CN114457082A - 一种辣椒NaCl诱导启动子、重组载体及其应用 - Google Patents

一种辣椒NaCl诱导启动子、重组载体及其应用 Download PDF

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CN114457082A
CN114457082A CN202210266513.1A CN202210266513A CN114457082A CN 114457082 A CN114457082 A CN 114457082A CN 202210266513 A CN202210266513 A CN 202210266513A CN 114457082 A CN114457082 A CN 114457082A
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颜嘉雯
宋宇
王玲玉
许一丰
郑敏
钱皆
吕陈生
徐文艳
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Abstract

本发明中公开了一种辣椒NaCl诱导启动子，所述启动子的核苷酸序列如SEQ ID N0.1所示。本发明同时公开了包含上述辣椒NaCl诱导启动子的重组载体。本发明还公开了上述辣椒NaCl诱导启动子在耐盐植物培育上的应用。本发明从辣椒CaRH20基因启动子区域克隆得到诱导启动子proCaRH20，通过对该启动子区的分析，其中含有多个能参与响应生物及非生物胁迫的顺式作用元件。当对含有启动子proCaRH20和报告基因GUS(proCaRH20::GUS)的拟南芥转基因植株用150mM NaCl(高盐条件下)处理时，发现启动子proCaRH20可以增强下游基因GUS的表达，致使转基因植株染色加深，证明启动子proCaRH20明显受到盐的诱导。

Description

一种辣椒NaCl诱导启动子、重组载体及其应用
技术领域
本发明涉及基因工程技术领域，具体涉及一种辣椒NaCl诱导启动子、重组载体及其应用。
背景技术
植物在自然界中经常由于其固着生长而遭受各种生物或非生物胁迫的影响，例如干旱、盐渍、高温或虫害、病害等。这些外界胁迫限制了植物的生长，进一步导致农作物减产。为了应对这些不利的环境因素，植物进化出了一套防御机制来提高自身对外界胁迫的耐受性，这其中重要的一点就是基因的表达调控。
基因的表达调控涉及DNA、RNA等多个层面的调节。而启动子是一段参与起始和调控基因表达的DNA序列，其本身是不被转录的，它能够通过本身所含有转录起始所需的保守序列与RNA聚合酶特异结合来发挥功能。根据作用方式及功能可将启动子分为3类：组成型启动子、诱导型启动子和组织特异型启动子。本专利所涉及的诱导型启动子能够在某些特定的刺激下，该类型启动子可以大幅提高下游基因的转录水平，使目的基因的表达产物在一定时空积累，从而在分子或生理水平上产生对特定刺激的响应。基因工程育种中通常使用诱导型启动子改善植株的抗逆性，这一定程度上解决了转基因应用中食品安全性问题。
发明内容
本发明的目的在于解决现有技术中存在的受到盐诱导的启动子在异源植物中对下游基因的表达水平较低的问题，提供一种辣椒NaCl诱导启动子、重组载体及其应用。
为解决上述技术问题，本发明采用的技术方案如下：一种辣椒NaCl诱导启动子，所述启动子的核苷酸序列如SEQ ID N0.1所示。
优选地，扩增所述启动子的引物序列如SEQ ID N0.2和SEQ ID N0.3所示。
本发明同时提供了一种重组载体，所述重组载体包含上述辣椒NaCl诱导启动子。
优选地，所述重组载体还包含报告基因GUS，所述重组载体的核苷酸序列如SEQ IDN0.4所示。
本发明还提供了上述辣椒NaCl诱导启动子在耐盐植物培育上的应用。
本发明从辣椒CaRH20基因启动子区域克隆得到诱导启动子proCaRH20，通过对该启动子区的分析，其中含有多个可能参与响应生物及非生物胁迫的顺式作用元件。当对含有启动子proCaRH20和报告基因GUS(proCaRH20::GUS)的拟南芥转基因植株用150mM NaCl(高盐条件下)处理时，发现启动子proCaRH20可以增强下游基因GUS的表达，致使转基因植株染色加深，证明启动子proCaRH20明显受到盐的诱导。
本发明所具有的有益效果：本发明从辣椒CaRH20基因启动子区域克隆得到的诱导启动子proCaRH20，该启动子在高盐条件下能够显著增强下游基因的表达水平，且能够稳定的在异源植物中发挥作用，其实际应用对于基因工程育种具有重要价值。
附图说明
图1为实施例1中proCaRH20启动子片段电泳图；
图2为实施例1中proCaRH20启动子的序列分析；
图3为实施例4中proCaRH20::GUS转基因拟南芥植株经NaCl处理后的GUS染色分析图。
具体实施方式
下面结合附图和具体实施例对本发明作进一步的说明。
T-Vector(TaKaRa)；含有报告基因GUS的双元表达载体、农杆菌和野生型拟南芥(Col-0)由西南大学植物分子实验室提供；LEAGENE GUS染色试剂盒购自LEAGENE。
实施例1proCaRH20启动子克隆及序列分析
1.proCaRH20启动子克隆
从PepperHub(http://pepperhub.hzau.edu.cn/)数据库中下载辣椒CaRH20基因DNA序列，选择ATG上游约902bp长度作为CaRH20启动子区域，以序列SEQ ID N0.2和SEQ IDN0.3为引物，通过PCR扩增获得proCaRH20启动子片段，连入T-vector，通过测序获得正确的proCaRH20启动子片段，proCaRH20启动子的核苷酸序列如SEQ ID N0.1所示；proCaRH20启动子片段电泳图如附图1所示，片段长度为902bp；本实施例中PCR扩增所用引物详见表1；proCaRH20启动子的PCR扩增体系详见表2：
表1 proCaRH20启动子扩增所用的引物序列
Primername Primer sequences(5`-3`)
proCaRH20-F GGTACCAACGAGGGATGCACATC(SEQ ID No.2)
proCaRH20-R GTCGACGGCAAATTCGTAGGAGG(SEQ ID No.3)
表2 proCaRH20启动子的PCR扩增体系
Figure BDA0003552041660000031
Figure BDA0003552041660000041
2.proCaRH20启动子序列分析
利用PLACE数据库(http://www.dna.affrc.go.jp/PLACE/)进行启动子调控元件分析，结果见表3：
表3 CaRH20启动子所含有的启动子必需元件及响应环境胁迫的元件汇总表
Figure BDA0003552041660000042
实施例2构建含有proCaRH20启动子和报告基因GUS的重组载体
在实施例1的基础上，将实施例1获得的proCaRH20启动子片段，通过双酶切的方法接入含有报告基因GUS的双元表达载体中，酶切位点为KpnⅠ和SalⅠ；获得含有proCaRH20启动子和报告基因GUS的重组载体，即proCaRH20::GUS表达载体，其核苷酸序列如SEQ IDN0.4所示。
实施例3构建转基因拟南芥植株
在实施例2的基础上，将实施例2中构建成功的proCaRH20::GUS表达载体转入农杆菌中，再通过花序浸染法转入野生型拟南芥(Col-0)，收取T0代种子，以含有潮霉素的1/2MS培养基进行培养筛选，得到拥有潮霉素抗性的转基因拟南芥植株proCaRH20::GUS。以序列SEQ ID N0.2和SEQ ID N0.3为引物对转基因拟南芥植株proCaRH20::GUS进行PCR鉴定，可扩增出长度为902bp的基因片段，测序结果与proCaRH20的核苷酸序列一致；同时对转基因拟南芥植株proCaRH20::GUS进行GUS染色鉴定，可检测到GUS的组织化学染色情况，说明检测到GUS基因的表达，证明proCaRH20启动子序列是具有启动子功能的，并能在异源植物中有效启动外源基因的表达。
实施例4 NaCl诱导实验
以实施例3中获得的proCaRH20::GUS转基因拟南芥植株为NaCl诱导实验对象，设置对照组标记为CK，实验组标记为150mM NaCl 12h。实验组将转基因拟南芥幼苗用150mMNaCl处理12h；而对照组的转基因拟南芥幼苗在液体1/2MS培养基中培养12h，再对实验组和对照组进行GUS染色，结果如图3所示。附图3显示，相对于对照组CK的幼苗，经过NaCl处理后的幼苗染色后颜色加深，证明proCaRH20启动子在高盐条件下能够显著增强外源基因GUS的表达，是一种积极响应NaCl诱导表达的诱导型启动子。
综上所述，本发明构建了含有诱导型启动子proCaRH20和外源基因(如GUS报告基因)的表达载体，并且转入拟南芥，获得proCaRH20::GUS转基因拟南芥植株。实施例3说明本发明构建的proCaRH20启动子能有效启动外源基因(GUS报告基因)在拟南芥植株中的表达。实施例4的NaCl诱导实验进一步说明本发明构建的proCaRH20启动子在高盐条件下能显著增强外源基因(GUS报告基因)的表达，是一种响应外源NaCl诱导表达的诱导型启动子，其在基因工程植物育种中有着非常重要的应用价值。
本发明的说明书和附图被认为是说明性的而非限制性的，在本发明基础上，本领域技术人员根据所公开的技术内容，不需要创造性的劳动就可以对其中一些技术特征做出一些替换和变形，均在本发明的保护范围内。
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tcagaacccg agggcgacgc tgcccgcgag gtccaggcgc tggccgctga aattaaatca 2220
aaactcattt gagttaatga ggtaaagaga aaatgagcaa aagcacaaac acgctaagtg 2280
ccggccgtcc gagcgcacgc agcagcaagg ctgcaacgtt ggccagcctg gcagacacgc 2340
cagccatgaa gcgggtcaac tttcagttgc cggcggagga tcacaccaag ctgaagatgt 2400
acgcggtacg ccaaggcaag accattaccg agctgctatc tgaatacatc gcgcagctac 2460
cagagtaaat gagcaaatga ataaatgagt agatgaattt tagcggctaa aggaggcggc 2520
atggaaaatc aagaacaacc aggcaccgac gccgtggaat gccccatgtg tggaggaacg 2580
ggcggttggc caggcgtaag cggctgggtt gtctgccggc cctgcaatgg cactggaacc 2640
cccaagcccg aggaatcggc gtgagcggtc gcaaaccatc cggcccggta caaatcggcg 2700
cggcgctggg tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac 2760
gcatcgaggc agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca 2820
aagaatcccg gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg 2880
acgagcaacc agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca 2940
gcatcatgga cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga 3000
tccgctacga gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca 3060
gtgtgtggga ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc 3120
gataccggga agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg 3180
tactcaagtt ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct 3240
gcattcggtt aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc 3300
gcctggtgac ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg 3360
aaaccgggcg gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca 3420
cagaaggcaa gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg 3480
gcatcggccg ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat 3540
ggttgttcaa gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt 3600
tcaccgtgcg caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg 3660
cggggcaggc tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat 3720
ccgccggttc ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag 3780
gtcgaaaagg tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg 3840
ggaaccggaa cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt 3900
aagtgactga tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta 3960
ttaaaactct taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag 4020
agctgcaaaa agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc 4080
ggcctatcgc ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag 4140
ggcgcggaca agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc 4200
ctcgcgcgtt tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc 4260
acagcttgtc tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt 4320
gttggcgggt gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact 4380
ggcttaacta tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa 4440
taccgcacag atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca 4500
ctgactcgct gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg 4560
taatacggtt atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc 4620
agcaaaaggc caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc 4680
cccctgacga gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac 4740
tataaagata ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc 4800
tgccgcttac cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata 4860
gctcacgctg taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc 4920
acgaaccccc cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca 4980
acccggtaag acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag 5040
cgaggtatgt aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta 5100
gaaggacagt atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg 5160
gtagctcttg atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc 5220
agcagattac gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt 5280
ctgacgctca gtggaacgaa aactcacgtt aagggatttt ggtcatgcat tctaggtact 5340
aaaacaattc atccagtaaa atataatatt ttattttctc ccaatcaggc ttgatcccca 5400
gtaagtcaaa aaatagctcg acatactgtt cttccccgat atcctccctg atcgaccgga 5460
cgcagaaggc aatgtcatac cacttgtccg ccctgccgct tctcccaaga tcaataaagc 5520
cacttacttt gccatctttc acaaagatgt tgctgtctcc caggtcgccg tgggaaaaga 5580
caagttcctc ttcgggcttt tccgtcttta aaaaatcata cagctcgcgc ggatctttaa 5640
atggagtgtc ttcttcccag ttttcgcaat ccacatcggc cagatcgtta ttcagtaagt 5700
aatccaattc ggctaagcgg ctgtctaagc tattcgtata gggacaatcc gatatgtcga 5760
tggagtgaaa gagcctgatg cactccgcat acagctcgat aatcttttca gggctttgtt 5820
catcttcata ctcttccgag caaaggacgc catcggcctc actcatgagc agattgctcc 5880
agccatcatg ccgttcaaag tgcaggacct ttggaacagg cagctttcct tccagccata 5940
gcatcatgtc cttttcccgt tccacatcat aggtggtccc tttataccgg ctgtccgtca 6000
tttttaaata taggttttca ttttctccca ccagcttata taccttagca ggagacattc 6060
cttccgtatc ttttacgcag cggtattttt cgatcagttt tttcaattcc ggtgatattc 6120
tcattttagc catttattat ttccttcctc ttttctacag tatttaaaga taccccaaga 6180
agctaattat aacaagacga actccaattc actgttcctt gcattctaaa accttaaata 6240
ccagaaaaca gctttttcaa agttgttttc aaagttggcg tataacatag tatcgacgga 6300
gccgattttg aaaccgcggt gatcacaggc agcaacgctc tgtcatcgtt acaatcaaca 6360
tgctaccctc cgcgagatca tccgtgtttc aaacccggca gcttagttgc cgttcttccg 6420
aatagcatcg gtaacatgag caaagtctgc cgccttacaa cggctctccc gctgacgccg 6480
tcccggactg atgggctgcc tgtatcgagt ggtgattttg tgccgagctg ccggtcgggg 6540
agctgttggc tggctggtgg caggatatat tgtggtgtaa acaaattgac gcttagacaa 6600
cttaataaca cattgcggac gtttttaatg tactgaatta acgccgaatt aattcggggg 6660
atctggattt tagtactgga ttttggtttt aggaattaga aattttattg atagaagtat 6720
tttacaaata caaatacata ctaagggttt cttatatgct caacacatga gcgaaaccct 6780
ataggaaccc taattccctt atctgggaac tactcacaca ttattatgga gaaactcgag 6840
cttgtcgatc gacagatccg gtcggcatct actctatttc tttgccctcg gacgagtgct 6900
ggggcgtcgg tttccactat cggcgagtac ttctacacag ccatcggtcc agacggccgc 6960
gcttctgcgg gcgatttgtg tacgcccgac agtcccggct ccggatcgga cgattgcgtc 7020
gcatcgaccc tgcgcccaag ctgcatcatc gaaattgccg tcaaccaagc tctgatagag 7080
ttggtcaaga ccaatgcgga gcatatacgc ccggagtcgt ggcgatcctg caagctccgg 7140
atgcctccgc tcgaagtagc gcgtctgctg ctccatacaa gccaaccacg gcctccagaa 7200
gaagatgttg gcgacctcgt attgggaatc cccgaacatc gcctcgctcc agtcaatgac 7260
cgctgttatg cggccattgt ccgtcaggac attgttggag ccgaaatccg cgtgcacgag 7320
gtgccggact tcggggcagt cctcggccca aagcatcagc tcatcgagag cctgcgcgac 7380
ggacgcactg acggtgtcgt ccatcacagt ttgccagtga tacacatggg gatcagcaat 7440
cgcgcatatg aaatcacgcc atgtagtgta ttgaccgatt ccttgcggtc cgaatgggcc 7500
gaacccgctc gtctggctaa gatcggccgc agcgatcgca tccatagcct ccgcgaccgg 7560
ttgtagaaca gcgggcagtt cggtttcagg caggtcttgc aacgtgacac cctgtgcacg 7620
gcgggagatg caataggtca ggctctcgct aaactcccca atgtcaagca cttccggaat 7680
cgggagcgcg gccgatgcaa agtgccgata aacataacga tctttgtaga aaccatcggc 7740
gcagctattt acccgcagga catatccacg ccctcctaca tcgaagctga aagcacgaga 7800
ttcttcgccc tccgagagct gcatcaggtc ggagacgctg tcgaactttt cgatcagaaa 7860
cttctcgaca gacgtcgcgg tgagttcagg ctttttcata tctcattgcc ccccgggatc 7920
tgcgaaagct cgagagagat agatttgtag agagagactg gtgatttcag cgtgtcctct 7980
ccaaatgaaa tgaacttcct tatatagagg aaggtcttgc gaaggatagt gggattgtgc 8040
gtcatccctt acgtcagtgg agatatcaca tcaatccact tgctttgaag acgtggttgg 8100
aacgtcttct ttttccacga tgctcctcgt gggtgggggt ccatctttgg gaccactgtc 8160
ggcagaggca tcttgaacga tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc 8220
accttccttt tctactgtcc ttttgatgaa gtgacagata gctgggcaat ggaatccgag 8280
gaggtttccc gatattaccc tttgttgaaa agtctcaata gccctttggt cttctgagac 8340
tgtatctttg atattcttgg agtagacgag agtgtcgtgc tccaccatgt tatcacatca 8400
atccacttgc tttgaagacg tggttggaac gtcttctttt tccacgatgc tcctcgtggg 8460
tgggggtcca tctttgggac cactgtcggc agaggcatct tgaacgatag cctttccttt 8520
atcgcaatga tggcatttgt aggtgccacc ttccttttct actgtccttt tgatgaagtg 8580
acagatagct gggcaatgga atccgaggag gtttcccgat attacccttt gttgaaaagt 8640
ctcaatagcc ctttggtctt ctgagactgt atctttgata ttcttggagt agacgagagt 8700
gtcgtgctcc accatgttgg caagctgctc tagccaatac gcaaaccgcc tctccccgcg 8760
cgttggccga ttcattaatg cagctggcac gacaggtttc ccgactggaa agcgggcagt 8820
gagcgcaacg caattaatgt gagttagctc actcattagg caccccaggc tttacacttt 8880
atgcttccgg ctcgtatgtt gtgtggaatt gtgagcggat aacaatttca cacaggaaac 8940
agctatgacc atgattacg 8959

Claims (5)

1.一种辣椒NaCl诱导启动子，其特征在于，所述启动子的核苷酸序列如SEQ ID N0.1所示。
2.根据权利要求1所述的辣椒NaCl诱导启动子，其特征在于，扩增所述启动子的引物序列如SEQ ID N0.2和SEQ ID N0.3所示。
3.一种重组载体，其特征在于，所述重组载体包含权利要求1或2所述的辣椒NaCl诱导启动子。
4.根据权利要求3所述的重组载体，其特征在于，所述重组载体还包含报告基因GUS，所述重组载体的核苷酸序列如SEQ ID N0.4所示。
5.如权利要求1或2所述的辣椒NaCl诱导启动子在耐盐植物培育上的应用。
CN202210266513.1A 2022-03-17 2022-03-17 一种辣椒NaCl诱导启动子、重组载体及其应用 Pending CN114457082A (zh)

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