CN110607307A - 佛甲草耐盐基因SlNAC及其应用 - Google Patents
佛甲草耐盐基因SlNAC及其应用 Download PDFInfo
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- CN110607307A CN110607307A CN201910820423.0A CN201910820423A CN110607307A CN 110607307 A CN110607307 A CN 110607307A CN 201910820423 A CN201910820423 A CN 201910820423A CN 110607307 A CN110607307 A CN 110607307A
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Abstract
本发明公开了佛甲草耐盐基因SlNAC及其应用，佛甲草耐盐基因SlNAC的核苷酸序列如SEQ ID NO.1所示，实验证明，采用SlNAC基因转染的拟南芥和杨树增强的对盐的耐受力，说明本发明提供的佛甲草对耐盐基因SlNAC增强在改良作物抗盐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)耐盐基因SlNAC及其应用，属于分子生物学和生物技术领域。
背景技术
土壤次生盐渍化是影响农业生产和生态环境的重要因素之一，主要是由于土壤得不到雨水的淋溶，施肥不合理、水分蒸发快和不合理灌溉等问题造成的。土壤中盐分的大量积累严重阻碍了农业的可持续发展。土壤中硝酸盐的积累能提高土壤溶液的盐浓度和渗透压，使作物出现生理性干旱，影响植物对营养元素的吸收，引起作物营养失调。因此，亟需一种能转入植物中，增强植物对盐的耐受性的基因，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，提供一种佛甲草耐盐基因SlNAC。
本发明的第二个目的是提供含佛甲草耐盐基因SlNAC的克隆载体pJET1.2_SlNAC。
本发明的第三个目的是提供含佛甲草耐盐基因SlNAC的表达载体pBI121_SlNAC。
本发明的第四个目的是提供含有表达载体pBI121_SlNAC的宿主细胞。
本发明的第五个目的是提供佛甲草耐盐基因SlNAC增强植物对盐的耐受性的应用。
本发明的技术方案概述如下：
佛甲草耐盐基因SlNAC，所述基因的核苷酸序列如SEQ ID NO.1所示。
含上述佛甲草耐盐基因SlNAC的克隆载体pJET1.2_SlNAC。
含上述佛甲草耐盐基因SlNAC的表达载体pBI121_SlNAC。
含有表达载体pBI121_SlNAC的宿主细胞。
上述佛甲草耐盐基因SlNAC增强植物对盐的耐受性的应用。
所述植物优选拟南芥或杨树。
本发明的优点：
实验证明，采用SlNAC基因转染的拟南芥和杨树表现出对盐的耐受力，说明本发明提供的耐盐基因SlNAC在改良作物耐盐方面起着重要的作用。从而为培育耐盐转基因物种提供了一种有效的方法，
附图说明
图1为佛甲草耐盐基因SlNAC克隆电泳示意图。
图2为佛甲草耐盐基因SlNAC插入表达载体后示意图。
图3为pBI121_SlNAC转化拟南芥后，转化子基因组PCR筛选结果(1-7号分别代表的是pBI121_SlNAC的单菌落菌液)。
图4为pBI121_SlNAC转化拟南芥后，T3纯合体半定量PCR测定表达水平结果(4、6代表中表达的拟南芥；2，3号代表高表达量的拟南芥；1，5号代表低表达量的拟南芥)。
图5为佛甲草耐盐基因SlNAC转基因拟南芥T3纯合体耐盐的实验效果照片。
图6为佛甲草耐盐基因SlNAC转基因杨树耐盐的实验效果照片。
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pJET1.2pJET1.2: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)SlNAC基因的克隆
从50mM NaNO3水溶液进行耐盐处理的佛甲草(取自天津市滨海新区)中，使用植物RNeasy Plant Mini Kit(Transgene Code#E101-0150rxns)提取总RNA，并且利用EasyScript Frist-Strand cDNA SynSgesis SuperMix(Transgene Code#AE301-03100rxns)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SlNAC基因的3’端序列，使用RACE技术(Takara-RACE kit)获得SlNAC基因的全长cDNA序列将扩增得到的SlNAC基因进行测序分析，得到完整的SlNAC基因全长为1767bp。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 ID No.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TaKaRaRNAPCR 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：
2).佛甲草SlNAC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SlNAC基因片段PCR扩增
利用Takara RACE kit扩增得到的SlNAC基因进行测序分析，得到完整的佛甲草SlNAC基因全长为1767bp(SEQ ID No.1)。由佛甲草SlNAC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SlNAC基因上下游引物:
SEQ ID No.7:5'-TCTTTGCCTGTACCTTTCCAAT-3'
SEQ ID No.8:5'-CACCACCAACAACATCATCATC-3'，其PCR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(见图1)，其余用作产物的纯化回收。
4).构建含有佛甲草SlNAC基因的克隆载体
构建含有佛甲草SlNAC基因的载体pJET1.2_SlNAC
胶回收纯化后的佛甲草SlNAC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_SlNAC。
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_SlNAC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SlNAC基因的表达载体
构建含有佛甲草SlNAC基因的表达载体pBI121_SlNAC，
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'-ACGGGGGACTCTAGAGGATCCTCTTTGCCTGTACCTTTCCAAT-3'
SEQ ID No.10:5'-CGATCGGGGAAATTCGAGCTCCACCACCAACAACATCATCATC-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SlNAC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SlNAC基因的大肠杆菌表达载体pBI121_SlNAC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(商品)，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；1-7号分别代表的是pBI121_SlNAC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；其中4、6代表中表达的拟南芥；2，3号代表高表达量的拟南芥；1，5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3号和表达水平低的独立转化株系5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3号和5号的T3代纯合体种子。
(3)对转基因拟南芥进行耐盐处理
将3号T3代纯合体种子、5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，用50mM NaNO3水溶液进行耐盐处理(浇灌)。每隔3天处理一次，共处理15天后植物照相(见图5)。
实施例3
1.转化杨树
供阳性克隆菌转化的杨树为欧洲山杨×银白杨(Populustremula×P.albaINRAclone N7171-B4，以下简称717杨树)组培苗。(商品)
(1)将717杨树腋芽或顶芽置于基本培养基(MS盐2.2g，蔗糖30g，定容至1L，pH＝5.7，琼脂7.2g)上继代繁殖，培养6周获得组培苗；切取所述组培苗1cm不带腋芽的茎段，划伤口后于24℃黑暗条件下预培养3天；
(2)将选取的实施例1获得的阳性克隆菌菌液(OD600＝0.8)在室温、4000rpm离心10min，弃去上清液，将沉淀用等体积的M液(M液：MS盐4.4g，蔗糖30g，生长素NAA1.86mg，细胞分裂素2ip1.02mg，乙酰丁香酮As19.86mg，定容至1L，pH＝5.7)重悬，24℃，100rpm活化1h得到侵染液；按40个，25mL的比例将步骤(1)预培养的茎段放入所述侵染液中，24℃条件下100rpm侵染1h。经过共培养(M1固体培养基：MS盐4.4g，蔗糖30g，琼脂7.2g，生长素NAA1.86mg，细胞分裂素2ip1.02mg，乙酰丁香酮As19.86mg，定容至1L，pH＝5.7；培养条件：26℃，暗条件下共培养36小时)、延迟选择(CIM延迟选择培养基：MS盐4.4g，蔗糖30g，生长素NAA1.86mg，细胞分裂素2ip1.02mg，头孢霉素500mg，琼脂7.2g，定容至1L，pH＝5.7；培养条件：800Lux弱光下培养8天，光照/黑暗为16h/8h)、诱导不定芽(SIM筛选培养基中：MS盐4.4g，蔗糖30g，细胞分裂素TDZ0.05mg，头孢霉素500mg，卡那霉素500mg，琼脂7.2g，定容至1L，pH＝5.7；培养条件：26℃，2000Lux，光照/黑暗为16h/8h)、伸长培养(SEM筛选培养基：MS盐4.4g，蔗糖30g，细胞分裂素2ip1.02mg，头孢霉素500mg，卡那霉素500mg，琼脂7.2g，定容至1L，pH＝5.7；培养条件：26℃，2000Lux光照，光照/黑暗为16h/8h，培养4周)、诱导生根(RM培养基的组成为：MS盐2.2g，蔗糖30g，头孢霉素500mg，卡那霉素500mg，琼脂7.2g，定容至1L，pH＝5.7；培养条件：26℃，2000Lux光照，光照/黑暗为16h/8h)多步后，待再生杨树生长正常后，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717杨树进行盐处理
将生长2个月的长势均匀的转基因高表达杨树、低表达转基因杨树、野生型717杨树移栽至土盆中，待土盆苗生长30d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，用盐分处理液(50mM NaNO3水溶液)进行浇灌处理。3天浇一次，每次浇灌量为土壤质量的0.5倍，以保持盆中处理液浓度的恒定，共处理30天后观察植株并照相(见图6)。
序列表
<110> 天津大学
<120> 佛甲草耐盐基因SlNAC及其应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1767
<212> DNA
<213> 佛甲草(Sedum lineare)
<400> 1
atgtttaata acaaaataaa atgatattgt tttgactaaa aatattacat agtttaatag 60
taaaacactt taaaactata tcgtgttgta tatcataatc atagtatttg actgcattgt 120
tccggatgcg ttaaccatga tatttatgaa actactaatg ttcactgtca tatgtcagat 180
aattacaacc agtgtgttat taaatactct ctaatgtaca acaaaacctt catttaaagc 240
aaacaaaata tgtgtttaag aaaaaccatc aacgctagtg cagcagcatt tgtgtcttcc 300
ccaacaattt gacggccgga aagccaacaa aaacagtaaa caaaacagcc gcaaatgctg 360
tccatatctt catctttttg cttaccagtt tattctcata aacttctacc tttgacatca 420
tagattttgc ttcagtttga tagcttaaaa catggttgca atggtacacc ggagcaagtt 480
tcttctgcaa aagtatccgt ctaactgcat ttccttgatt ttgaacatca gttacttcta 540
gttcttcgtc gtcgtcgtcg gcccgaaacc ttaatctgat tccagtctct atactcgtcg 600
gactgcaggt atttacatct ccaactgttt catctttact tgaacttcca aattgtttaa 660
acatttcatc atcaaaatca aacattccat catcgaaaat gtcagttgct aggtcattat 720
tgtttaaaac acaagtcccg gtttctactc ttgattctga aaccattgaa acatcatcgt 780
tccaacagtt ccgttcgagc tccgcgaaga tttgttcaag tgaaacctcc gtatcgattt 840
cactaggaaa tccactttta tttttaccca tgtaacgata cgaatcatcc gattccaccg 900
gagccaaaac ggtactactt accatcgatc gagtttcatc attgacaaat cgatagtcaa 960
actggtcgag agttgtaccc gcttgattta acacattttc aatctcatca tcatgtaaag 1020
aatcttggac actttcatcg ttttttttaa acaatcgaca taagacaaac ttactttgat 1080
cgattaaatc gttgagagtt gtacgatact cgtgcattac ccaattagtt cgattaccat 1140
tcggagcacg gccggtgtaa aaaactaatg tttttttaat gccaatttgt tgtccgacat 1200
ggttcttgat tttacgatct ttgcctgtac ctttccaata tcctctgttt gttgctctgt 1260
ttagtctctg actgcttgga aatttttgat ctcttccggc gaagaaaaac cactctccgt 1320
catcggattt tatcgctgat aaacctggta gatcccaagg ctcccattta caaacatcaa 1380
tttcacgaat cacttcatta acgtgacgat gatgatgacc agtaatcttg ttacgcaagt 1440
aaaaatcaat aagttcttga tcagtgggtc gaaaacggta accgagtggc aatgaatcaa 1500
gactcatagc tgcagtcata aatttcaaaa caagaaaatt gcgaaaaaaa agtgatgatc 1560
agaaaatcga cgtcatgagg tttttattta tatgttacga tgaattgatg atctgatgat 1620
gatgttgttg gtggtggtgg tttctgggaa gtagcgattt gggatcagag tgaaatgcga 1680
tgtttcgatt gagtgaaaat ttgtgttgtt ttctggaaga gtggaaagtt cgaggaagat 1740
ggattcactg ggtgttgaag aaactaa 1767
<210> 2
<211> 588
<212> PRT
<213> 佛甲草(Sedum lineare)
<400> 2
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Asn Cys Glu Lys Lys Val Met Ile Arg Lys Ser Thr Ser His Gly Phe
515 520 525
Tyr Leu Tyr Val Thr Met Asn Leu Asp Ser Asp Asp Asp Val Val Gly
530 535 540
Gly Gly Gly Phe Trp Glu Val Ala Ile Trp Asp Gln Ser Glu Met Arg
545 550 555 560
Cys Phe Asp Glu Val Lys Ile Cys Val Val Phe Trp Lys Ser Gly Lys
565 570 575
Phe Glu Glu Asp Gly Phe Met Gly Val Glu Glu Thr
580 585
<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> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tctttgcctg tacctttcca at 22
<210> 8
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caccaccaac aacatcatca tc 22
<210> 9
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
acgggggact ctagaggatc ctctttgcct gtacctttcc aat 43
<210> 10
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cgatcgggga aattcgagct ccaccaccaa caacatcatc atc 43
<210> 11
<211> 1767
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
atgtttaata acaaaataaa atgatattgt tttgactaaa aatattacat agtttaatag 60
taaaacactt taaaactata tcgtgttgta tatcataatc atagtatttg actgcattgt 120
tccggatgcg ttaaccatga tatttatgaa actactaatg ttcactgtca tatgtcagat 180
aattacaacc agtgtgttat taaatactct ctaatgtaca acaaaacctt catttaaagc 240
aaacaaaata tgtgtttaag aaaaaccatc aacgctagtg cagcagcatt tgtgtcttcc 300
ccaacaattt gacggccgga aagccaacaa aaacagtaaa caaaacagcc gcaaatgctg 360
tccatatctt catctttttg cttaccagtt tattctcata aacttctacc tttgacatca 420
tagattttgc ttcagtttga tagcttaaaa catggttgca atggtacacc ggagcaagtt 480
tcttctgcaa aagtatccgt ctaactgcat ttccttgatt ttgaacatca gttacttcta 540
gttcttcgtc gtcgtcgtcg gcccgaaacc ttaatctgat tccagtctct atactcgtcg 600
gactgcaggt atttacatct ccaactgttt catctttact tgaacttcca aattgtttaa 660
acatttcatc atcaaaatca aacattccat catcgaaaat gtcagttgct aggtcattat 720
tgtttaaaac acaagtcccg gtttctactc ttgattctga aaccattgaa acatcatcgt 780
tccaacagtt ccgttcgagc tccgcgaaga tttgttcaag tgaaacctcc gtatcgattt 840
cactaggaaa tccactttta tttttaccca tgtaacgata cgaatcatcc gattccaccg 900
gagccaaaac ggtactactt accatcgatc gagtttcatc attgacaaat cgatagtcaa 960
actggtcgag agttgtaccc gcttgattta acacattttc aatctcatca tcatgtaaag 1020
aatcttggac actttcatcg ttttttttaa acaatcgaca taagacaaac ttactttgat 1080
cgattaaatc gttgagagtt gtacgatact cgtgcattac ccaattagtt cgattaccat 1140
tcggagcacg gccggtgtaa aaaactaatg tttttttaat gccaatttgt tgtccgacat 1200
ggttcttgat tttacgatct ttgcctgtac ctttccaata tcctctgttt gttgctctgt 1260
ttagtctctg actgcttgga aatttttgat ctcttccggc gaagaaaaac cactctccgt 1320
catcggattt tatcgctgat aaacctggta gatcccaagg ctcccattta caaacatcaa 1380
tttcacgaat cacttcatta acgtgacgat gatgatgacc agtaatcttg ttacgcaagt 1440
aaaaatcaat aagttcttga tcagtgggtc gaaaacggta accgagtggc aatgaatcaa 1500
gactcatagc tgcagtcata aatttcaaaa caagaaaatt gcgaaaaaaa agtgatgatc 1560
agaaaatcga cgtcatgagg tttttattta tatgttacga tgaattgatg atctgatgat 1620
gatgttgttg gtggtggtgg tttctgggaa gtagcgattt gggatcagag tgaaatgcga 1680
tgtttcgatt gagtgaaaat ttgtgttgtt ttctggaaga gtggaaagtt cgaggaagat 1740
ggattcactg ggtgttgaag aaactaa 1767
Claims (6)
1.佛甲草耐盐基因SlNAC，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的佛甲草耐盐基因SlNAC的克隆载体pJET1.2_SlNAC。
3.含权利要求1的佛甲草耐盐基因SlNAC的表达载体pBI121_SlNAC。
4.含有权利要求3的表达载体pBI121_SlNAC的宿主细胞。
5.权利要求1佛甲草耐盐基因SlNAC增强植物对盐的耐受性的应用。
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杨树。
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| CN110551733A (zh) * | 2019-09-01 | 2019-12-10 | 天津大学 | 佛甲草耐盐基因SlR2R3-MYB及其应用 |
| CN110564736A (zh) * | 2019-09-01 | 2019-12-13 | 天津大学 | 佛甲草耐盐基因SlWRKY及其应用 |
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| CN110564736A (zh) * | 2019-09-01 | 2019-12-13 | 天津大学 | 佛甲草耐盐基因SlWRKY及其应用 |
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