CN110129336B - 铁线莲clhsp18基因编码序列及其应用 - Google Patents
铁线莲clhsp18基因编码序列及其应用 Download PDFInfo
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- CN110129336B CN110129336B CN201910472571.8A CN201910472571A CN110129336B CN 110129336 B CN110129336 B CN 110129336B CN 201910472571 A CN201910472571 A CN 201910472571A CN 110129336 B CN110129336 B CN 110129336B
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
Abstract
本发明公开了一种如SEQ ID NO.1所示的氨基酸序列。本发明的铁线莲ClHSP18基因在拟南芥中可以实现异源表达,提高拟南芥的热激胁迫抗性。
Description
技术领域
本发明涉及植物分子生物学领域,具体的说涉及铁线莲CLHSP18基因编码序列及其应用。
背景技术
由于“温室效应”现象日益明显,全球气温持续升高,夏季高温已成为制约植物生长和发育的主要环境因子,植物生长发育面临高温逆境的严峻挑战。热激蛋白(heat shockprotein,HSPs)是一类在有机体受到高温等逆境刺激后大量表达的蛋白,根据分子质量大小将其分成HSP110s、HSP90s、HSP70s、HSP60s和小分子热激蛋白(smHSPs)。热激蛋白是植物对逆境胁迫短期适应的必需组成成分,对减轻逆境胁迫引起的伤害有很大的作用。有机体在受到逆境胁迫后,体内变性蛋白急剧增加,热激蛋白可以与变性蛋白结合,维持它们的可溶状态, 在有Mg2 +和ATP的存在下使解折叠的蛋白质重新折叠成有活性的构象。已有研究证明HSPs的主要功能是参与新生肽的折叠以及蛋白质变性后的复性、降解,维持胞内环境的稳定,起分子伴侣(molecular chaperones)的作用。
虽然植物热激蛋白的研究起步较晚,但已成为当今分子生物学、蛋白质生物化学和植物抗逆生理的一个重要研究内容,其中心问题是HSPs的生物学功能。许多研究表明,HSPs的生成量与生物耐热性呈正相关。Alvaro等(1999)实验证明,将栗子 ( Castaneasativa) CsHSP17.5基因转入大肠杆菌显著提高转基因菌株对高温(50℃)及低温(4℃)的耐受性。
随着生活水平的提高,园林观赏植物越来越受到人们关注,研究观赏植物对温度逆境抗性的遗传机制有广泛的应用前景。
铁线莲属(Clematis florida Thunb)为毛茛科(Ranunculaceae)直立灌木、草本,木质或草质藤本,是一类观赏价值高、具有多种抗逆性的藤本植物。全世界约有铁线莲属植物335种,广泛分布于除南极洲以外的各大洲。中国铁线莲属植物极其丰富,约有147种,全国各地均有分布,大部分种类分布于华中和西南地区。铁线莲喜凉爽气候,在野生环境中常与灌木伴生。上海地区近5年(2014—2018年)夏季持续的高温(35℃以上)天气多达1个月左右,严重影响了铁线莲的生长,如叶片变黄、萎蔫、脱落,茎干还易感枯萎病,限制了铁线莲在上海地区的大面积推广应用。因此,如何提高铁线莲品种耐热性是园林工作者面临的重要任务。已有研究表明小分子热激蛋白基因是一类胁迫诱导表达基因,它在耐热品种中诱导表达可以明显提高品种的耐热性,该类基因的挖掘与应用在园林植物及其它植物的抗逆研究中具有非常重大的应用前景。
发明内容
本发明的目的首先在于提供一种铁线莲CLHSP18基因;
该铁线莲CLHSP18基因是一种小分子热激蛋白基因,该基因长度为471bp。
本发明还提供了一种如SEQ ID NO.1所示的氨基酸序列;
其编码的蛋白质分子量为17959.49道尔顿,pI为8.113。
本发明还提供了上述铁线莲ClHSP18基因的克隆方法,包括如下步骤:
①提取铁线莲的总RNA,将总RNA反转录成cDNA;
②根据同源序列设计简并引物,正向引物:5’-CATTCRCTGAATHTCCTG-3’( SEQ IDNO.2)反向引物:5’-AACMTCCTGGAGKACTTG-3’( SEQ ID NO.3);
利用简并引物,采用RT-PCR方法从cDNA中扩增出一条247bp的条带;进行测序分析获得碱基序列;
③利用步骤②中得到的片段序列,设计三对反向PCR引物;
第一对反向PCR引物:
SEQ ID NO.4正向引物5’-TGTCAACGTTGAGATTGA-3’,
SEQ ID NO.5反向引物5’- TGATGAAAGCCGTATTCT-3’;
第二对反向PCR引物:
SEQ ID NO.6正向引物5’- TGCTGCAGATCAGCGGCC-3’,
SEQ ID NO.7反向引物5’- AATTCAGGGAATGTGGCG-3’;
第三对反向PCR引物:
SEQ ID NO.8正向引物5’-CAAGTTCTCCAGGAGGTT-3’,
SEQ ID NO.9反向引物5’-AGGGAATTGGAAATCCCT-3’;
④提取铁线莲的基因组DNA;
⑤用限制性内切酶EcoRI对基因组DNA进行酶切,纯化并回收酶切后的片段,以T4连接酶催化片段首尾自连成环;以自连后的片段作反向PCR模板,以三对反式PCR引物的反向引物分别进行三轮巢式PCR,获得一427bp片段为5’ 端;
⑥3’-RACE获得ClHSP18基因3’ 端
用3’-RACE反转录试剂盒将铁线莲总RNA反转录成cDNA作3’-RACE模板,以上述3对反式PCR引物的正向引物(5’-3’) 分别与3’-RACE引物AUAP配对进行三轮巢式PCR;回收获得的327bp片段;
⑦全长基因的克隆
根据上述②、⑤、⑥获得的序列片段进行拼接,获得该基因的cDNA全长序列;依据基因5’、3’端序列设计基因全长引物(SEQ ID NO.10和SEQ ID NO.11的引物),采用RT-PCR方法从铁线莲cDNA中扩增出一条471bp的条带;
将该471bp的PCR产物回收,连接到T-Vector上,转化大肠杆菌,进行测序。将测序结果提交至NCBI非冗余数据库,BLAST结果表明该序列和月季小分子热激蛋白基因RcHSP17.8序列高度保守。
本发明还提供了上述铁线莲ClHSP18基因在拟南芥中异源表达应用,该应用可以提高拟南芥的热激胁迫抗性。
本发明提供了铁线莲中表达的ClHSP18(铁线莲小分子热激蛋白,clematis smallHeat shock protein,ClHSP18)基因序列,其编码的核苷酸序列,转基因载体的构建,拟南芥的转化,转基因植株的获得及其耐热性能鉴定。本发明提供了从铁线莲中克隆小分子热激蛋白基因,并转入拟南芥后提高转基因拟南芥对高温抗性的技术。
附图说明
图1铁线莲ClHSP18编码的蛋白质序列与月季RcHSP17.8蛋白的氨基酸序列同源性比较
铁线莲ClHSP18与月季(Rosa chinensis)RcHSP17.8氨基酸序列(GenBankAccession No.ABK32539.1)的同源性比较(FASTA)表。其中,相同的氨基酸在两个序列之间用氨基酸单字符标出,相似的氨基酸用“+”标出
图2 半定量RT-PCR检测铁线莲ClHSP18的表达
其中上图为RT-PCR扩增产物,下图为相应的actin对照
lane1: ‘金斯托韦克’25℃常温;lane2: ‘波兰精神’25℃常温;
lane3: ‘金斯托韦克’38℃热激;lane4: ‘波兰精神’38℃热激
图3 转基因拟南芥热激胁迫抗性比对
左边为野生拟南芥,右边为转基因拟南芥。
具体实施方式
下面结合具体实施例,进一步阐明本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法,通常按照常规条件,例如Sambrook等分子克隆:实验手册(New York:Cold Spring Harbor LabortaryPress,1989)中所叙述的条件,或按照制造厂商所建议的条件。
实施例1
铁线莲CLHSP18基因的克隆
1.将耐热的铁线莲品种‘波兰精神’(Polish Spirit)(上海植物园)进行38℃热激3小时,提取嫩叶片的总RNA,提取试剂盒为植物RNAout试剂盒(四川绵阳高新区天泽基因工程有限公司),利用反转录试剂盒(TaKaRa,中国大连)将总RNA反转录成cDNA 。
2.根据月季RcHSP17.8(登录号:EF053229.1)与菟丝子CjHSP 17.6(登录号:AB017273)同源序列设计简并引物,正向引物:5’-CATTCRCTGAATHTCCTG-3’( SEQ ID NO.2)反向引物:5’-AACMTCCTGGAGKACTTG-3’( SEQ ID NO.3)。
采用RT-PCR方法从‘波兰精神’cDNA中扩增出一条247bp的条带。将PCR产物回收,连接到T-Vector(TaKaRa,中国大连)上,转化大肠杆菌 DH5α,((TaKaRa,D9057A)),进行测序。将测序结果提交至NCBI非冗余数据库,BLAST结果表明该序列和月季小分子热激蛋白基因RcHSP17.8的核苷酸序列高度保守。
利用片段序列中设计3对反向PCR引物。
第一对反向PCR引物:
SEQ ID NO.4正向引物5’-TGTCAACGTTGAGATTGA-3’,
SEQ ID NO.5反向引物5’- TGATGAAAGCCGTATTCT-3’;
第二对反向PCR引物:
SEQ ID NO.6正向引物5’- TGCTGCAGATCAGCGGCC-3’,
SEQ ID NO.7反向引物5’- AATTCAGGGAATGTGGCG-3’;
第三对反向PCR引物:
SEQ ID NO.8正向引物5’-CAAGTTCTCCAGGAGGTT-3’,
SEQ ID NO.9反向引物5’-AGGGAATTGGAAATCCCT-3’;
3.基因组DNA的提取
取‘波兰精神’嫩叶片,研成液氮粉,立即加入预热的提取缓冲液,65℃保温25min,离心,上清以等体积的氯仿/异戊醇(24/1)抽提2次,以乙醇沉淀DNA,70%乙醇洗涤两次,最后用TE溶解DNA;加入RNaseA(0.5μg/ml)(TaKaRa,中国大连),65℃消化RNA 1h;用0.8%琼脂糖凝胶检测DNA的质量。
4.用限制性内切酶EcoRI(TaKaRa,中国大连)对基因组DNA进行充分酶切,纯化并回收酶切后的片段,以T4连接酶(TaKaRa,中国大连)催化片段首尾自连成环。以自连后的片段作反向PCR模板,以3对反式PCR引物进行三轮巢式PCR,获得一427bp片段,回收、纯化,连接到T-Vector上,测序,序列分析后获得基因的5’端序列。
5.3’-RACE获得ClHSP18基因3’ 端
用3’-RACE反转录试剂盒(TaKaRa,中国大连)将‘波兰精神’嫩叶总RNA反转录成cDNA作3’-RACE模板,以上述3对反式PCR引物的正向引物(5’-3’) 分别与3’-RACE引物AUAP(TaKaRa,中国大连)配对进行三轮巢式PCR;回收获得的327bp片段,纯化,连接到T-Vector上,测序,序列分析后获得基因的3’端序列。
6.全长基因的克隆
根据上述2、4、5获得的序列进行拼接,获得该基因的cDNA全长序列,即开放阅读框(ORF)。依据基因5’、3’端序列设计基因全长引物(SEQ ID NO.10和SEQ ID NO.11的引物),采用RT-PCR方法从‘波兰精神’cDNA中扩增出一条471bp的条带。将PCR产物回收,连接到T-Vector上,转化大肠杆菌,进行测序。将测序结果提交至NCBI非冗余数据库,BLAST结果表明该序列和月季小分子热激蛋白基因RcHSP17.8序列高度保守。
其中全长引物:
SEQ ID NO.10 正向引物5’-ATGTCGCTTATCCTAAGT-3’,
SEQ ID NO.11 反向引物5’-TTACGCAGTAAGATCAAT-3’
实施例2
铁线莲ClHSP18基因的序列信息与同源性分析
实施例1得到的铁线莲ClHSP18基因长度为471 bp。该基因编码的多肽由156个氨基酸残基组成,分子量为17959.49道尔顿,pI为8.113。详细序列见SEQ ID NO.1。
将铁线莲ClHSP18全长基因的编码区序列及其编码的蛋白质序列用BLAST程序在Non-redundant GeneBank+EMBL+DDBJ+PDB 和Non-redundant GeneBank CDStranslations+PDB+SwissPort+Superdate+PIR数据库中进行核苷酸和蛋白质同源性检测,结果发现它与月季(Rosa chinensis)RcHSP17.8存在较高的同源性。在核苷酸水平上,它与月季(Rosa chinensis)RcHSP17.8基因的mRNA编码序列(GeneBankAccessionNo.EF053229.1)有一定的同源性,在氨基酸水平上,它与月季(Rosa chinensis)RcHSP17.8氨基酸序列(GenBank Accession No.ABK32539.1)的氨基酸残基有87.74%的相似性(见图1)。说明该铁线莲ClHSP18基因与月季(Rosa chinensis)RcHSP17.8基因存在较高的同源性。
实施例3
铁线莲ClHSP18表达模式分析
用RT-PCR的方法检测
分别取25℃(常温)及38℃热激3h的‘波兰精神’(PolishSpirit )和 ‘金斯托韦克’(StolwijkGold)(上海植物园)嫩叶提取总RNA,反转录成cDNA,然后根据铁线莲ClHSP18基因全长序列设计一对引物,正向引物5’-ATGTCGCTTATCCTAAGT-3’,反向引物5’-TTACGCAGTAAGATCAAT-3’,进行PCR。电泳之后,用相对定量软件(天程公司)进行分析。结果表明,常温下,铁线莲ClHSP18基因在两个铁线莲品种中均不表达,在热激后‘波兰精神’表达而‘金斯托韦克’不表达(图2)。
实施例4
铁线莲ClHSP18基因在拟南芥中异源表达提高了转基因拟南芥热激胁迫抗性
(1)含目的基因(铁线莲ClHSP18)植物表达载体pCAMBIA1300- ClHSP18的构建
以铁线莲ClHSP18基因的ORF设计引物:正向引物5’-ATGTCGCTTATCCTAAGT-3’,反向引物5’-TTACGCAGTAAGATCAAT-3’。正向引物引入EcoRI酶切位点,反向引物引入XbaI酶切位点。以实施例1中获得的耐热品种‘波兰精神’cDNA为模板,进行PCR扩增;割胶回收PCR产物,连接到T-Vector(TaKaRa,中国大连)构建成重组质粒T- ClHSP18;转化大肠杆菌DH5α(TaKaRa,D9057A),PCR检测阳性克隆,提取质粒T- ClHSP18;EcoRI、XbaI双酶切质粒T-ClHSP18,电泳分离,割胶回收酶切产物的小片段,与同样经EcoRI、XbaI双酶切的植物表达载体pCAMBIA1300(TaKaRa,中国大连)连接,构建pCAMBIA1300- ClHSP18重组质粒;转化大肠杆菌DH5α,PCR检测阳性克隆,并测序证明插入片段ClHSP18序列正确,无移码发生;提取质粒pCAMBIA1300- ClHSP18。
将重组表达载体质粒pCAMBIA1300- ClHSP18转化农杆菌GV3101(TaKaRa,中国大连),以农杆菌浸染法(浸花法)转化拟南芥(野生型Columbia,Arabidopsis BiologicalResource Center,美国俄亥俄州立大学)花蕾;潮霉素(15mg/L)(德国进口,上海索来宝生物科技有限公司)筛选阳性植株,获得T3代转基因(pCAMBIA1300- ClHSP18)拟南芥植株。选取花期的转基因(pCAMBIA1300- ClHSP18)拟南芥植株土培盆苗,进行42℃热激12小时后,转基因拟南芥植株(pCAMBIA1300- ClHSP18)未发生明显的形态变化而野生型拟南芥(CK)出现严重萎蔫(图3),结果显示转基因拟南芥(pCAMBIA1300-ClHSP18)比野生型拟南芥(CK)具有更强的耐热性。表明铁线莲ClHSP18基因在拟南芥中异源过表达能提高转基因拟南芥对热激胁迫的耐性。
序列表
<110> 上海植物园
<120> 铁线莲CLHSP18基因及其编码蛋白和应用
<160> 11
<170> SIPOSequenceListing 1.0
<210> 1
<211> 155
<212> PRT
<213> 铁线莲 (Clematis florida Thunb)
<400> 1
Met Ser Leu Ile Leu Ser Tyr Arg Arg Asn Ser Val Phe Asp Leu Asp
1 5 10 15
Leu Trp Asp Pro Phe Arg Asp Phe Gln Phe Pro Ser Ser Ser Leu Ala
20 25 30
Thr Phe Pro Glu Phe Pro Gly Glu Asn Thr Ala Phe Ile Asn Thr Arg
35 40 45
Ile Ala Asp Trp Lys Gln Thr Pro Glu Ala His Val Phe Lys Val Asp
50 55 60
Leu Pro Ala Leu Lys Ile Glu Asp Val Asn Val Glu Ile Glu Asn Asp
65 70 75 80
Arg Val Leu Gln Ile Ser Gly Leu Arg Lys Ile Glu Lys Glu Asp Lys
85 90 95
Asn Asp Lys Trp His Arg Val Asp Arg Ser Ser Cys Lys Phe Ser Arg
100 105 110
Arg Phe Arg Leu Pro Glu Asn Ala Lys Leu Asp Glu Ile Lys Ala Ala
115 120 125
Met Glu Asn Gly Val Leu Arg Val Thr Val Pro Lys Ala Asn Val Lys
130 135 140
Arg Pro Asp Val Lys Ala Ile Asp Leu Thr Ala
145 150 155
<210> 2
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
cattcrctga athtcctg 18
<210> 3
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
aacmtcctgg agkacttg 18
<210> 4
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
tgtcaacgtt gagattga 18
<210> 5
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
thathaaahc chtattct 18
<210> 6
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
thctgcagat cagcggcc 18
<210> 7
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aattcaggga atgtggcg 18
<210> 8
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
caagttctcc aggaggtt 18
<210> 9
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
agggaattgg aaatccct 18
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
atgtcgctta tcctaagt 18
<210> 11
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ttacgcagta agatcaat 18
Claims (1)
1.一种氨基酸序列,其序列如SEQ ID NO.1所示。
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