CN110643614A - 佛甲草抗旱基因SlDREB及其应用 - Google Patents
佛甲草抗旱基因SlDREB及其应用 Download PDFInfo
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Abstract
本发明公开了佛甲草抗旱基因SlDREB及其应用，佛甲草抗旱基因SlDREB的核苷酸序列如SEQ ID NO.1所示，实验证明，采用SlDREB基因转染的拟南芥和烟草杨增强了对旱的耐受力，说明本发明提供的抗旱基因SlDREB在改良作物抗旱能力方面起着重要的作用。
Description
技术领域
本发明涉及一种佛甲草(Sedum lineare)抗旱基因SlDREB及其应用，属于分子生物学和生物技术领域。
背景技术
环境的非生物胁迫，如干旱，极端温度，寒冷，重金属，或高盐，严重损害植物生长和生产力。干旱是最严重的环境胁迫，超过任何其他环境因素，严重损害植物的生长和发育，限制植物生产和作物的性能。全球约有43％的耕地为干旱、半干旱地区，严重影响植物的生长发育，造成树木和农作物严重减产。干旱已经成为世界范围内严重影响农作物产量的逆境胁迫之一，对抗旱作物的育种及抗旱机理的研究具有深远重大的意义。因此，亟需能转入植物中，增强植物对旱的耐受性，对修复生态环境和提高土地利用率具有重要战略意义。
发明内容
本发明的目的是克服现有技术的不足，提供一种佛甲草抗旱基因SlDREB。
本发明的第二个目的是提供含佛甲草抗旱基因SlDREB的克隆载体pJET1.2_SlDREB。
本发明的第三个目的是提供含佛甲草抗旱基因SlDREB的表达载体pBI121_SlDREB。
本发明的第四个目的是提供含有表达载体pBI121_SlDREB的宿主细胞。
本发明的第五个目的是提供佛甲草抗旱基因SlDREB增强植物对旱的耐受性的应用。
本发明的技术方案概述如下：
佛甲草抗旱基因SlDREB，所述基因的核苷酸序列如SEQ ID NO.1所示。
含佛甲草抗旱基因SlDREB的克隆载体pJET1.2_SlDREB。
含佛甲草抗旱基因SlDREB的表达载体pBI121_SlDREB。
含表达载体pBI121_SlDREB的宿主细胞。
佛甲草抗旱基因SlDREB增强植物对旱的耐受性的应用。
所述植物优选拟南芥或烟草。
本发明的优点：
实验证明，采用SlDREB基因转染的拟南芥和烟草表现出对旱的耐受力，说明本发明提供的抗旱基因SlDREB在改良作物抗旱能力方面起着重要的作用。
附图说明
图1为佛甲草抗旱基因SlDREB克隆电泳示意图。
图2为佛甲草抗旱基因SlDREB插入表达载体后示意图。
图3为pBI121_SlDREB转化拟南芥后，转化子基因组PCR筛选结果(1-8号分别代表的是pBI121_SlDREB的单菌落菌液)。
图4为pBI121_SlDREB转化拟南芥后，T3纯合体半定量PCR测定表达水平结果(7，8代表中表达的拟南芥；4，5号代表高表达量的拟南芥；1，2，3，6号代表低表达量的拟南芥)。
图5为佛甲草抗旱基因SlDREB转基因拟南芥T3纯合体抗旱的实验效果照片。
图6为佛甲草抗旱基因SlDREB转基因烟草抗旱的实验效果照片。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例中未注明具体条件的实验方法，通常按照常规条件以及手册中所述的条件，或按照制造厂商所建议的条件。
载体pJET1.2 pJET1.2:Thermo,Clone JET PCR Cloning Kit#K1231。
载体pBIl21购于中国质粒载体菌株细胞株基因保藏中心，http://biovector.blog.163.com/
实施例1
1.佛甲草(Sedum lineare,简称Sl)SlDREB基因的克隆
从用100g/L聚乙二醇(HO[CH2CHO]nH相对分子质量：697.661)水溶液进行抗旱处理的佛甲草(取自天津市滨海新区)中，使用植物RNeasy Plant Mini Kit(TransgeneCode#E101-0150rxns)提取总RNA，并且利用EasyScript Frist-Strand cDNA SynSgesisSuperMix(Transgene Code#AE301-03100 rxns)反转录出cDNA。对cDNA进行高通量测序获得78407转录本(诺禾致源公司进行高通量测序)，通过与GO数据库进行对比分析，获得SlDREB基因的3’端序列，使用RACE技术(Takara-RACE kit)获得SlDREB基因的全长cDNA序列将扩增得到的SlDREB基因进行测序分析，得到完整的SlDREB基因全长为1314bp。构建超表达载体时，则分别在特异性引物的5’端添加pBI121重组位点，上游5'–ACGGGGGACTCTAGAGGATCC-3'(SEQ ID No.3)，下游5'-CGATCGGGGAAATTCGAGCTC-3'(SEQ IDNo.4)，以利于后期表达载体的构建。
其具体步骤如下：
1).cDNA第一链的合成
用反转录试剂盒TaKaRa RNA PCR Kit(AMV)Ver.3.0，以总RNA为模板，Oligo(dT)为引物，在AMV逆转录酶的作用下合成cDNA第一链，反转录体系如下：
反应条件：42℃60min，99℃5min。
2).佛甲草SlDREB基因反转录质量PCR扩增检测
用佛甲草Actin基因特异引物SEQ ID No.5:5'-GAACTTACTAGCCGACTG-3',SEQ IDNo.6：5'-CCTCAAGCCTTATACGCAA-3'，PCR扩增，以验证反转录反应及RNA质量。
PCR反应体系如下：
反应条件：94℃3min；94℃30s，40℃30s，72℃50s，35cycles；72℃5min。
3).佛甲草SlDREB基因片段PCR扩增
利用Takara RACE kit扩增得到的SlDREB基因进行测序分析，得到完整的佛甲草SlDREB基因全长为1314bp(SEQ ID No.1)。由佛甲草SlDREB基因编码的蛋白质，是SEQ IDNo.2所示的氨基酸序列。根据已知cDNA序列利用primer软件设计SlDREB基因上下游引物:
SEQ ID No.7:5'-CTCAGCTTGGCGGTTACCA-3'
SEQ ID No.8:5'-CAATTTCCCCTGCATTTTCTC-3'，其PCR反应程序如下：
反应条件：94℃3min；94℃30s，40℃30s，72℃50s，35cycles；72℃5min。
PCR反应结束后，取1μLPCR产物进行1.0％琼脂糖凝胶电泳，检测PCR产物的质量(见图1)，其余用作产物的纯化回收。
4).构建含有佛甲草SlDREB基因的克隆载体
构建含有佛甲草SlDREB基因的载体pJET1.2_SlDREB
胶回收纯化后的佛甲草SlDREB基因目的片段利用Clone JET PCR Cloning Kit(pJET1.2:Thermo,Clone JET PCR Cloning Kit#K1231)重组到载体pJET1.2上，得到载体pJET1.2_SlDREB。
其反应程序如下：
反应条件24℃,10min冰上静止30min，42℃热激1min30s冰上静止2min30s，
转入感受态细胞DH5α，37℃，180rpm，45min，此程序结束后将菌液涂到LB(添加抗生素Amp100uM)固体培养基(蛋白胨10g，酵母浸出物5g，氯化钠5g，琼脂15g，定容至1L，pH＝7)中，37℃过夜培养。
分别利用目的片段的上下游引物(SEQ ID No.7和SEQ ID No.8)对不同的菌落进行菌落PCR验证，筛选阳性菌落测序，得到含有克隆载体pJET1.2_SlDREB的宿主细胞。
注：pJET1.2:Thermo,Clone JET PCR Cloning Kit#K1231载体购于invitrogen；所用大肠杆菌为DH5α感受态细胞，TIANGEN，CB101-2。
5).构建含有佛甲草SlDREB基因的表达载体
构建含有佛甲草SlDREB基因的表达载体pBI121_SlDREB，
构建超表达载体时，则分别在特异性引物的5’端和3’添加pBI121重组位点，
SEQ ID No.3:5'–ACGGGGGACTCTAGAGGATCC-3',
SEQ ID No.4:5'-CGATCGGGGAAATTCGAGCTC-3'
得到：
SEQ ID No.9:5'-ACGGGGGACTCTAGAGGATCCCTCAGCTTGGCGGTTACCA-3'
SEQ ID No.10:5'-CGATCGGGGAAATTCGAGCTCCAATTTCCCCTGCATTTTCTC-3'
提取测序正确的pJET1.2-基因的质粒，作为模版，利用含有重组位点SEQ IDNo.9，SEQ ID No.10为引物进行PCR扩增，其反应程序如下
PCR反应结束后，取1μLPCR产物进行1.0％琼脂糖凝胶电泳，检测PCR产物的质量，其余用作产物的纯化回收。
提取pBI121质粒，其载体图谱(见图2)，对其进行双酶切线性化，程序如下：
反应条件：37℃，12h，80℃20min失活。
将基因和线性化的pBI121质粒使用Clone Express Entry One Step CloningKit试剂盒进行重组构建，反应程序如下：
(线性化pBI121质粒需50-200ng均可；胶回收基因片段需20-200ng均可；本实验线性化pBI121质粒浓度100ng/μL；胶回收基因片段浓度在100ng/μL)
反应程序：37℃，30min，冰上5min，42℃热激1min30s冰上静止2min30s，转入感受态细胞DH5α，37℃，180r，45min，此程序结束后将菌液涂到LB(添加抗生素kan 50uM)固体培养基中，37℃过夜培养。
分别利用载体和目的片段的上下游引物(见SEQ ID No.9和SEQ ID No.10)对同一个菌落进行菌落PCR双重验证，筛选阳性菌落测序(见SEQ ID No.11)。
注：本步骤使用Clon Express Entry One Step Cloning Kit购于vazyme，
6).含有佛甲草SlDREB基因的重组载体转化农杆菌感受态细胞
实验所用的农杆菌菌株为C58(购于中国质粒载体菌株细胞株基因保藏中心，http://biovector.blog.163.com/)，C58具有利福平抗性(Rif)，辅助质粒具有庆大霉素抗性(Gen)。
利用电击农杆菌转化法，将含有佛甲草SlDREB基因的大肠杆菌表达载体pBI121_SlDREB转化到农杆菌株C58(pMP90)感受态细胞中，28℃，培养36h，菌落PCR挑选阳性克隆菌落。
实施例2
1.转化拟南芥
(1)转化拟南芥。
转化拟南芥的具体操作步骤：
①实施例1获得的阳性克隆菌落活化与扩大培养
活化：挑保存的阳性克隆菌落置于3mLYEB液体培养基(蛋白胨5g，酵母浸出物1g，牛肉膏5g，蔗糖5g，定容至1L，pH＝7)中(添加Gen、Rift、kana抗生素，使浓度分别为30mg/L,、25mg/L、50mg/L)培养15小时左右(至OD600＝0.8左右)，180rpm，28℃。
阳性克隆菌的扩大培养：新鲜的10ml的YEB液体培养基中加入适量的抗生素(Gen、Rift、kana抗生素，浓度分别为30mg/L、25mg/L、50mg/L)，然后接种适量阳性克隆菌液到YEB液体培养基中进行培养，180rpm，在28℃培养至OD600＝0.6。
②转化
将菌液离心(3000rpm，15℃,10min)后弃上清，用体积两倍于所取菌液的质量浓度为5％的蔗糖水溶液重悬菌体(缓慢操作以保证菌体活力)，使菌体散开，调OD600＝0.8。
选取培养3-4周抽苔5-7cm的野生型拟南芥(商业)，倒置于装有转化液的容器中，使整个花序浸泡在菌液中15秒，取出拟南芥横躺在托盘中，用塑料薄膜罩住保湿，并暗处理12h，使拟南芥直立在温度25℃，光周期16h光照/8h黑暗，相对湿度为70％的培养条件下生长，直到种子成熟。种子采集后放到37℃烘箱烘干两周，以备后续试验使用。
(2)转基因拟南芥阳性转化子纯合体的筛选
将收取的T1代种子经过消毒以后，放置在冰箱4℃三天，然后在超净台上将转基因拟南芥种子均匀播种在含有50μg/mL卡那霉素的1/2MS固体筛选培养基(MS盐2.2g，蔗糖10g，定容至1L，pH＝5.7，琼脂7.2g)上，在1800Lux，光周期16h光照/8h黑暗，生长8-10天，叶子为深绿色即为转基因拟南芥的T1代阳性转化子。当T1代阳性转化子植株长到3-4片真叶时，将其移植到土壤(购于EPAGMA,荷兰，http://www.epagma.eu/)中，在温度25℃，1800Lux，光周期16h光照/8h黑暗，相对湿度为70％的培养条件下继续生长14天，先作阳性转化子的鉴定(见图3；1-8号分别代表的是pBI121_SlDREB的单菌落菌液)，再通过半定量PCR先对其转基因的表达水平进行鉴定(见图4；7，8代表中表达的拟南芥；4，5号代表高表达量的拟南芥；1，2，3，6号代表低表达量的拟南芥)，选取表达水平高的独立转化株系5号和表达水平低的独立转化株系2号。在上述条件下继续生长，约一个半月后收集种子即为T2代转化种子。重复上述步骤得到5号和2号的T3代纯合体种子。
(3)对转基因拟南芥进行抗旱处理
将5号T3代纯合体种子、2号T3代纯合体种子、野生拟南芥种子分别种植在土壤中，在温度25℃，1800Lux，光周期16h光照/8h黑暗，相对湿度为70％的培养条件下生长21天，每种植物保留21株长势一致的幼苗，随机分为三组平行实验，每组不同类型的植株各7株，通过用100g/L聚乙二醇(HO[CH2CHO]nH相对分子质量：697.661)水溶液进行干旱胁迫处理。每隔5天处理一次，共处理20天后植物照相(见图5)。
实施例3
1.转化烟草
供阳性克隆菌转化的烟草为NC89(6855-2×6772)组培苗(商业)。
(1)选取生长30天状况良好的烟草组培苗，挑选厚实叶片，减去叶边缘，把叶生剪成1cm×1cm的外植体碎生。将选取的实施例1获得的阳性克隆菌菌液(OD600＝0.8)将剪好的叶生材料放到农杆菌的LB液体培养基(蛋白胨10g，酵母浸出物5g，氯化钠5g，定容至1L，pH＝7)上，24℃条件下震荡培养10min。侵染后，将外植体吸干后放在MS培养基上(MS盐4.4g，蔗糖30g，定容至1L，pH＝5.7，琼脂7.2g)，黑暗培养3天。将经过暗培养的外植体用灭过菌的蒸馏水清洗2-3次，用滤纸吸干后，外植体放在选择培养基上(MS盐4.4g，蔗糖30g，生长素NAA 1.86mg，细胞分裂素2ip1.02mg，卡那霉素500mg，头孢霉素500mg，琼脂7.2g，定容至1L，pH＝5.7)；培养条件：2000Lux光下培养14天，光照/黑暗为16h/8h).
(2)经过选择培养基，外植体长出小苗子的时候，将整棵小苗子切下，将根原基竖直插到MS培养基上，诱导生根。待生根完成后，对每一棵独立转化子进行阳性鉴定。
通过半定量PCR选取表达水平高的独立转化株系和表达水平低的独立转化株系进行抗盐实验。
(3)将烟草进行抗旱处理
将生长7天的长势均匀的转基因高表达烟草、低表达转基因烟草、野生型烟草移栽至土盆中，待土盆苗生长5天后，每种植物保留21株长势一致的幼苗，随机分成三组，每组不同类型的植株各7株，通过用100g/L聚乙二醇(HO[CH2CHO]nH相对分子质量：697.661)水溶液进行干旱胁迫处理。每隔5天处理一次，共处理50天后植物照相(见图6)。
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<110> 天津大学
<120> 佛甲草抗旱基因SlDREB及其应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1314
<212> DNA
<213> 佛甲草(Sedum lineare)
<400> 1
atgagccact tcaaaaacac aacatacctt ttttcaacac aacatcttta cacacacgtg 60
tcttttaact ttaagttcat ttccctccta tatatacatg tttcaaatcc caccagactc 120
ttcatcttca aatatcttat agtcacatac acacttacaa aaaacacagc aactaaaaca 180
atggacaatt ttacacaatt ctccgatgat gtaccggaga attcagaagc gatgatcagc 240
ggtggtggtg gcggtcgcgg tggcggtgga attaattcaa aaacaaaaag tataagctat 300
tcagatgatg atatcataca attagcatca agtcatccta aaaaaaaagc cggtcggaaa 360
atcttcagag aaactaggca tccaatttac agaggtgtac gtcggagaaa tactaacaaa 420
tgggtatgtg aagtccgaga accgattaaa aaaaatcgaa tttggttagg aacatatcca 480
acaccagaaa tggcagctag agcacatgat gtagctgcat tagcttatag aggtttaaaa 540
acttgtctca attttgctga ctcagcttgg cggttaccaa aaccggcttc aactgacgcg 600
gcggatgttc aaaaggccgc cgctcaagct gctgaaatgt tcagaacaac aacatctcag 660
caactgaata ttaacaacga gagtaataga aatgttagca ttgttaaaga tggagatgaa 720
tcaacggcgg ccggagaaaa tgcaggggaa attgatggag gtgattataa ttatatggat 780
gaagaagcga tttttaacat gcctggatta ttgaataaca tggcggaagg actgttacta 840
ccgccgccgt gttatttacc ggattgggag tttgattcgg atatgaatgc ggatgttggt 900
gatgatttta ctgatctgtg gagtcattct atttgaggtt ggaattgtgt tggaagtgtg 960
aaatgctgtt cgtttaagta cgatgaagtg tagcgtttag ggtttgtaga cattattttt 1020
gagcatttat agtttgaagt cagtacaaga ttggatagtg ataggtatct gcgtgcgtca 1080
aattaagaac atacgtatcg atacaatctt tagaagacga tccccacgaa atggatatgt 1140
tagtaaaagt atcggtcgca atctttagaa gacggttctc acgtacctct caaaatgaaa 1200
gcaaatttaa gagattgttt tagaaaaaca tatcgcaaga acctctcgta gagattgcat 1260
cggtgaagat tccctaactc acggatggaa ggggttgaga gaaagttact ataa 1314
<210> 2
<211> 437
<212> PRT
<213> 佛甲草(Sedum lineare)
<400> 2
Met Ser His Phe Lys Asn Thr Thr Tyr Leu Phe Ser Thr Gln His Leu
1 5 10 15
Tyr Thr His Val Ser Phe Asn Phe Lys Phe Ile Ser Leu Leu Tyr Ile
20 25 30
His Val Ser Asn Pro Thr Arg Leu Phe Ile Phe Lys Tyr Leu Ile Val
35 40 45
Thr Tyr Thr Leu Thr Lys Asn Thr Ala Thr Lys Thr Met Asp Asn Phe
50 55 60
Thr Gln Phe Ser Asp Asp Val Pro Glu Asn Ser Glu Ala Met Ile Ser
65 70 75 80
Gly Gly Gly Gly Gly Arg Gly Gly Gly Gly Ile Asn Ser Lys Thr Lys
85 90 95
Ser Ile Ser Tyr Ser Asp Asp Asp Ile Ile Gln Leu Ala Ser Ser His
100 105 110
Pro Lys Lys Lys Ala Gly Arg Lys Ile Phe Arg Glu Thr Arg His Pro
115 120 125
Ile Tyr Arg Gly Val Arg Arg Arg Asn Thr Asn Lys Trp Val Cys Glu
130 135 140
Val Arg Glu Pro Ile Lys Lys Asn Arg Ile Trp Leu Gly Thr Tyr Pro
145 150 155 160
Thr Pro Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala Tyr
165 170 175
Arg Gly Leu Lys Thr Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu
180 185 190
Pro Lys Pro Ala Ser Thr Asp Ala Ala Asp Val Gln Lys Ala Ala Ala
195 200 205
Gln Ala Ala Glu Met Phe Arg Thr Thr Thr Ser Gln Gln Leu Asn Ile
210 215 220
Asn Asn Glu Ser Asn Arg Asn Val Ser Ile Val Lys Asp Gly Asp Glu
225 230 235 240
Ser Thr Ala Ala Gly Glu Asn Ala Gly Glu Ile Asp Gly Gly Asp Tyr
245 250 255
Asn Tyr Met Asp Glu Glu Ala Ile Phe Asn Met Pro Gly Leu Leu Asn
260 265 270
Asn Met Ala Glu Gly Leu Leu Leu Pro Pro Cys Tyr Leu Pro Asp Trp
275 280 285
Glu Phe Asp Ser Asp Met Asn Ala Asp Val Gly Asp Asp Phe Thr Asp
290 295 300
Leu Trp Ser His Ser Ile Gln Gly Trp Asn Cys Val Gly Ser Val Lys
305 310 315 320
Cys Cys Ser Phe Lys Tyr Asp Glu Val Ser Arg Leu Gly Phe Val Asp
325 330 335
Ile Ile Phe Glu His Leu Val Phe Glu Val Ser Thr Arg Leu Asp Ser
340 345 350
Asp Arg Tyr Leu Arg Ala Ser Asn Ala Glu His Thr Tyr Arg Tyr Asn
355 360 365
Leu Leu Lys Thr Ile Pro Thr Lys Trp Ile Cys Thr Arg Lys Tyr Arg
370 375 380
Ser Gln Ser Leu Glu Asp Gly Ser His Val Pro Leu Lys Met Lys Ala
385 390 395 400
Asn Leu Arg Asp Cys Phe Arg Lys Thr Tyr Arg Lys Asn Arg Leu Val
405 410 415
Glu Ile Ala Ser Val Lys Ile Pro Glu Leu Thr Asp Gly Arg Gly Gln
420 425 430
Glu Lys Val Thr Ile
435
<210> 3
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
acgggggact ctagaggatc c 21
<210> 4
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cgatcgggga aattcgagct c 21
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
gaacttacta gccgactg 18
<210> 6
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
cctcaagcct tatacgcaa 19
<210> 7
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ctcagcttgg cggttacca 19
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caatttcccc tgcattttct c 21
<210> 9
<211> 40
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acgggggact ctagaggatc cctcagcttg gcggttacca 40
<210> 10
<211> 42
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgatcgggga aattcgagct ccaatttccc ctgcattttc tc 42
<210> 11
<211> 1314
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgagccact tcaaaaacac aacatacctt ttttcaacac aacatcttta cacacacgtg 60
tcttttaact ttaagttcat ttccctccta tatatacatg tttcaaatcc caccagactc 120
ttcatcttca aatatcttat agtcacatac acacttacaa aaaacacagc aactaaaaca 180
atggacaatt ttacacaatt ctccgatgat gtaccggaga attcagaagc gatgatcagc 240
ggtggtggtg gcggtcgcgg tggcggtgga attaattcaa aaacaaaaag tataagctat 300
tcagatgatg atatcataca attagcatca agtcatccta aaaaaaaagc cggtcggaaa 360
atcttcagag aaactaggca tccaatttac agaggtgtac gtcggagaaa tactaacaaa 420
tgggtatgtg aagtccgaga accgattaaa aaaaatcgaa tttggttagg aacatatcca 480
acaccagaaa tggcagctag agcacatgat gtagctgcat tagcttatag aggtttaaaa 540
acttgtctca attttgctga ctcagcttgg cggttaccaa aaccggcttc aactgacgcg 600
gcggatgttc aaaaggccgc cgctcaagct gctgaaatgt tcagaacaac aacatctcag 660
caactgaata ttaacaacga gagtaataga aatgttagca ttgttaaaga tggagatgaa 720
tcaacggcgg ccggagaaaa tgcaggggaa attgatggag gtgattataa ttatatggat 780
gaagaagcga tttttaacat gcctggatta ttgaataaca tggcggaagg actgttacta 840
ccgccgccgt gttatttacc ggattgggag tttgattcgg atatgaatgc ggatgttggt 900
gatgatttta ctgatctgtg gagtcattct atttgaggtt ggaattgtgt tggaagtgtg 960
aaatgctgtt cgtttaagta cgatgaagtg tagcgtttag ggtttgtaga cattattttt 1020
gagcatttat agtttgaagt cagtacaaga ttggatagtg ataggtatct gcgtgcgtca 1080
aattaagaac atacgtatcg atacaatctt tagaagacga tccccacgaa atggatatgt 1140
tagtaaaagt atcggtcgca atctttagaa gacggttctc acgtacctct caaaatgaaa 1200
gcaaatttaa gagattgttt tagaaaaaca tatcgcaaga acctctcgta gagattgcat 1260
cggtgaagat tccctaactc acggatggaa ggggttgaga gaaagttact ataa 1314
Claims (6)
1.佛甲草抗旱基因SlDREB，其特征是所述基因的核苷酸序列如SEQ ID NO.1所示。
2.含权利要求1的佛甲草抗旱基因SlDREB的克隆载体pJET1.2_SlDREB。
3.含权利要求1的佛甲草抗旱基因SlDREB的表达载体pBI121_SlDREB。
4.含有权利要求3的表达载体pBI121_SlDREB的宿主细胞。
5.权利要求1的佛甲草抗旱基因SlDREB增强植物对旱的耐受性的应用。
6.根据权利要求5所述的应用，其特征是所述植物为拟南芥或烟草。
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