CN113957083A - 一种花生脂氧合酶基因AhLOX29及其克隆与功能表达方法、应用 - Google Patents
一种花生脂氧合酶基因AhLOX29及其克隆与功能表达方法、应用 Download PDFInfo
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- CN113957083A CN113957083A CN202111470353.4A CN202111470353A CN113957083A CN 113957083 A CN113957083 A CN 113957083A CN 202111470353 A CN202111470353 A CN 202111470353A CN 113957083 A CN113957083 A CN 113957083A
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- ahlox29
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- cloning
- lipoxygenase
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Abstract
本发明公开了一种花生脂氧合酶基因AhLOX29及其克隆与功能表达方法、应用，涉及生物工程的技术领域，其技术方案要点一是在于提供一种花生脂氧合酶基因AhLOX29，其开放阅读框为2754bp，共编码917个氨基酸；二是在于提供一种该基因的克隆与功能表达方法方法，包括以下步骤：S1、材料准备，S2、PCR扩增获得AhLOX29基因全序列，S3、构建AhLOX29基因超表达的载体，S4、农杆菌介导的拟南芥遗传转化、筛选及分子鉴定，S5、荧光定量PCR对AhLOX29基因表达进行分析，S6、转基因T2代拟南芥幼苗干旱胁迫处理及表型观察；三是在于提供该基因AhLOX29的应用。
Description
技术领域
本发明涉及生物工程技术领域，更具体地说，它涉及一种花生脂氧合酶基因AhLOX29及其克隆与功能表达方法、应用。
背景技术
花生是世界优质植物油脂和蛋白质的核心来源之一。我国是世界第一花生生产大国，种植面积近7000万亩，居世界第二位；年产约1700万吨，居全球首位，在我国油料作物中总产量、总产值、出口量均第一位。但是，花生的产量和品质受到干旱威胁较为严重。据统计，我国每年因干旱引起的花生减产达30％～50％；此外，干旱胁迫还会增加黄曲霉毒素污染几率。国际半干旱作物研究所(I-CRISAT)研究认为，由干旱造成的约一半的损失可以通过遗传改良提高花生抗旱能力而避免。传统育种方法虽然也能使花生的抗旱能力有所提高，但总的来说进展缓慢。随着现代分子生物学、生物技术的发展，近年来抗逆基因的研究已取得显著进展，但花生中可供有效利用的基因资源相对缺乏。因此，挖掘抗旱相关基因，阐明抗旱分子机理具有重要意义。
脂氧合酶(LOX)是一种含非血红素的双加氧酶，在植物、动物和真菌中广泛存在，并已形成多个亚家族。在植物中，LOX催化脂肪酸氧化形成氢过氧衍生物，催化的底物主要是不饱和脂肪酸，包括α-亚油酸(18:2)、α-亚麻酸(18:3)和十六碳三烯酸(16:3)等。根据催化底物位点的特异性(即在多不饱和脂肪酸碳9或13的位置加氧)，植物LOX可分为两个亚家族9-LOX和13-LOX。LOX是茉莉酸生物合成途径中的一种关键酶，有研究表明，水分胁迫下LOX活性与茉莉酸含量呈极显著正相关，抑制LOX酶活性后茉莉酸含量也显著减少。目前植物中只发现过表达拟南芥AtLOX6和玉米ZmLOX6可显著提高植物的抗旱性，关于花生LOX基因功能的报道较少，而关于其抗旱功能的研究还未有报道。
发明内容
针对现有技术存在的不足，本发明的第一个目的在于提供一种花生脂氧合酶基因AhLOX29；本发明的第二个目的在于提供一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法；本发明的第三个目的在于提供一种花生脂氧合酶基因AhLOX29的应用。
为实现上述第一个目的，本发明提供了如下技术方案：一种花生脂氧合酶基因AhLOX29，所述AhLOX29基因开放阅读框为2754bp，共编码917个氨基酸；所述AhLOX29基因的核苷酸序列如SEQ ID No:1所示，其氨基酸序列如SEQ ID No:2所示。
为实现上述第二个目的，本发明提供了如下技术方案：一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，包括以下步骤：
S1、材料准备；
S2、PCR扩增获得AhLOX29基因全序列；
S3、构建AhLOX29基因超表达的载体；
S4、农杆菌介导的拟南芥遗传转化、筛选及分子鉴定；
S5、荧光定量PCR对AhLOX29基因表达进行分析；
S6、转基因T2代拟南芥幼苗干旱胁迫处理及表型观察。
进一步地，步骤S2中，PCR扩增所述花生脂氧合酶AhLOX29的引物为AhLOX29-F和AhLOX29-R，其中：
AhLOX29-F：5’-TTTGGTACATCTTAAGTCAATAAG-3’，
AhLOX29-R：5’-CTATTCCGGAGTAAGTTTGATT-3’。
进一步地，步骤S4中，PCR检测筛选阳性转基因植株的引物为GUS-F和GUS-R，其中，GUS-F：GTGACAAAAACCACCCAAG，
GUS-R：CTTTCTTGTTACCGCCAAC。
进一步地，步骤S5中，使用荧光定量RT-PCR对AhLOX29基因干旱下的表达进行分析，所述的AhLOX29基因荧光定量引物序列如下：
AhLOX29-RT-F：5’-CACATAATTAGCCACTTGGACG-3’，
AhLOX29-RT-R：5’-TACGAGTTGCATATGCTTTTCG-3’。
进一步地，步骤S5中，AhLOX29基因定量PCR的正向引物序列为：
AhLOX29-RT-F：5’-CACATAATTAGCCACTTGGACG-3’，
AhLOX29-RT-R：5’-TACGAGTTGCATATGCTTTTCG-3’。
进一步地，步骤S5中，使用Actin作为RT-PCR的内参基因，所述的内参基因Actin所用引物序列为：
Actin-F:5’-TTGGAATGGGTCAGAAGGATGC-3’，
Actin-R:5’-AGTGGTGCCTCAGTAAGAAGC-3’。
为实现上述第三个目的，本发明提供了如下技术方案：一种花生脂氧合酶基因AhLOX29的应用，可提高植物的抗旱性。
综上所述，本发明具有以下有益效果：
第一、本发明首次发现了花生脂氧合酶基因AhLOX29，通过研究验证，该基因可提高植物的抗旱性。
第二、本发明的方法，通过荧光定量PCR验证了AhLOX29在干旱胁迫下的表达模式，该基因干旱胁迫下在叶片和根部的转录水平均有明显升高，干旱胁迫处理后叶片和根中AhLOX29基因的相对表达量一直处于上升趋势，在24小时达到峰值，初步验证了该基因能够响应干旱胁迫。
附图说明
图1是本发明20％PEG6000胁迫处理后花生AhLOX29基因在不同时间段和不同部位的表达模式分析。
图2是本发明中过表达花生AhLOX29基因T2代拟南芥种子在干旱胁迫下的表现。
具体实施方式
以下结合实施例对本发明作进一步详细说明。
实施例1花生脂氧合酶基因AhLOX29的克隆与功能表达
S1、材料准备
植物材料为花生品种“J11”和拟南芥(生态型Col0)。大肠杆菌感受态DH5α、克隆载体pEASY-Blunt Simple等购自北京全式金生物有限公司；高保真DNA聚合酶(PrimeSTARGXL DNA Polymerase)、RNA提取试剂盒(MiniBEST Universal RNA Extraction Kit)、反转录试剂盒(PrimeScriptTMRT Master Mix)、无缝克隆试剂盒(In-Fusion HD Cloning Kit)、荧光定量试剂盒(SYBR Premix Ex TaqTMII)购自宝生物工程(大连)有限公司(TaKaRa)；PEG6000、MS培养基培养基等购自青岛生工生物科技有限公司。超表达载体pCambia2300EC和农杆菌菌株GV3101由本实验室保存。
S2、PCR扩增获得AhLOX29基因全序列
用植物RNA提取试剂盒提取花生“J11”叶片和根的总RNA，使用反转录试剂盒获得cDNA，以cDNA为模板，利用特异扩增引物AhLOX29-F和AhLOX29-R进行PCR扩增，其中：
AhLOX29-F：5’-TTTGGTACATCTTAAGTCAATAAG-3’，
AhLOX29-R：5’-CTATTCCGGAGTAAGTTTGATT-3’。
PCR反应体系为：5μL 5×PrimeSTAR GXL Buffer、1μL dNTP Mixture、1μL总cDNA、1μL AhLOX29-F、1μL AhLOX29-R、1μL PrimeSTAR GXL DNA Polymerase和9μL无菌双蒸水；
PCR反应条件为：98℃1min；98℃10s,58℃15s,68℃1min；共30cycles；68℃3min。
用1％琼脂糖凝胶进行电泳，电泳后切取目的基因条带，用胶回收试剂盒回收PCR产物。将PCR产物连接至克隆载体pEASY-Blunt Simple，将阳性克隆测序，将测序后结果与PeanutBase网站公布序列进行比对，比对正确的序列命名为pEASY-AhLOX29。
S3、构建AhLOX29基因超表达的载体
用一步法克隆试剂盒将pEASY-AhLOX29中的AhLOX29基因序列连接入超表达载体pCambia2300EC，得到质粒OE-AhLOX29。
S4、农杆菌介导的拟南芥遗传转化、筛选及分子鉴定
利用热激法将构建好的质粒OE-AhLOX29转入农杆菌GV3101，将拟南芥花序浸到侵染液中进行转化。待拟南芥荚果变黄开裂时收获T0代种子。将收获的种子在超净工作台中进行消毒，70％酒精处理5min，2.6％的NaClO处理10min，无菌水冲洗3-5次，然后将种子均匀播种到含有50μg/mL卡那霉素的MS固体培养基中于培养箱中培养筛选。待培养基上T1代卡那霉素抗性的拟南芥幼苗长至两片子叶后，选取绿色、根系发达的转基因植物移栽到基质中。待生长至大约3周后，取叶片提取基因组DNA，以GUS-F和GUS-R为引物，进行PCR检测筛选阳性转基因植株。其中，
GUS-F：GTGACAAAAACCACCCAAG，
GUS-R：CTTTCTTGTTACCGCCAAC。
S5、荧光定量PCR对AhLOX29基因表达进行分析
S51、用20％PEG6000处理花生“J11”的幼苗，生长至三叶期(14天)后施加干旱胁迫处理，分别在处理0h，6h，12h，24h和48h时取花生的叶片和根用液氮速冻，保存于-80℃超低温冰箱中备用。使用RNA提取试剂盒提取花生组织总RNA，抽提后用DNase I处理，反转录后将产物于-20℃冰箱中保存备用。
S52、用ABI 7500FAST型荧光定量PCR仪进行荧光定量反应。20μL PCR反应体系为：10μL SYBRGreen Mix，正反向引物(10μM)各0.8μL；；cDNA 2μL(20ng)。
PCR反应程序如下：95℃5min；95℃10s，60℃30s，共40个循环；循环结束，每10s升高0.5℃至95℃，绘制溶解曲线。采用2-ΔΔCT法计算相对表达量。
S53、AhLOX29基因定量PCR的正向引物序列为：
AhLOX29-RT-F：5’-CACATAATTAGCCACTTGGACG-3’，
AhLOX29-RT-R：5’-TACGAGTTGCATATGCTTTTCG-3’；
花生Actin作为内参基因，扩增的正向引物序列为：
Actin-F：5’-TTGGAATGGGTCAGAAGGATGC-3’；
反向引物序列为：
Actin-R：5’-AGTGGTGCCTCAGTAAGAAGC-3’。
S6、转基因T2代拟南芥幼苗干旱胁迫处理及表型观察
T2代转基因幼苗与野生型每次浇相同量的水，生长至30天后开始不浇水，干旱20天后复水，复水两天后拍照记录。
通过荧光定量PCR验证了AhLOX29在干旱胁迫下的表达模式，从图1(图中，CK为对照，Drought为干旱胁迫处理)可以看出：在正常情况下，该基因在叶片和根部的转录水平较为稳定，相对表达量无明显变化；干旱胁迫下在叶片和根部的转录水平均有明显升高，干旱胁迫处理后叶片和根中AhLOX29基因的相对表达量一直处于上升趋势，24h到达峰值，初步验证了该基因能够响应干旱胁迫。以上结果表明AhLOX29基因在花生对干旱胁迫的适应性中发挥重要作用。
如图2显示，转基因幼苗与野生型在表型上无明显差别；20天不浇水，分别同时进行干旱胁迫处理后，转基因幼苗和野生型均植株枯萎，叶片干瘪萎缩；同时复水两天后，转基因植株与野生型对比明显，野生型植株叶片干枯、无存活植株，转基因株系植株大部分叶片变绿，生长状态良好。将该基因通过转基因手段转入拟南芥，转基因植株比野生型具有明显的抗旱性，说明AhLOX29基因能显著提高植物的抗旱性。
实施例2AhLOX29基因序列信息与特征分析
通过PCR扩增及测序，得到了目的基因，AhLOX29基因全长为3231bp,开放阅读框为2754bp，共编码917个氨基酸。AhLOX29基因的核苷酸序列如SEQ ID No:1所示，其氨基酸序列如SEQ ID No:2所示。
本具体实施例仅仅是对本发明的解释，其并不是对本发明的限制，本领域技术人员在阅读完本说明书后可以根据需要对本实施例做出没有创造性贡献的修改，但只要在本发明的权利要求范围内都受到专利法的保护。
序列表
<110> 山东省花生研究所
山东省花生研究所
<120> 一种花生脂氧合酶基因AhLOX29及其克隆与功能表达方法、应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3231
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
tttggtacat cttaagtcaa taagctacta ctctataact atagcacttg cattgtgtat 60
aaattaatag taacaaatac acactttagg gtttgttaga gttgaaattg agaattgaaa 120
tgaggatggt taaggaaatt ttgggacaaa gtgtagtggt tgaagcattt tgttttgatg 180
cttctagaag atgtcacact catcaaaata ataataagaa acacaaaatt tcaatgtttc 240
ccagtttctc ttgctcatca attctgttgc ccattgtgaa gaggagaacc acctttgatg 300
atttgattag tagtaataag agtctccatg atggtggtgg cgtaacgttg acgataagtg 360
caagtgttac tataaagaat agtaaggagt tcatggcaat gatgcttcat tactttgata 420
gtttcagcca taatacaact ggtggggata gaggaatcat catgcagcta gttagcactc 480
aaattgatcc cacaacaatg gagggaaagc tgagcaagag agccgtactg gattggacaa 540
gagaattatt atttaatgga ggtggggata gtgagaggtc cactcacaaa gttgaatttg 600
agattgactc taattttgga tttccgggag ccattacagt cacaaacaaa tatgacaaag 660
agatcttctt ggaaaccatt tccattgaag gaggctttgt ggaattttct tgcaattctt 720
gggttcaacc tgaaaaggtt cacccagaca agagaatttt cttcaccaac aaggcatgct 780
taccatgtga cacagcagag ggtgtgaagg aactacgaaa agaagagctg agacaaaaga 840
ggggagatgg aaatggaatg aggaaggtga tgagtgatag ggtttatgac tatgatgtct 900
acaatgactt gggaaatcta gacaagggca ttcaacatgt taggccaatt ttgggaacca 960
aacaacatcc taatcctagg cgttgtcgaa ctggccgtcc acctgtcata actaacgaga 1020
aatatgagtc gtgtgtgaat gcattcatgg aaacatatgt gccaagagat gaagtgtttg 1080
aaggagttag aaaggaggca cttgatgtgg agagagtgaa aggtgtaaca agaaatttga 1140
ttcccttcat aagaacttat ataactaagt gtggagtctt taaggagcta ttagagatta 1200
agaacatata caaaagaagg catcaagtgg atgcaatgaa tcatactaat gaggagaatg 1260
ggacactttc tattgatgtt aacaagttat tccaagattc cttcgaagag tacttcaaat 1320
ttagcactcc acacattata agtgggaatg gtggttgctg cataacagat gaagaacttg 1380
ggcgtcaggc cttggctggc gtcaaccctt taagcataaa aaggcttcag acttttccac 1440
cggtgagtga tttggacccc tccatgtatg gtccacaaga gtccgctcta aaagcagagc 1500
acataattag ccacttggac ggcatgtccg tacaacaggc tatagtggaa aagaagctat 1560
tcattttgga ttaccatgat gcttatatgc cctttctaaa agggatcaat gctcaagagg 1620
accgaaaagc atatgcaact cgtaccatat tgtacttgac tagaatggga actttgaaac 1680
ctattgccat agagctgagt cttcctgagg gggagcacca acaacaacaa tccaaacagg 1740
ttctaacacc tccattggat gccacttctt attggttgtg gcaacttgcc aaagcacatg 1800
tttgctccaa tgatgctgga gttcaccaac ttgttcatca ctgcgttagg ttgaggaccc 1860
acgcgtgcat ggagccattc atcatagcag cacatcgcca gatgagtgtc acgcacccta 1920
tcttcaagct cctaaagcct catatgaaat acacactgca gataaatgca ttggctcgca 1980
aggctcttat caatggtgga ggcatcattg aatctgactt ctctaccggc agatacagca 2040
ctgagatcgt ctcggccgct tacaaagatt ggtggcgttt tgacatggaa gccctcccag 2100
ctgaccttat tagaagggga ctagcagaac cagacagagc acaacctctt gggataaaac 2160
ttacaattga agattaccct tatgcaaatg atgggcttct tatttggttt gctttggaga 2220
ggatggttag aacttatgtg aactactact accataatgg cctaatggta cggtcggaca 2280
ctgaactcca agggtggtac aatgaggtca tcaacgtagg ccatggtgat catgcacatg 2340
ctacttggtg gccaacactt gccactccca gagatcttat ctcagtcctc accacactca 2400
tttggattgc ttcagttcag cattcagcag tgaattttgg gcagtaccct cttggcgggt 2460
acgtcccgat gcgcccgccg ctggtgaaga agctgctacc caaggaagga gatccagagt 2520
acaaggagtt cttggaggat cccgaaggtt tcttgtgttc ttccctgccg aatatgtttc 2580
aaaccaccaa gtttcttgca gtgcttaaca tactctctca gcatccggag gatgaagaat 2640
acatagggca gagaagggac ttgtcagatt ggattggtga ccctaaaatc atagaggcat 2700
tctatgattt ctccatagag ctaaagagaa tagagaagga gattgagaag aggaataaag 2760
accagattct tagaaacaga tgcggtgctg ggattccacc atatgaattg cttatagcta 2820
gctctggtcc tggagttact tgtagagggg tgcctaatag cataagtaca tagatatcac 2880
acaattgtgg cgtagagtaa acgaggtgga tgttaaatca aatgaatcaa tcatatttct 2940
ccctctaaga gtaaacgagg aagaaacccc aagaaagggc caagacaatg tcaccgacgg 3000
tttgcgggag ccaatcccag tataagcagg gcattagcta aagcttaagg tgccgtgctt 3060
tatgttaagg taaggaatgt ctctaggtta tttatgcctc taagctgtgt gtggttcaaa 3120
tattacatta ttataaagat tctattctca tgggaattaa agatatccaa aaagatggga 3180
attgaaatga tggtattttg atttcctgaa atcaaactta ctccggaata g 3231
<210> 2
<211> 917
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
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1 5 10 15
Phe Cys Phe Asp Ala Ser Arg Arg Cys His Thr His Gln Asn Asn Asn
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Lys Lys His Lys Ile Ser Met Phe Pro Ser Phe Ser Cys Ser Ser Ile
35 40 45
Leu Leu Pro Ile Val Lys Arg Arg Thr Thr Phe Asp Asp Leu Ile Ser
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Ser Asn Lys Ser Leu His Asp Gly Gly Gly Val Thr Leu Thr Ile Ser
65 70 75 80
Ala Ser Val Thr Ile Lys Asn Ser Lys Glu Phe Met Ala Met Met Leu
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His Tyr Phe Asp Ser Phe Ser His Asn Thr Thr Gly Gly Asp Arg Gly
100 105 110
Ile Ile Met Gln Leu Val Ser Thr Gln Ile Asp Pro Thr Thr Met Glu
115 120 125
Gly Lys Leu Ser Lys Arg Ala Val Leu Asp Trp Thr Arg Glu Leu Leu
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Phe Asn Gly Gly Gly Asp Ser Glu Arg Ser Thr His Lys Val Glu Phe
145 150 155 160
Glu Ile Asp Ser Asn Phe Gly Phe Pro Gly Ala Ile Thr Val Thr Asn
165 170 175
Lys Tyr Asp Lys Glu Ile Phe Leu Glu Thr Ile Ser Ile Glu Gly Gly
180 185 190
Phe Val Glu Phe Ser Cys Asn Ser Trp Val Gln Pro Glu Lys Val His
195 200 205
Pro Asp Lys Arg Ile Phe Phe Thr Asn Lys Ala Cys Leu Pro Cys Asp
210 215 220
Thr Ala Glu Gly Val Lys Glu Leu Arg Lys Glu Glu Leu Arg Gln Lys
225 230 235 240
Arg Gly Asp Gly Asn Gly Met Arg Lys Val Met Ser Asp Arg Val Tyr
245 250 255
Asp Tyr Asp Val Tyr Asn Asp Leu Gly Asn Leu Asp Lys Gly Ile Gln
260 265 270
His Val Arg Pro Ile Leu Gly Thr Lys Gln His Pro Asn Pro Arg Arg
275 280 285
Cys Arg Thr Gly Arg Pro Pro Val Ile Thr Asn Glu Lys Tyr Glu Ser
290 295 300
Cys Val Asn Ala Phe Met Glu Thr Tyr Val Pro Arg Asp Glu Val Phe
305 310 315 320
Glu Gly Val Arg Lys Glu Ala Leu Asp Val Glu Arg Val Lys Gly Val
325 330 335
Thr Arg Asn Leu Ile Pro Phe Ile Arg Thr Tyr Ile Thr Lys Cys Gly
340 345 350
Val Phe Lys Glu Leu Leu Glu Ile Lys Asn Ile Tyr Lys Arg Arg His
355 360 365
Gln Val Asp Ala Met Asn His Thr Asn Glu Glu Asn Gly Thr Leu Ser
370 375 380
Ile Asp Val Asn Lys Leu Phe Gln Asp Ser Phe Glu Glu Tyr Phe Lys
385 390 395 400
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405 410 415
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Ile Lys Arg Leu Gln Thr Phe Pro Pro Val Ser Asp Leu Asp Pro Ser
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450 455 460
His Leu Asp Gly Met Ser Val Gln Gln Ala Ile Val Glu Lys Lys Leu
465 470 475 480
Phe Ile Leu Asp Tyr His Asp Ala Tyr Met Pro Phe Leu Lys Gly Ile
485 490 495
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500 505 510
Leu Thr Arg Met Gly Thr Leu Lys Pro Ile Ala Ile Glu Leu Ser Leu
515 520 525
Pro Glu Gly Glu His Gln Gln Gln Gln Ser Lys Gln Val Leu Thr Pro
530 535 540
Pro Leu Asp Ala Thr Ser Tyr Trp Leu Trp Gln Leu Ala Lys Ala His
545 550 555 560
Val Cys Ser Asn Asp Ala Gly Val His Gln Leu Val His His Cys Val
565 570 575
Arg Leu Arg Thr His Ala Cys Met Glu Pro Phe Ile Ile Ala Ala His
580 585 590
Arg Gln Met Ser Val Thr His Pro Ile Phe Lys Leu Leu Lys Pro His
595 600 605
Met Lys Tyr Thr Leu Gln Ile Asn Ala Leu Ala Arg Lys Ala Leu Ile
610 615 620
Asn Gly Gly Gly Ile Ile Glu Ser Asp Phe Ser Thr Gly Arg Tyr Ser
625 630 635 640
Thr Glu Ile Val Ser Ala Ala Tyr Lys Asp Trp Trp Arg Phe Asp Met
645 650 655
Glu Ala Leu Pro Ala Asp Leu Ile Arg Arg Gly Leu Ala Glu Pro Asp
660 665 670
Arg Ala Gln Pro Leu Gly Ile Lys Leu Thr Ile Glu Asp Tyr Pro Tyr
675 680 685
Ala Asn Asp Gly Leu Leu Ile Trp Phe Ala Leu Glu Arg Met Val Arg
690 695 700
Thr Tyr Val Asn Tyr Tyr Tyr His Asn Gly Leu Met Val Arg Ser Asp
705 710 715 720
Thr Glu Leu Gln Gly Trp Tyr Asn Glu Val Ile Asn Val Gly His Gly
725 730 735
Asp His Ala His Ala Thr Trp Trp Pro Thr Leu Ala Thr Pro Arg Asp
740 745 750
Leu Ile Ser Val Leu Thr Thr Leu Ile Trp Ile Ala Ser Val Gln His
755 760 765
Ser Ala Val Asn Phe Gly Gln Tyr Pro Leu Gly Gly Tyr Val Pro Met
770 775 780
Arg Pro Pro Leu Val Lys Lys Leu Leu Pro Lys Glu Gly Asp Pro Glu
785 790 795 800
Tyr Lys Glu Phe Leu Glu Asp Pro Glu Gly Phe Leu Cys Ser Ser Leu
805 810 815
Pro Asn Met Phe Gln Thr Thr Lys Phe Leu Ala Val Leu Asn Ile Leu
820 825 830
Ser Gln His Pro Glu Asp Glu Glu Tyr Ile Gly Gln Arg Arg Asp Leu
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Ser Asp Trp Ile Gly Asp Pro Lys Ile Ile Glu Ala Phe Tyr Asp Phe
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Ser Ile Glu Leu Lys Arg Ile Glu Lys Glu Ile Glu Lys Arg Asn Lys
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Asp Gln Ile Leu Arg Asn Arg Cys Gly Ala Gly Ile Pro Pro Tyr Glu
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Leu Leu Ile Ala Ser Ser Gly Pro Gly Val Thr Cys Arg Gly Val Pro
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Asn Ser Ile Ser Thr
915
Claims (8)
1.一种花生脂氧合酶基因AhLOX29，其特征在于，所述AhLOX29基因开放阅读框为2754bp，共编码917个氨基酸；所述AhLOX29基因的核苷酸序列如SEQ ID No:1所示，其氨基酸序列如SEQ ID No:2所示。
2.一种如权利要求1所述的花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，包括以下步骤：
S1、材料准备；
S2、PCR扩增获得AhLOX29基因全序列；
S3、构建AhLOX29基因超表达的载体；
S4、农杆菌介导的拟南芥遗传转化、筛选及分子鉴定；
S5、荧光定量PCR对AhLOX29基因表达进行分析；
S6、转基因T2代拟南芥幼苗干旱胁迫处理及表型观察。
3.根据权利要求2所述的一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，步骤S2中，PCR扩增所述花生脂氧合酶AhLOX29的引物为AhLOX29-F和AhLOX29-R，其中：
AhLOX29-F：5’-TTTGGTACATCTTAAGTCAATAAG-3’，
AhLOX29-R：5’-CTATTCCGGAGTAAGTTTGATT-3’。
4.根据权利要求2所述的一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，步骤S4中，检测筛选阳性转基因植株的引物为GUS-F和GUS-R，其中，GUS-F：GTGACAAAAACCACCCAAG，
GUS-R：CTTTCTTGTTACCGCCAAC。
5.根据权利要求2所述的一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，步骤S5中，使用荧光定量RT-PCR对AhLOX29基因干旱下的表达进行分析，所述的AhLOX29基因荧光定量引物序列如下：
AhLOX29-RT-F：5’-CACATAATTAGCCACTTGGACG-3’，
AhLOX29-RT-R：5’-TACGAGTTGCATATGCTTTTCG-3’。
6.根据权利要求2所述的一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，步骤S5中，AhLOX29基因定量PCR的正向引物序列为：
AhLOX29-RT-F：5’-CACATAATTAGCCACTTGGACG-3’，
AhLOX29-RT-R：5’-TACGAGTTGCATATGCTTTTCG-3’。
7.根据权利要求2所述的一种花生脂氧合酶基因AhLOX29的克隆与功能表达方法，其特征在于，步骤S5中，使用Actin作为RT-PCR的内参基因，所述的内参基因Actin所用引物序列为：
Actin-F:5’-TTGGAATGGGTCAGAAGGATGC-3’，
Actin-R:5’-AGTGGTGCCTCAGTAAGAAGC-3’。
8.根据权利要求1所述的一种花生脂氧合酶基因AhLOX29，其特征在于，所述基因AhLOX29可以提高植物的抗旱性。
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| US20070083954A1 (en) * | 2002-03-27 | 2007-04-12 | Priti Sharma | Novel genes from drought stress tolerant tea plant and a method of introducing water-stress tolerance |
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