CN117305330A - 一种与耐盐碱胁迫相关的苹果酸过氧化物酶基因及其用途 - Google Patents
一种与耐盐碱胁迫相关的苹果酸过氧化物酶基因及其用途 Download PDFInfo
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Abstract
本发明属于基因工程技术领域，具体涉及一种与耐盐碱胁迫相关的苹果酸过氧化物酶基因及其用途。与耐盐碱胁迫相关的苹果酸过氧化物酶基因(HVUL2H37793.2基因)的核苷酸序列如SEQ ID NO.1所示。本发明研究发现在盐碱胁迫后，HVUL2H37793.2基因可显著调控青稞抗性品种中苹果酸的积累水平从而提高青稞的耐盐碱能力。将基因HVUL2H37793.2在烟草中进行过表达后，烟草叶片中苹果酸的含量显著增加，烟草耐盐碱能力提高。因此，HVUL2H37793.2基因在植物抵御盐碱胁迫中具有重要的意义。
Description
技术领域
本发明属于基因工程技术领域，具体涉及一种与耐盐碱胁迫相关的苹果酸过氧化物酶基因及其用途。
背景技术
盐胁迫是农业生产力中最有害的非生物胁迫之一。跟据统计，全球盐渍化土地的面积约为9.54×108hm2，约占世界陆地总面积的7％。由于灌溉土地排水不足，施肥管理不善和全球变暖，使得盐害农田的范围和严重程度将进一步恶化。盐胁迫的主要原因是钠离子(Na+)和氯离子(Cl-)的增加，这是自然界中来源中最丰富的盐离子。高盐度会引起植物的水分胁迫、离子毒性、营养障碍、氧化应激、代谢过程的改变、膜破坏、细胞分裂和扩张的减少和遗传毒性等。这些效应共同影响了植物生长和生存。
与大麦属的其他植物物种相比，青稞(Hordeum vuglare L.var.nudum Hook.f)具有一套强大的耐受系统来应对环境胁迫。由于海拔极高，青藏高原的作物必须遭受各种类型的非生物胁迫，如寒冷，干旱和盐胁迫。青稞作为西藏自治区及其附近地区人口最重要的主食和经济作物，提高其抗逆性和产量尤为重要。
苹果酸在耐逆性中起着至关重要的作用。盐碱胁迫可以诱导苹果酸的富集。青稞在遭受盐碱胁迫后，苹果酸积累水平迅速得以富集。但是有关青稞中苹果酸的积累的分子机制尚待进一步研究。对青稞响应盐碱胁迫的苹果酸富集进行解析，将会成为青稞抗性育种的亮点，且具有重要的市场应用价值。
发明内容
本发明的目的是提供一种与耐盐碱胁迫相关的苹果酸过氧化物酶基因及其用途。
本发明提供了一种基因片段，所述基因片段的核苷酸序列如SEQ ID NO.1所示。
本发明还提供了一种重组载体，所述重组载体包含核苷酸序列如SEQ ID NO.1所示的基因片段。
进一步地，所述重组载体是重组pEAQ或重组pCXSN。
本发明还提供了一种重组菌，所述重组菌包含前述的重组载体。
进一步地，所述重组菌为转化农杆菌。
本发明还提供了一种苹果酸过氧化物酶，所述苹果酸过氧化物酶是序列如SEQ IDNO.1所示的基因片段表达而得。
本发明还提供了前述的基因片段、前述的重组载体、前述的重组菌或前述的苹果酸过氧化物酶在制备耐盐碱的植物的用途；
优选地，所述植物为青稞、烟草。
本发明还提供了一种表达苹果酸过氧化物酶的转基因植物的构建方法，取核苷酸序列如SEQ ID NO.1所示的基因片段，转入植物中，获得表达苹果酸过氧化物酶的植株，即可。
进一步地，所述转基因植物为转基因烟草。
进一步地，所述转入植物的方法是农杆菌法、基因枪法、电转法、PEG介导法、脂质体法和磷酸钙-DNA共沉淀法中的一种。
本发明研究发现在盐碱胁迫后，HVUL2H37793.2基因可显著调控青稞抗性品种中苹果酸的积累水平从而提高青稞的耐盐碱能力。将基因HVUL2H37793.2在烟草中进行过表达后，烟草叶片中苹果酸的含量显著增加，烟草耐盐碱能力提高。因此，HVUL2H37793.2基因在植物抵御盐碱胁迫中具有重要的意义。
显然，根据本发明的上述内容，按照本领域的普通技术知识和惯用手段，在不脱离本发明上述基本技术思想前提下，还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式，对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为盐碱胁迫不同时间，青稞叶片中苹果酸积累水平及HVUL2H37793.2表达水平变化分析。
图2为与野生型烟草株系(CK)和叶片中表达HVUL2H37793.2后的过表达烟草株系中苹果酸含量结果以及目标产物二级谱图分析结果图：A为苹果酸含量结果图；B为目标产物二级谱图分析结果图。
图3为HVUL2H37793.2过表达后相关生长指标分析结果图：A为正常和盐胁迫环境下野生型和T2代转基因种子萌发率统计结果图；B为在正常条件下和盐胁迫处理下的主根长统计数据结果图；数据为三个独立重复的平均±SD；*p<0.05，**p<0.01(t-检验)。
具体实施方式
本发明所用原料与设备均为已知产品，通过购买市售产品所得。
青稞中与耐盐碱胁迫相关的苹果酸过氧化物酶基因(HVUL2H37793.2)，该基因序列如SEQ ID NO.1所示，该基因片段可以通过直接合成的方式获得，也可以通过其他本领域常规制备方法获得。
SEQ ID NO.1：
ATGGAGGTTGATCACCGCATCAGGGTGAGCGACGGCGACGGCGAGACGACGGCCGGACAAGGAGGCGTTGTTGCCGGCGTCTCGTTCGCGGGCTGCTGGCAGCGGCTCCGGTCGGTGCTCGTGGGGCTGTGGTGTTGGGTC GCCGTGTTCGCGAGGAAGGTGGGCAGGATCGCGAGGGAGGACCCACGGCGGGTGGCGCACTCGCTCAAGGTCGGCCTGGCGCTCACCCTGGTGTCCGTCCTCTACTACGTCACGCCGCTCTTCAAGGGCTTCGGGGTCTCCACGATGTGGGCCGTGCTCACCGTCGTCGTCGTCATGGAGTACACCGTCGGTGGCACGCTGAGCAAAGGCTTGAATAGAGCTTTCGCGACGCTGGTGGCTGGGTTCATCGCCGTGGGAGCTCATCAGGTAGCTAACCGCTGTGGTGCACAAGGGGAGCCCATACTGCTCGCCATCTTCGTCTTCTTCCTAGCGTCGGCGGCGACGTTCTCGCGCTTCATCCCGGAGATCAAGGCGAGGTACGACTACGGCGTGACCATCTTCATACTCACCTTCAGCCTGGTGGCCGTGTCCAGCTACCGCGTGGAGGAGCTCATTCAGCTGGCGCACCAGCGCTTCTCCACCATCGTCATCGGCGTCCTTACCTGCCTCTGCACCACCATCTTCGTCTTCCCCGTCTGGGCCGGCGAGGATCTCCACAAGCTCACCGCCGCCAACCTCGATAAGCTGGCGCAGTTTCTTCAAGGATTGGAATCCGAATGTTTTGGAGAGAAGGCTGCTAGCGAGAATTTGGAGGACAAAGCCTTTCTGCAAGTGTACAAGAGCGTCCTCAACTCCAAGGCCAGTGAGGACTCTCTGAGCAATTTTGCCAAGTGGGAGCCCGGTCATGGCAAATTCGGCTTCCGGCACCCATGGAGCCAATACCAGAAGCTCGGAGCTCTTTGTCGTCAGTGCGCATCTTCAATGGAGGCTCTTGCTTCCTATGTCATCACACTGCAAAAATCCCAGTACCCTGAGGCCAATCCAGAGCTAACCTTCAAGGTCCGAATGGCGTGTGGGGAAATGAGCTCACACTCGGCCAAGGCGCTCAAGGACCTATCAACAGCCATTCGGACAATGATAGTACCATCTCCAGCCAACATCACCATGTCCTCAGCCATCAAAGTTGCAAAAGACCTCAGGAATGAATTATCAGAGGACGCAGCTGTGTTGCAAGTGATGCATGTGGCTGTTACTGCCACACTTATCTCAGACTTGGTTACGACAATAGTGAAAATTGCAGAAACTACTGACAACCTAGCACGGCTTGGCCACTTCAAGAACCCTGAAAAAACTCAGAAAGATGTCGCTATCAACATTGCGAGTTGA
实施例1、转基因烟草中HVUL2H37793.2基因的表达
1.方法
1.1转基因烟草的构建
①将含有目标基因(基因序列如SEQ ID NO.1所示)的瞬时表达载体(瞬时表达载体pEAQ，来自John Innes Centre)转化农杆菌(EHA105)；
②挑取阳性农杆菌克隆于500ul含有相应抗生素(kn)的LB中，培养20-24小时；
③转接200ul于5ml含有相应抗生素(kn)的LB中，28℃摇床220rpm至OD＝2.0左右。
④10000rpm常温离心2min收集菌体，用提前配制转化缓冲液进行菌体的重悬，摇床震荡3h；缓冲液工作液成分以及浓度如下：10mM MES(pH5.7)，10mM MgCl2，100μUDP-葡萄糖。
⑤拿准备好的1ml的注射器，去掉针头，选取出口光滑的注射器吸入菌液，取1月龄的本氏烟草(Nicotiana benthamiana)，用手按住叶片，从叶片反面注射，使农杆菌渗透进去。给每株打过的烟草做好标记，可在叶片上圈出农杆菌渗透的区域，并选取转化缓冲液打烟草做对照。
⑥注射了农杆菌的烟草黑暗培养24h，然后移至烟草培养箱中光照培养24-48小时即可取样(注意打过的烟草不可直接在叶片上喷洒水)。
1.2产物收集和纯化
剪取农杆菌渗透区域的叶片，放在已称重的装有钢珠的EP管中，做好标记，迅速放置液氮中，进行冻干。冻干后的样本，利用研磨仪(MM 400,Retsch)在30Hz条件下研磨60s，将研磨好的样本粉末装入2mlEP管内。用电子天平称取每个EP管的重量并记录；将已研磨好的样本取适量(范围30-60mg)于EP管中，称量并记录，算出所有EP管中样本的净重。已知每份样本的净重，按体积V＝样本净重(mg)*12μL/mg在4℃冰上操作加入70％MeOH溶液。混匀，涡旋15s，每隔半小时涡旋一次，共涡旋4次，放4℃冰箱内提取12h以上。后离心。先将离心机开机预冷到4℃，设置时间10min和转速12000rpm，将样本涡旋后放入离心，使用离心机注意对称平衡，离心后吸取上清液。将上清液用微孔滤膜(0.22μm pore size)过滤，装入上样瓶中，准备LC-MS检测。
1.3目的产物检测
将装有样本提取液的进样瓶放入自动进样器内的样品盘，并记录每个进样瓶编号所对应的进样孔位置。同时打开软件Analyst Software，双击Hardware Configuration，选择LCMS-V(有切换阀模式)，点击Activate Profile，并选择Acquire Mode模式，点击Acquire，点击图上方的Equilibrate键，一般设定时间为3min，此操作目的是预热仪器，使高压输液泵、色谱柱、柱温箱、离子源温度等达到方法中设置的条件。待各仪器部件状态Ready后，功能区Start Sample键成为可点击状态，此时表明仪器正常，分析条件正常，然后点击Start Sample开始跑样，首次跑样前先提交4针空白样。
通过RT-PCR技术鉴定HVUL2H16977.2的表达，所用的引物为：
F：tatccagtcactatggtcgacatggaggttgatcaccgca(SEQ ID NO.2)
R：gatattattgacacgcccgggtcaactcgcaatgttgatagcga(SEQ ID NO.3)
2.结果
实验结果如图1所示，从结果中可以看到，本发明将基因HVUL2H37793.2转移到烟草中，使得烟草植株可以表达苹果酸过氧化物酶，并诱导烟草积累苹果酸。
实施例2、烟草瞬时表达的研究
1、烟草瞬时表达
①将含有目标基因(基因序列如SEQ ID NO.1所示)的瞬时表达载体(瞬时表达载体pEAQ，来自John Innes Centre)转化农杆菌(EHA105)；
②挑取阳性农杆菌克隆于500μl含有相应抗生素(kn)的LB中，培养20-24小时；
③转接200μl于5ml含有相应抗生素(kn)的LB中，28℃摇床220rpm至OD600＝1.0左右。
④10,000rpm常温离心15min收集菌体，用提前配制转化缓冲液进行菌体的重悬至OD600＝1.0，摇床震荡3h；转化缓冲液含10mmol/L MgCl2，10mmol/L MES，150μmol/L乙酰丁香酮，pH＝5.6；
⑤拿准备好的1ml的注射器，去掉针头，选取出口光滑的注射器吸入菌液，取1月龄的本氏烟草(Nicotiana benthamiana)，用手按住叶片，从叶片反面注射，使农杆菌渗透进去。给每株打过的烟草做好标记，可在叶片上圈出农杆菌渗透的区域，并选取转化缓冲液打烟草做对照。
⑥注射了农杆菌的烟草黑暗培养24h，然后移至烟草培养箱中光照培养24-48小时即可取样(注意打过的烟草不可直接在叶片上喷洒水)。
2、产物收集和纯化
剪取农杆菌渗透区域的叶片，放在已称重的装有钢珠的EP管中，做好标记，迅速放置液氮中，进行冻干。冻干后的样本，利用研磨仪(MM 400,Retsch)在30Hz条件下研磨60s，将研磨好的样本粉末装入2ml EP管内。用电子天平称取每个EP管的重量并记录；将已研磨好的样本取适量(范围30-60mg)于EP管中，称量并记录，算出所有EP管中样本的净重。已知每份样本的净重，按体积V＝样本净重(mg)*12μL/mg在4℃冰上操作加入70％MeOH溶液。混匀，涡旋15s，每隔半小时涡旋一次，共涡旋4次，放4℃冰箱内提取12h以上。后离心。先将离心机开机预冷到4℃，设置时间10min和转速12000rpm，将样本涡旋后放入离心，使用离心机注意对称平衡，离心后吸取上清液。将上清液用微孔滤膜(0.22μm pore size)过滤，装入上样瓶中，准备LC-MS检测。
3、目的产物检测
将装有样本提取液的进样瓶放入自动进样器内的样品盘，并记录每个进样瓶编号所对应的进样孔位置。同时打开软件Analyst Software，双击Hardware Configuration，选择LCMS-V(有切换阀模式)，点击Activate Profile，并选择Acquire Mode模式，点击Acquire，点击图上方的Equilibrate键，一般设定时间为3min，此操作目的是预热仪器，使高压输液泵、色谱柱、柱温箱、离子源温度等达到方法中设置的条件。待各仪器部件状态Ready后，功能区Start Sample键成为可点击状态，此时表明仪器正常，分析条件正常，然后点击Start Sample开始跑样，首次跑样前先提交4针空白样。
瞬时表达的的烟草取三个样本，分别为OX-1、OX-2和OX-3；野生型(CK)本氏烟草作为对照。
4、结果
实验结果说明(图2A)：本发明将基因HVUL2H16977.2转移到烟草中，使得烟草植株可以表达苹果酸过氧化物酶(与野生型烟草相比，表达量显著提高)，并诱导烟草积累苹果酸。制备得到的烟草耐盐碱能力得到提高，提高了烟草的应用价值。图2B通过对过表达的代谢物进行二级谱图分析，说明了显著富集的化合物就是苹果酸。
实施例3、转基因烟草的构建
HVUL2H37793.2转基因烟草的获得方法如下：
首先，构建包含HVUL2H37793.2基因的重组表达载体pCXSN：HVUL2H37793.2。其次，利用农杆菌转化法将前述重组表达载体转化入本氏烟草(WT)中，将浸泡过农杆菌液的外植体叶片转入培养基(MS+As)上进行暗培养3d，再更换分化培养基进行培养至长出不定芽1-2cm，后转入生长培养基。最后，将获得的T0代种子播种于1/2MS(含有25mg·L-1Hyg)的培养基筛选。在潮霉素筛选下观察烟草的生长情况，选取转基因阳性种子进行种植，通过RT-PCR技术鉴定HVUL2H37793.2在T1代转基因烟草中的表达水平，从而获得阳性T2代转基因种子。
以下通过试验例的方式来说明本发明的有益效果。
试验例1、HVUL2H37793.2基因与耐盐碱胁迫相关的研究
1.方法
本发明主要使用的植物材料包括本氏烟草(Nicotiana benthamiana)。
种子萌发试验：将按照实施例3所述方法制备的转基因烟草的T2代种子(三个样本：OE1、OE2和OE3)和野生型(WT)本氏烟草种子用10％的84消毒液进行灭菌2～3次，用无菌水清洗后分别均匀播种在含有0、100和150mM氯化钠的培养基上。在温室(室温25℃，连续光照)中培养2周后，统计三次生物学重复的种子发芽率。
根长实验统计：野生型(WT)本氏烟草种子和按照实施例3所述方法制备的转基因烟草的T2代种子(三个样本：OE1、OE2和OE3)在MS培养基中分别培养2周后，再转移到含有氯化钠(0、100和150mM)的培养基上，10天后用游标卡尺测定幼苗的主根长度。
2.结果
如图3所示。在烟草中构建HVUL2H37793.2的转基因过表达植株。随后进行了种子萌发试验和根系伸长试验来评估过表达株系对盐胁迫处理的反应。结果表明，过表达株系和野生型株系在正常条件下，萌发率和根长没有明显的差异，在盐胁迫处理下(100和150mM)下，过表达株系的萌发率和根长均显著高于野生型，表明过表达HVUL2H37793.2的烟草对盐胁迫的抗性。说明HVUL2H37793.2基因与耐盐碱胁迫相关，可以提高植物的耐盐碱能力。
综上，本发明研究发现在盐碱胁迫后，HVUL2H37793.2基因可显著调控青稞抗性品种中苹果酸的积累水平从而提高青稞的耐盐碱能力。将基因HVUL2H37793.2在烟草中进行过表达后，烟草叶片中苹果酸的含量显著增加，烟草耐盐碱能力提高。因此，HVUL2H37793.2基因在植物抵御盐碱胁迫中具有重要的意义。
Claims (10)
1.一种基因片段，其特征在于：所述基因片段的核苷酸序列如SEQ ID NO.1所示。
2.一种重组载体，其特征在于：所述重组载体包含核苷酸序列如SEQ ID NO.1所示的基因片段。
3.根据权利要求2所述的重组载体，其特征在于：所述重组载体是重组pEAQ或重组pCXSN。
4.一种重组菌，其特征在于：所述重组菌包含权利要求2或3所述的重组载体。
5.根据权利要求4所述的重组菌，其特征在于：所述重组菌为转化农杆菌。
6.一种苹果酸过氧化物酶，其特征在于：所述苹果酸过氧化物酶是序列如SEQ ID NO.1所示的基因片段表达而得。
7.权利要求1所述的基因片段、权利要求2或3所述的重组载体、权利要求4或5所述的重组菌或权利要求6所述的苹果酸过氧化物酶在制备耐盐碱的植物的用途；
优选地，所述植物为青稞、烟草。
8.一种表达苹果酸过氧化物酶的转基因植物的构建方法，其特征在于：取核苷酸序列如SEQ ID NO.1所示的基因片段，转入植物中，获得表达苹果酸过氧化物酶的植株，即可。
9.根据权利要求8所述的构建方法，其特征在于：所述转基因植物为转基因烟草。
10.根据权利要求8所述的构建方法，其特征在于：所述转入植物的方法是农杆菌法、基因枪法、电转法、PEG介导法、脂质体法和磷酸钙-DNA共沉淀法中的一种。
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979551A (zh) * | 2010-10-09 | 2011-02-23 | 山东大学 | 玉米苹果酸脱氢酶基因启动子序列克隆和应用 |
| CN105039354A (zh) * | 2015-08-31 | 2015-11-11 | 哈尔滨师范大学 | 紫花苜蓿MsSOS3基因及其编码蛋白和应用 |
| CN109517827A (zh) * | 2018-10-25 | 2019-03-26 | 华中科技大学 | 二穗短柄草抗旱抗盐基因及其编码蛋白质与应用 |
| CN111304197A (zh) * | 2018-12-11 | 2020-06-19 | 东北农业大学 | 一种甜菜耐碱胁迫的长链非编码rna基因及其制备方法与应用 |
| CN112457386A (zh) * | 2021-01-19 | 2021-03-09 | 中国科学院遗传与发育生物学研究所 | 一种控制玉米穗长和行粒数相关的蛋白ead1及其编码基因与应用 |
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101979551A (zh) * | 2010-10-09 | 2011-02-23 | 山东大学 | 玉米苹果酸脱氢酶基因启动子序列克隆和应用 |
| CN105039354A (zh) * | 2015-08-31 | 2015-11-11 | 哈尔滨师范大学 | 紫花苜蓿MsSOS3基因及其编码蛋白和应用 |
| CN109517827A (zh) * | 2018-10-25 | 2019-03-26 | 华中科技大学 | 二穗短柄草抗旱抗盐基因及其编码蛋白质与应用 |
| CN111304197A (zh) * | 2018-12-11 | 2020-06-19 | 东北农业大学 | 一种甜菜耐碱胁迫的长链非编码rna基因及其制备方法与应用 |
| CN112457386A (zh) * | 2021-01-19 | 2021-03-09 | 中国科学院遗传与发育生物学研究所 | 一种控制玉米穗长和行粒数相关的蛋白ead1及其编码基因与应用 |
Non-Patent Citations (2)
| Title |
|---|
| EUKARYOTA等: "PREDICTED: Hordeum vulgare subsp. vulgare aluminum-activated ma(LOC123430267), mRNA NCBI Reference Sequence: XM_045114145.1", GENBANK, 12 November 2021 (2021-11-12) * |
| 巴桑玉珍: "青稞新光合作用相关基因的克隆和序列分析", 大麦与谷类科学, vol. 34, no. 6, 31 December 2017 (2017-12-31), pages 1 - 7 * |
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