵母质粒，提取方法参照Method I：QuickPlasmid DNA Preparations from Yeast(Christine Guthrie 1991)。将酵母质粒转化E.Coli DH5α，在从中提取质粒，酶切分析，测序可知所获得的阳性克隆的DNA序列，再对序列进行分析。
ABP9全长cDNA序列的获得：
采用5’RACE的方法得到了ABP9基因的全长cDNA序列，按GibcoBRL公司5’RACE System for Rapid Amplification of cDNA Ends，Version2.0kit操作。
用于反转录的引物：ABP9rv2：5’-CCT TCA CCA GGA AGT CCT CCA-3’
用于PCR的引物：ABP9rv3：5’-AAC CAA TCC TCC GTT CTC ACC-3’
扩增条件是：PCR：94℃，3min；94℃，30sec；60℃，30sec；72℃，1min；72℃，5min；35个循环。把扩增出DNA片段从琼脂糖胶中回收并连接到pGEM-T easy vector，转化E.coli.JM109，酶切鉴定后测序，获得ABP9基因的全长cDNA序列(图1)，根据cDNA序列推测出其蛋白序列(图2)。
实施例2
ABP2和ABP9的ABRE体内结合特异性分析：
将yA4和ymA4转化到yWAM2酵母中，得到yA4和ymA4菌株，把筛库得到的ABP9质粒分别转化yA4和ymA4酵母，28℃在His-选择培养基上培养3~5天，只有用ABP9质粒转化的yA4酵母能够生长，而ABP9转化的ymA4酵母不能够生长，说明ABP9在酵母体内能够特异结合ABRE元件，激活了报告基因HIS3基因的表达，故能够在His-选择培养基上生长(图3B)，相反由于ABP9不能与mABRE结合，不能激活了报告基因HIS3基因的表达，在His-选择培养基上也不能生长(图3A)，因此，ABP9在酵母体内具有ABRE结合特异性。
ABP9的ABRE体外结合特异性分析(EMSA实验)：
ABP9蛋白的纯化：把ABP2和ABP9的全长基因克隆到pGEX4T-1原核表达载体上，将克隆到pGEX4T-1载体上的ABP9转化到BL21菌株中，用37℃，0.3mM IPTG诱导表达2~3小时，SDS-PAGE电泳，有特异的ABP9表达条带。ABP9蛋白纯化的操作方法按照Pharmacia公司的MicroSpinTMGST Purification ModuLe protocol进行，纯化的蛋白用于EMSA实验。
同位素标记ABRE和mABRE：采用Promega公司的DNA 5’End-LabelingSystem，反应体系是：ABRE(或mABRE)1μL，T4PNK 10×buffer 5μL，γ-32P-ATP 3μL，T4PNK(10U/μL)2μL，H2O 39μL；37℃20分钟；加2μL 0.5M EDTA，68℃10分钟灭活；37℃10分钟；4℃保存备用。
ABP9蛋白与DNA结合反应体系：5×binding buffer(125mMHEPES-KOH ph7.6；50％甘油；250mM KCl)4μL；ABF2、ABF9和GST蛋白各约4μg(9μL)；1M DTT 1μL；上述标记的ABRE(或mABRE)探针1μL；H2O 4μL；冰上结合30分钟，加3μL上样缓冲液(含0.025％溴酚蓝的无菌水)，进行聚丙烯酰胺电泳分析。
非变性聚丙烯酰胺电泳：配制聚丙烯酰胺胶(5.4％)：30％丙烯酰胺9ml；10×电泳缓冲液5ml(甘氨酸142.7g/L，EDTA 3.92g/L，Tris30.28g/L)；50％甘油2.5ml；去离子水33ml；10％APS 400μL；TEMED 25μL。待胶凝后，用1×电泳缓冲液电泳，预电泳10分钟(300V)；上样，电泳1小时(300V)；用滤纸粘下胶，保鲜膜封好后，压X光片1小时；洗片，显影2分钟，定影5分钟。结果表明有明显的被ABP9蛋白阻滞ABRE的电泳条带(图4)，相反，mABRE则不被阻滞，说明ABP9基因产物在体外同样具有ABRE结合特异性。
实施例3
酵母细胞中转录激活试验
把ABP9和YepGAP(Trp+)用Xba I和Xho I双切、回收连接，转化E.coliDH5α；得到含有ABP9基因全长cDNA的酵母表达载体YepGAPABP-9质粒，将其转化到yA4和ymA4酵母中，在His-选择培养基上28℃培养3~5天，结果表明用YepGAPABP-9质粒转化的yA4能够生长(图5B)，而ymA4不能生长(图5A)，因此ABP9在酵母细胞内不但具有ABRE结合特异性，而且具有转录激活功能。
玉米细胞中的转录激活功能试验
报告载体的构建：pIG46载体用Xho I酶切，T4 DNA聚合酶补平，pBluescript II SK+载体上的4mer ABRE用Sma I、Ecl136 II酶切，回收约80bp的DNA片段与载体连接，转化E.coli DH5α，提质粒，酶切鉴定，测序表明ABRE已连在35S mini promoter上游。
ABP9瞬时表达载体的构建：把ABP9(Xba I，Xho I)基因的全长cDNA构建到植物瞬时表达载体pBI221上，得到pBI221-ABP9质粒，采用基因枪的方法共转化报告质粒和目的基因，转化材料为玉米悬浮细胞，转化方法参照B.R.格利克和J.E.汤普森主编的《植物分子生物学及生物技术的实用方法》，结果表明单独转化pIG46质粒，没有报告基因的表达(图6A)，把pIG46和pBI221-ABP9共转化时，有明显的报告基因的表达(图6B，C)，因此ABP9在玉米细胞内不但具有ABRE结合特异性，而且具有转录激活功能。
ABP9在非生物逆境条件下的表达特异性分析
玉米材料的处理：取玉米种子，吸胀24hr，种植于花盆中，28℃，12hr光照培养约20天，待长出三叶一芯，进行不同条件的处理。
1).冷害处理：将玉米苗置于2℃培养箱，12hr光照培养48hr，取出并洗去根上的土，用液氮速冻，-80℃保存备用。
2).盐渍处理：分别将玉米苗置于0.6％，0.8％，1％的NaCl溶液中，12hr光照培养3天，取出并洗去根上的土，用液氮速冻，-80℃保存备用。
3).干旱处理：分别将玉米苗置于含水量为8％(混和920g干土和80mL水)，10％，13％的土壤中12hr光照培养3天，取出并洗去根上的土，用液氮速冻，-80℃保存备用。
4).ABA处理：分别将玉米苗置于10-4M、10-5M、10-6M的ABA溶液中(取5mgABA用0.1N KOH溶解后，定容至95mL水中即为10-4M)，12hr光照培养24hr，取出并洗去根上的土，用液氮速冻，-80℃保存备用。
5).H2O2处理：分别将玉米苗置于10mM H2O2(1.13mL 30％H2O2/L)，60mM H2O2(6.78mL 30％H2O2/L)，150mM H2O2(14.95mL 30％H2O2/L)的水溶液中，12hr光照培养24hr，取出并洗去根上的土，用液氮速冻，-80℃保存备用。
6).水处理：将玉米苗直接置于水中12hr光照培养24hr，-80℃冻存。
7).对照的处理：直接取未经任何处理的苗-80℃冻存作为对照。
RNA的提取及DNA的去除：
1).取约200mg处理的玉米材料，液氮研磨，提取RNA的方法按照Promega公司的RNAgents Total RNA Isolation System kit进行。
2).将RNA溶于85μL水中，加入10X buffer 10μL和5μL RQ1 RNAFree DNase(1U/μL)，37℃，15min，以去除DNA的污染。
3).加入100μL的酚氯仿抽提一次，取上清，用等体积的异丙醇沉淀RNA，70％的乙醇洗一次，溶于50μL的水中。
4).定量RNA的浓度为1μg/μL。
RT-PCR：
取Oligo dT18 1μL(0.5μg/μL)，RNA 5μL(1μg/μL)，dNTP 1μL(10mM)，水27μL；65℃，5min；0℃，2min；加入5X buffer 10μL，DTT(100mM)5μL，RNase Inhibitor 10U(40U/μL)；42℃，2~5min；加入SuperScipt II 1μL(200u/μL)；42℃，50min；70℃，15min灭活，备用。
模板cDNA的相对定量及内标引物的设计：根据GenBank上登录的玉米肌动蛋白基因(Maize Actin1 gene：Accession NO.J01238)设计如下引物：
mAct1 F：5’-CAC CTT CTA CAA CGA GCT CCG-3’
mAct1 R：5’-TAA TCA AGG GCA ACG TAG GCA-3’
用该引物进行扩增，如果是cDNA可扩增出405bp的条带，如果是基因组DNA可扩增出512bp的条带(有107bp的内含子)。
PCR反应体系：模板1μL，2X PCR buffer 10μL，10mM dNTP 1μL，10μM mAct1 F 1μL，10μM mAct1 R 1μL，Taq 1U，灭菌水6μL。
PCR条件：94℃，2min；94℃，30sec；55℃，30sec；72℃，30sec；30cycle；72℃，5min。
根据PCR产物的电泳结果对模板DNA进行稀释，调整模板DNA的用量，直到用mAct1 F和mAct1 R引物扩增出的DNA条带的量基本一致，使每微升溶液中模板cDNA的含量基本一致。
ABP9基因的PCR扩增。
2).ABP9，设计PCR扩增的引物如下(扩增937bp)：
FW1 5’-CAT GAC GCT GGA GGA CTT CCT-3’
RV 5’-TTG ACG AAA ACA CAG AGC-3’
PCR体系：模板1μL，2X PCR buffer 10μL，10mM dNTP 1μL，10uMmAct1 F 1μL，10uM mAct1 R 1μL，Taq 1U，灭菌水6μL。
PCR条件：94℃，2min；94℃，30sec；55℃，30sec；72℃，50sec；30cycle；72℃，5min。电泳结果表明ABP9基因受盐渍(图7A，B，C)、干旱(图7J，K)、ABA(L，M，N)、过氧化氢(F，G)等逆境条件的诱导表达。
实施例4
ABP9基因的拟南芥转化
载体构建及农杆菌转化：把ABP9用Xho I酶切，T4DNA聚合酶补平，再用Xba I酶切，回收目的片段；载体pBI121用Xba I和Ecl136II酶切，纯化回收相应大小的片段，连接(图8)，转化JM109，提质粒，酶切鉴定，挑出所需克隆，测序，并将其转化到农杆菌LBA4404中。
拟南芥转化
1).拟南芥的培养。拟南芥种子在4℃进行2-3天的春化处理，每盆播种7~10粒种子(营养土和蛭石按2∶1)；放于温室中培养(22℃，光照16h)；待拟南芥抽出初生苔后，剪去初生苔，待其抽出更多的次生苔，且少数开始结荚时，可用于转化。
2).农杆菌的培养。挑农杆菌单菌落接种在3mlYEB(Kan50mg/L，利福平50mg/L)，28℃，250rpm培养30小时；按1∶400转接入200ml新鲜的YEB(Kan50mg/L，利福平50mg/L)中，28℃，250rpm培养约14小时，测OD600≈1.5；7500rpm，4℃，10min离心收集菌体；重悬菌体于二倍体积(400mL)的渗透液(1/2MS盐+5％蔗糖，pH5.7，121℃，15min灭菌；用之前加6-BA至终浓度为0.044uM，VB6终浓度为1mg/L，VB1终浓度为10mg/L，SILWET0.02％。
3).转化。把拟南芥的花蕾浸入渗透液中，抽真空(25IN Hg，保持5分钟)；转化完毕后，花盆外套上塑料袋，水平放置，使其在低光强度下生长24-48小时后，即可正常培养。
4).收集种子，进行筛选。称25-30mg种子放入1.5mL离心管，加1mL 75％的乙醇(含0.05％Tween 20)，于摇床上摇10分钟(300rpm)，离心，去上清；加1ml 95％的乙醇洗一次，离心，去上清；重复一次；在超净台中加0.3ml的100％乙醇，移到无菌滤纸上，吹干；把吹干的种子撒到1/2MS平板(Kan50mg/L)上；4℃，放2天后，22℃，16h光照培养；将阳性植株(T0代)移栽到盆中培养，并收集种子进行T1代筛选。
转基因拟南芥基因组DNA的提取
1).在液氮中研磨0.1-0.2g植物叶片，转移至1.5ml离心管中。
2).加入0.7ml CTAB(Tris 100mM，NaCl 1.4M，20mM EDTA，CTAB 2％，巯基乙醇0.1％)，60℃，30分钟。每隔10分钟，颠倒一次。
3).加0.7ml酚∶氯仿(1∶1)，颠倒几次，10000rpm离心5分钟，转移上清至一新的离心管，加等体积的氯仿∶异戊醇(24∶1)，混匀，10000rpm离心5分钟。转移上清至一新的离心管。
4).加等体积的异丙醇，颠倒混匀，10000rpm离心10分钟，除去上清，70％乙醇洗一次，抽干，溶于50μL的无菌水中，用于PCR检测。
转基因拟南芥PCR检测。
正向引物：35S promoter：5’-TCTGCCGACAGTGGTCCCAA
反向引物：ABP9 rv3：5’-CCACTCTTGCCTCCTAACCAA
反应体系(20μL)：转基因植株DNA 1μL(20ng~50ng)；10×buffer 2μL；MgCl2(2.5mM)2μL；Taq酶0.2μL；dNTP(2.5mM)2μL；引物各加10μM；加无菌水至20μL。
反应条件：94℃，5分钟；94℃，45秒；60℃，45秒；72℃，45秒；35个循环；72℃延伸5分钟。筛选出PCR阳性植株。
ABP9转基因植株的生理分析实验。
1).抗寒实验：把转基因植株与未转基因植株置于-6℃，保持6小时；转移到正常的生长条件下继续培养，结果表明：转基因植株的存活率为80％，未转基因的植株存活率为10％，ABP9能够明显提高植株的抗寒性能(图9)。
2).抗盐实验：把转基因植株与未转基因植株置于600mM的NaCl中浸泡3小时；22℃，光照培养24小时；转移到拟南芥正常的生长条件下继续培养，结果表明：转基因植株的存活率为80％，未转基因的植株存活率为15％，ABP9能够明显提高植株的抗盐性能(图10)。
3).抗旱实验：把转基因植株与未转基因植株置于拟南芥正常的生长条件下不给水连续培养15~20天。结果表明：转基因植株的存活率为90％，未转基因的植株存活率为5％，ABP9能够明显提高植株的抗旱性能(图11)
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<110>中国农业科学院生物技术研究所
<120>一个玉米bZIP类转录因子及其编码基因与应用
<130>I020275
<160>2
<170>PatentIn version 3.1
<210>1
<211>1510
<212>DNA
<213>maize
<400>1
taaaaggagt ctttgcagtt gcaggtcgag tgtctagata tgatatggcg tcggagacga 60
ccaagaacgt gaaggccacc ctcgacgagc aggaggtaac ctcgcatcag cgcgaccaga 120
gcgctgccga ggaggaggag gtggtggtag tggtggatcc gctggcgcgg cagtcgtccg 180
tcatgtcgct tacgctggag gagctgcaga gctcgctctg cgagccgggg cgcaacttcg 240
ggtccatgaa catggacgag ttcatggcca acatatggaa cgccgaggag ttccaggccg 300
ccaccgccac cgccaccggc ggctgcagca agcaggaggg cacgcagcgg gagccgatga 360
tgcccgtggc gaatggaaca ggtgagaacg gaggattggt tcggcaggcc aacgcggcgg 420
cgcaggccgt ggtgcagccc cagatgggga gcggcggtgg cggcggcggc gtcgccgcca 480
gcgggcggca gcaggtgacg ctggccgaca tgacgctgga ggacttcctg gtgaaggctg 540
gcgtcgtgcg aggagccttc gccggccacg gccacgcggt cgtcggcatg gccccaatcc 600
cagccgggcg gatgggcatc cagcagcagc acgcggctcc cacgatgtcg taccaagtgg 660
cagcgccggc gcccaacgcc gtgtacccgg ttatgggcaa cggcacgggg taccacaacg 720
ggtaccccag ggccatcgcg gtggtgccgc cgtctcagtg cgtgacggcc gccgtgagcc 780
cggggtcgtc ggacggggtg agcgcgatga cgcaggcgga gatgatgagc tgcattggca 840
acgaaggggc agggacggtc cggaactacg gcggcggcgg cggcggcggc agcgcgcgga 900
agcgcgactc ccccgaggac gcgtgcaccg agaagaccgt ggagcgccgg cagcggcgga 960
tgatcaagaa ccgtgagtcc gcggcccggt cacgcgccag gaagcaggcg tatacggtgg 1020
agctcgaagc tgaactgaac cacctcaaag aggagaacga tcgcctcaga gcagagcaga 1080
agacgattct gctatcgaag aaaaagaagc tggtggagaa gatggtggag caggcaaggg 1140
agaatgtgag cgccaagaag ggcggtcgcg ggctgcgccg ctcgggcagc gccatgtggt 1200
gaactgtgac gactgacgag tgtctcgaat tgtgcagttc cagtccgctt gcttagttgc 1260
ttgtatggga aaaaaaatcc tgtacctgtc ggtagcagca ccgttagtaa cagtatgcca 1320
tgtggtcgac acaaacgtct gcgagggacg atatggggca tgggcgagag cttccgcgtt 1380
ggcctgctgc tgttcatgag cctgggttgt acaaacaata aagcaactgt aagatagtag 1440
taattaaaca caaagctctg tgttttcgtc aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 1500
aaaaaaaaaa
1510
<210>2
<211>385
<212>PRT
<213>maize
<400>2
Met Ala Ser Glu Thr Thr Lys Asn Val Lys Ala Thr Leu Asp Glu Gln
1 5 10 15
Glu Val Thr Ser His Gln Arg Asp Gln Ser Ala Ala Glu Glu Glu Glu
20 25 30
Val Val Val Val Val Asp Pro Leu Ala Arg Gln Ser Ser Val Met Ser
35 40 45
Leu Thr Leu Glu Glu Leu Gln Ser Ser Leu Cys Glu Pro Gly Arg Asn
50 55 60
Phe Gly Ser Met Asn Met Asp Glu Phe Met Ala Asn Ile Trp Asn Ala
65 70 75 80
Glu Glu Phe Gln Ala Ala Thr Ala Thr Ala Thr Gly Gly Cys Ser Lys
85 90 95
Gln Glu Gly Thr Gln Arg Glu Pro Met Met Pro Val Ala Asn Gly Thr
100 105 110
Gly Glu Asn Gly Gly Leu Val Arg Gln Ala Asn Ala Ala Ala Gln Ala
115 120 125
Val Val Gln Pro Gln Met Gly Ser Gly Gly Gly Gly Gly Gly Val Ala
130 135 140
Ala Ser Gly Arg Gln Gln Val Thr Leu Ala Asp Met Thr Leu Glu Asp
145 150 155 160
Phe Leu Val Lys Ala Gly Val Val Arg Gly Ala Phe Ala Gly His Gly
165 170 175
His Ala Val Val Gly Met Ala Pro Ile Pro Ala Gly Arg Met Gly Ile
180 185 190
Gln Gln Gln His Ala Ala Pro Thr Met Ser Tyr Gln Val Ala Ala Pro
195 200 205
Ala Pro Asn Ala Val Tyr Pro Val Met Gly Asn Gly Thr Gly Tyr His
210 215 220
Asn Gly Tyr Pro Arg Ala Ile Ala Val Val Pro Pro Ser Gln Cys Val
225 230 235 240
Thr Ala Ala Val Ser Pro Gly Ser Ser Asp Gly Val Ser Ala Met Thr
245 250 255
Gln Ala Glu Met Met Ser Cys Ile Gly Asn Glu Gly Ala Gly Thr Val
260 265 270
Arg Asn Tyr Gly Gly Gly Gly Gly Gly Gly Ser Ala Arg Lys Arg Asp
275 280 285
Ser Pro Glu Asp Ala Cys Thr Glu Lys Thr Val Glu Arg Arg Gln Arg
290 295 300
Arg Met Ile Lys Asn Arg Glu Ser Ala Ala Arg Ser Arg Ala Arg Lys
305 310 315 320
Gln Ala Tyr Thr Val Glu Leu Glu Ala Glu Leu Asn His Leu Lys Glu
325 330 335
Glu Asn Asp Arg Leu Arg Ala Glu Gln Lys Thr Ile Leu Leu Ser Lys
340 345 350
Lys Lys Lys Leu Val Glu Lys Met Val Glu Gln Ala Arg Glu Asn Val
355 360 365
Ser Ala Lys Lys Gly Gly Arg Gly Leu Arg Arg Ser Gly Ser Ala Met
370 375 380
Trp ***
385
1
1
Claims (8)
1、玉米的bZIP类转录因子ABP9，它具有序列表中序列2的氨基酸残基序列。
2、玉米bZIP类转录因子ABP9的编码基因，是序列表中序列1的DNA片段。
3、根据权利要求2所述的基因，其特征在于：该基因的读码框为自5’端第45位到第1202位碱基。
4、含有权利要求2所述基因的表达载体。
5、根据权利要求4所述的表达载体，其特征在于：所述表达载体为pBI121ABP9。
6、转入权利要求2所述基因的转基因细胞系。
7、权利要求2所述基因在培育抗非生物逆境植物品种中的应用。
8、根据权利要求7所述的应用，其特征在于：所述非生物逆境为寒害、旱害和盐害。
Priority Applications (7)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021271879A CN1273483C (zh) | 2002-07-30 | 2002-07-30 | 一个玉米bZIP类转录因子及其编码基因与应用 |
| PCT/CN2003/000599 WO2004011493A1 (fr) | 2002-07-30 | 2003-07-28 | Facteur de transcription de type bzipm du mais et gene codant, et utilisation de ceux-ci |
| CNB038178079A CN1296382C (zh) | 2002-07-30 | 2003-07-28 | 玉米bZIP类转录因子及其编码基因与应用 |
| AU2003257774A AU2003257774A1 (en) | 2002-07-30 | 2003-07-28 | The bzip type transcription factor of corn and its coding gene and the usage thereof |
| US11/046,255 US7323339B2 (en) | 2002-07-30 | 2005-01-28 | Maize bZIP transcription factors and genes encoding the same and use thereof |
| US11/810,784 US20080060097A1 (en) | 2002-07-30 | 2007-06-07 | Maize bZIP transcription factors and genes encoding the same and use thereof |
| US11/810,795 US20080060098A1 (en) | 2002-07-30 | 2007-06-07 | Maize bZIP transcription factors and genes encoding the same and use thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021271879A CN1273483C (zh) | 2002-07-30 | 2002-07-30 | 一个玉米bZIP类转录因子及其编码基因与应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1472223A CN1472223A (zh) | 2004-02-04 |
| CN1273483C true CN1273483C (zh) | 2006-09-06 |
Family
ID=30121518
Family Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021271879A Expired - Lifetime CN1273483C (zh) | 2002-07-30 | 2002-07-30 | 一个玉米bZIP类转录因子及其编码基因与应用 |
| CNB038178079A Expired - Lifetime CN1296382C (zh) | 2002-07-30 | 2003-07-28 | 玉米bZIP类转录因子及其编码基因与应用 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB038178079A Expired - Lifetime CN1296382C (zh) | 2002-07-30 | 2003-07-28 | 玉米bZIP类转录因子及其编码基因与应用 |
Country Status (4)
| Country | Link |
|---|---|
| US (3) | US7323339B2 (zh) |
| CN (2) | CN1273483C (zh) |
| AU (1) | AU2003257774A1 (zh) |
| WO (1) | WO2004011493A1 (zh) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1273483C (zh) | 2002-07-30 | 2006-09-06 | 中国农业科学院生物技术研究所 | 一个玉米bZIP类转录因子及其编码基因与应用 |
| CN100395266C (zh) * | 2005-03-10 | 2008-06-18 | 中国农业科学院生物技术研究所 | 一个玉米抗逆转录调控因子及其编码基因与应用 |
| CN102382182B (zh) * | 2010-09-01 | 2013-07-24 | 中国科学院遗传与发育生物学研究所 | 一种与植物耐逆性相关的蛋白nek6及其编码基因与应用 |
| CN102911262B (zh) * | 2011-08-03 | 2014-05-07 | 中国农业科学院作物科学研究所 | 植物耐逆性相关蛋白及其编码基因和应用 |
| CN103044534B (zh) * | 2011-10-14 | 2014-07-02 | 中国农业科学院北京畜牧兽医研究所 | 紫花苜蓿抗旱相关基因及其编码的蛋白和应用 |
| CN104017061B (zh) * | 2013-03-01 | 2016-08-03 | 中国科学院植物研究所 | 转录因子ZmbZIP17及编码基因与其在响应逆境中的应用 |
| CN103320448B (zh) * | 2013-06-26 | 2014-09-03 | 昆明理工大学 | 一种岷江百合bZIP转录因子基因LrbZIP1及应用 |
| WO2015042737A1 (zh) * | 2013-09-26 | 2015-04-02 | 创世纪转基因技术有限公司 | 一种小盐芥亮氨酸拉链蛋白bZIP-4及其编码基因与应用 |
| CN104513823B (zh) * | 2014-11-24 | 2017-06-20 | 扬州大学 | 一种耐旱耐盐的转基因植物的制备方法 |
| CN107298701B (zh) * | 2017-04-18 | 2020-10-30 | 上海大学 | 玉米转录因子ZmbZIP22及其应用 |
| CN107488667B (zh) * | 2017-08-15 | 2019-12-03 | 四川农业大学 | 玉米ZmbZIP107基因在培育耐铅胁迫的植物中的应用 |
| CN111206031A (zh) * | 2020-03-11 | 2020-05-29 | 中国农业科学院生物技术研究所 | 一种用于检测玉米植物naz-4的核酸序列及其检测方法 |
| CN118652958A (zh) * | 2024-05-06 | 2024-09-17 | 蓝景科信河北生物科技有限公司 | 一种基于双重Pull down的DNA亲和纯化测序建库方法 |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US72159A (en) * | 1867-12-17 | bostw-ick | ||
| US20110093981A9 (en) * | 1999-05-06 | 2011-04-21 | La Rosa Thomas J | Nucleic acid molecules and other molecules associated with transcription in plants and uses thereof for plant improvement |
| US20100293669A2 (en) * | 1999-05-06 | 2010-11-18 | Jingdong Liu | Nucleic Acid Molecules and Other Molecules Associated with Plants and Uses Thereof for Plant Improvement |
| BR0013337A (pt) * | 1999-08-19 | 2002-04-02 | Kumiai Chemical Industry Co | Gene regulador da ramificação em plantas, vetor contendo o referido gene, microorganismo transformado pelo referido vetor, método para a regulagem da ramificação em plantas por meio do uso do referido microorganismo e planta contendo o referido gene regulador de ramificação em plantas |
| JP4028956B2 (ja) * | 2000-10-11 | 2008-01-09 | 独立行政法人農業生物資源研究所 | イネ貯蔵タンパク質の発現を制御するｂＺＩＰ型転写因子 |
| CN1273483C (zh) | 2002-07-30 | 2006-09-06 | 中国农业科学院生物技术研究所 | 一个玉米bZIP类转录因子及其编码基因与应用 |
-
2002
- 2002-07-30 CN CNB021271879A patent/CN1273483C/zh not_active Expired - Lifetime
-
2003
- 2003-07-28 AU AU2003257774A patent/AU2003257774A1/en not_active Abandoned
- 2003-07-28 CN CNB038178079A patent/CN1296382C/zh not_active Expired - Lifetime
- 2003-07-28 WO PCT/CN2003/000599 patent/WO2004011493A1/zh active Search and Examination
-
2005
- 2005-01-28 US US11/046,255 patent/US7323339B2/en not_active Expired - Lifetime
-
2007
- 2007-06-07 US US11/810,784 patent/US20080060097A1/en not_active Abandoned
- 2007-06-07 US US11/810,795 patent/US20080060098A1/en not_active Abandoned
Also Published As
| Publication number | Publication date |
|---|---|
| CN1472223A (zh) | 2004-02-04 |
| US20060021091A1 (en) | 2006-01-26 |
| CN1671738A (zh) | 2005-09-21 |
| WO2004011493A1 (fr) | 2004-02-05 |
| US7323339B2 (en) | 2008-01-29 |
| US20080060098A1 (en) | 2008-03-06 |
| AU2003257774A1 (en) | 2004-02-16 |
| CN1296382C (zh) | 2007-01-24 |
| US20080060097A1 (en) | 2008-03-06 |
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Application publication date: 20040204 Assignee: Origin Agritech Ltd. Assignor: BIOTECHNOLOGY Research Institute CHINESE ACADEMY OF AGRICULTURAL SCIENCES Contract record no.: X2022990000469 Denomination of invention: A maize bZIP transcription factor and its encoding gene and application Granted publication date: 20060906 License type: Exclusive License Record date: 20220810 |