CN116200412A - 小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1及其应用 - Google Patents
小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1及其应用 Download PDFInfo
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Abstract
本发明公开了一种小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1,所述基因cDNA的核苷酸序列如SEQ ID No.1所示。本发明还公开了含有所述基因TaPP2C.D1的植物表达载体pGA3426‑TaPP2C.D1或pTCK303‑TaPP2C.D1以及所述基因和植物表达载体在培育抗碱植物中的应用。经过实验比较分析证明,本发明所述转基因TaPP2C.D1干涉促进了小麦对碱胁迫的抗性,转基因植株的耐碱能力明显提高。
Description
技术领域
本发明属于生物基因工程技术领域,尤其涉及耐碱基因——小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1及其应用。
背景技术
土壤盐碱化严重影响农作物产量。特别是随着工业的发展,土壤盐碱化愈来愈严重,已成为一个全球关注的社会问题。我国人口众多,而土壤盐碱化更为严重,已经成为制约我国经济和社会发展的重要因素。因此,除了缓解土壤盐碱化以外,培育耐盐碱农作物新品种已成为当前一个十分迫切的任务。
利用转基因改良植物技术将新的性状转入高生物量植物中,以此开发高效的转基因植物新品种并用于在盐碱地种植,是一项具有广阔应用前景的技术。
利用基因工程技术开展植物耐碱方面研究已取得了较大的进展,克隆了大量相关基因,并将这些基因转入植物中,用于耐碱机制研究。一些实验表明,将植物本身以及其它生物中与耐碱相关的基因转入植物中,其异源转录和翻译产物可以使转基因植物的抗碱能力得到提高。
目前,已发现了一些能显著提高植物耐碱能力的基因,但检索发现小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1及其在作物中调控小麦耐碱性未见报道。
发明内容
针对现有技术的不足,本发明的目的是提供一种调控小麦在碱胁迫下生长的基因——小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1及其应用。
本发明所述的小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1,其特征在于:所述基因cDNA的核苷酸序列如SEQ ID No.1所示。
本发明还提供了含上述基因TaPP2C.D1的植物表达载体,其特征在于:所述植物表达载体是pGA3426-TaPP2C.D1或pTCK303-TaPP2C.D1。
本发明的技术方案在于从小麦中分离得到小麦基因TaPP2C.D1,然后将该基因转化到普通小麦YM20中以实现研究TaPP2C.D1基因的功能以及植物的碱胁迫响应机理。
本发明所述小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1在培育抗碱植物中的应用。
本发明所述植物表达载体pGA3426-TaPP2C.D1或pTCK303-TaPP2C.D1在培育抗碱植物中的应用。
其中:所述植物优选是普通小麦。
将本发明所述小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1在植物细胞中干扰表达,植物就可以获得碱胁迫的耐受能力。为了便于对转基因植物或细胞系进行筛选,可以对含有所述基因TaPP2C.D1的植物表达载体(pGA3426-TaPP2C.D1或pTCK303-TaPP2C.D1)进行加工,如可以加入选择标记(GUS等)或者具有抗性的抗生素标记物(潮霉素,卡那霉素,庆大霉素等)等。
事实上,任何一种可以将外源基因导入植物中表达的载体都可以应用,本发明优选的载体是pTCK303。
本发明提供了一种小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1,所述基因可广泛用于培育耐碱农作物品种。本发明的有益效果是:利用现有的植物基因工程技术,本发明克隆得到了小麦碱胁迫应答基因TaPP2C.D1,并通过根瘤农杆菌介导的方法将该基因在普通小麦中干扰表达,经过实验比较分析证明,TaPP2C.D1干涉促进了小麦对碱胁迫的抗性(见图3),转基因植株的耐碱能力明显提高。
附图说明
图1山融4号和济南177碱胁迫TaPP2C.D1的RT-PCR分析。
图2TaPP2C.D1转基因小麦表达量鉴定。
图3TaPP2C.D1转基因小麦碱胁迫下的表型。
其中:(A)TaPP2C.D1转基因小麦碱胁迫下的表型;(B)TaPP2C.D1-OE碱胁迫下的鲜重统计;(C)TaPP2C.D1-OE碱胁迫下与对照的相对鲜重;(D)TaPP2C.D1-RNAi碱胁迫下的鲜重统计;(E)TaPP2C.D1-RNAi碱胁迫下与对照的相对鲜重。
具体实施方式
下面结合附图和具体实施例对本发明内容进行详细说明。如下所述例子仅是本发明的较佳实施方式而已,应该说明的是,下述说明仅仅是为了解释本发明,并非对本发明作任何形式上的限制,凡是依据本发明的技术实质对实施方式所做的任何简单修改,等同变化与修饰,均属于本发明技术方案的范围内。
下述实施例中,所使用的材料、试剂、载体、菌株等,如无特殊说明,均从商业途径得到。
实施例1、TaPP2C.D1的克隆
1.1提取小麦Total RNA
1.将组织材料与钢株放入2.0的EP管中,液氮速冻并用组织研磨机研磨成粉末;
2.待液氮挥发干,每100mg材料约加入1ml的Invitrogen公司的TRIzol提取液,融化后,用加样枪反复吸吹,剧烈振荡混匀样品,使样品充分裂解,室温放置5分钟;
3.加入0.2ml氯仿(chloroform),剧烈振荡混匀15秒,室温放置10分钟;
4.4℃,12000rpm离心15分钟;
5.用移液器小心吸出上层水相,加入新的1.5ml的离心管中,加入500μl的异丙醇(1:1体积),充分混匀,-20℃,沉淀30min或过夜;
6.4℃,12000rpm离心10min,小心弃去上清液;
7.RNA沉淀用1ml的75%乙醇洗涤;4℃,12000rpm离心10min收集沉淀;
8.重复用75%乙醇洗涤一次RNA沉淀;
9.去上清,RNA沉淀于无菌操作台上晾干约10-15分钟,RNA略显透明,加入适当体积(30-50μl)的RNase-free水充分溶解(可放于-80℃长期保存);
10.紫外分光光度计及1%Agrose凝胶电泳检测RNA浓度及质量。
注:a)用紫外分光光度计检测RNA的产量,在260nm处的吸光度,1OD=40μg/ml。根据在260nm和280nm处的吸光值,检测RNA的纯度,纯RNA的OD260/OD280比值应接近2.0(比值最好在1.9~2.1之间)。
b)用1%Agrose凝胶电泳检侧RNA的质量及大小。吸取1μl RNA加入3μl的RNase-free水,加1μl上样缓冲液65℃变性5分钟。电泳后用EB染色,另取3μl的1kb DNAMarker作为对照。
1.2cDNA反转录
反转录酶:M-MLV Reverse Transcriptase(Invitrogen)。
1.12μl体系:
Oligo(dT) 1μl
Total RNA 100ng-5μg
dNTP 1μl
DEPC水补至12μl
2.65℃变性5min,迅速插入冰中,然后依次加入:
5×First-Strand Buffer 4μl
0.1M DTT 2μl
RNaseOUT(Invitrogen) 1μl
3.轻轻混匀,37℃反应2min;
4.加入1μl M-MLV RT,混匀,37℃反应50min;
5.70℃温育15min使M-MLV RT失活;
6.加入1μl RNase H(Invitrogen),37℃反应20min;
7.用超纯水稀释至合适浓度,作为PCR模板。
1.3开放阅读框的克隆和序列测定
1.引物序列:根据测序结果,设计基因上下游引物TaPP2C.D1-F、TaPP2C.D1-R,扩增基因的开放阅读框。
TaPP2C.D1-F:5’-ATGGCGGCGGTGATGGACTA-3’
TaPP2C.D1-R:5’-TCAGTAGGTGCTGCTCACCAT-3’
2.PCR反应体系(50μl):
3.PCR反应程序为:94℃预变性5min;94℃变性45sec,55℃复性45sec,72℃延伸1.5min,循环35次;72℃延伸7min。
4.扩增片段回收后与pEASY-T1载体连接并转化大肠杆菌Trans1 T1,测序由青岛擎科公司完成。
1.4基因表达分析(RT-PCR和real-time PCR)
a.胁迫下RNA的提取
山融4号和济南177种子正常萌发,Hangload培养液培养至株高约10cm时(约2周时间),开始施加碱性盐胁迫100mM NaHCO3:Na2CO3=9:1。分别在处理后的0、1、12、24,48小时取幼嫩的叶片和根系提取RNA。
b.反转录(RT)产生cDNA
反转录产生cDNA,方法同上。
c.PCR反应及电泳
1.以cDNA为模板,进行PCR反应。引物如下:
TaPP2C.D1-RT-F:5’-CTGAACCTGCGATTGCTG-3’
TaPP2C.D1-RT-R:5’-GATCTTCTTCAGGTCCGAG-3’
2.PCR体系:
ddH2O 13.5μl
10×Taq buffer(Mg2+free)2μl
MgCl2(25mM)1.2μl
TaPP2C.D1-RT-F(10μM)1μl
TaPP2C.D1-RT-R(10μM)1μl
dNTP(10mM each)0.2μl
rTaq polymerase(5U/μl)0.1μl
逆转录cDNA模板 1μl
Total Volume 20μl
3.PCR程序:
95℃5min,25~30cycles 95℃20s,57℃60s,72℃60s;72℃7min。
根据内参Actin的扩增情况确定PCR的循环数,调整cDNA模板的加入量。
结果见图1。
实施例2、植物表达载体的构建(Ubi启动子)
2.1Ubi启动子植物表达载体的构建
利用植物表达载体pGA3426,选用KpnⅠ和HindⅢ分别对pGA3426和含有目的基因的pEASY-T1载体进行双酶切,分别回收载体大片段和目的基因小片段,用T4 DNA连接酶连接后转化大肠杆菌Trans1 T1感受态细胞,鉴定重组子后即得到带有目的基因的植物表达载体pGA3426-TaPP2C.D1。
(1)质粒pGA3426空载体和pEASY-T1的KpnⅠ和HindⅢ双酶切
碱裂解法提取pGA3426空载体和pEASY-T1质粒,各取10μg酶切,酶切体系如下:
KpnⅠ1μl
HindⅢ1μl
pGA3426载体/pEASY-T1质粒 1~2μl
10×Buffer K 1μl
ddH2O补充至20μl
于30℃恒温水浴锅酶切2小时以上。双酶切后以1×TAE为电泳缓冲液,将酶切产物进行1%琼脂糖凝胶电泳。在紫外透射仪下用洁净刀片切下pGA3426中11kb的载体大片段和pEASY-T1中大约1kb的目的基因条带,回收该条带。
(2)经酶切和脱磷的pGA3426载体片段(约11kb)和pEASY-T1双酶切回收片段(约1kb)以摩尔比1:4的比例进行16℃连接过夜。
(3)连接产物热激法转化大肠杆菌Trans1 T1感受态细胞,转化菌在含Kan 50μg/ml的LB固体平板上37℃培养16小时左右。
(4)重组子的鉴定
①质粒的PCR验证
挑取单菌落分别接种于5ml含Kan的LB液体培养基中37℃振荡培养过夜,碱变性法提取质粒,用基因特异引物(TaPP2C.D1-F,TaPP2C.D1-R)进行PCR扩增,引物如下:
TaPP2C.D1-F:5’-ATGGCGGCGGTGATGGACTA-3’
TaPP2C.D1-R:5’-TCAGTAGGTGCTGCTCACCAT-3’
体系如下:
PCR反应条件如下:预变性94℃3min,35个循环为:94℃30sec,55℃30sec,72℃1min,最后,72℃延伸10min。PCR产物用1.0%琼脂糖凝胶电泳鉴定。
②质粒酶切鉴定
提质粒进行KpnⅠ和HindⅢ双酶切,酶切体系同上。
1%琼脂糖凝胶电泳,检测是否含有预期分子量大小的片段,验证载体的正确构建。
实施例3、农杆菌感受态的制备与转化
3.1农杆菌EHA105感受态的制备
(1)从YEP平板(含50μg/ml利福平)上挑取根癌农杆菌单菌落,接种于含50μg/ml利福平的YEP液体培养基中,200rpm/min,28℃培养过夜。
(2)取2ml过夜培养液接种于50ml含相同抗生素的YEP液体培养基中在相同条件下培养至OD600达0.5。
(3)菌液冰浴30min,4℃,5000rpm离心10min,收集菌体。
(4)将菌体重悬于冰浴的10ml 0.15mol/L的NaCl中,离心收集菌体。
(5)再悬浮于1ml 20mmol/L冰预冷的CaCl2溶液中,以200μl/管将菌液分装在1.5ml Eppendorf管中,置液氮中速冻1min,-70℃保存备用。
3.2冻融法转化根癌农杆菌EHA105
(1)在室温下融化农杆菌感受态细胞,加入1μg表达载体质粒DNA,混匀后冰浴30min。
(2)置液氮速冻1min,迅速移至37℃保温3min。
(3)加入无抗生素的YEP 800μl,28℃震荡培养3小时。
(4)7000rpm离心30s收集菌体,涂于含50μg/ml利福平、50μg/ml Kan的YEP平板上,28℃倒置暗培养2-3天。
3.3菌体PCR鉴定
实施例4、转基因功能验证-小麦转化及筛选
小麦转化
(1)挑选饱满的YM20种子,70%乙醇中浸泡5min,然后清洁剂(20%漂水(白猫,上海),0.1% Triton)洗涤10-15min,无菌水冲洗4次,并浸泡于无菌水中过夜,将泡发的YM20种子转移至垫有浸湿滤纸的无菌培养皿中暗培养3天。
(2)转化前一天,取2ml活化的农杆菌EHA105加到含相应抗生素的200ml YEP培养基中,过夜培养至OD600=1.0-1.2。
(3)离心收集菌体,并重悬于浸染液中,使OD600=0.8。
(4)采用茎尖法进行小麦转化,用解剖刀切开小麦嫩芽,暴露其生长点并浸泡在侵染液中抽真空15min并保持压力15min。
(5)待抽真空完毕后倒掉侵染液并沥干侵染液,将小麦幼苗平铺于垫有浸湿滤纸的无菌培养皿中暗培养3天。
(6)暗处理完成后将小麦幼芽转移到培养土中正常生长。
(7)将正常生长的小麦幼苗取叶片于填装钢株的2.0EP管中,采用CTAB法提取小麦全基因组DNA,并采用TaPP2C.D1特异性引物(Ubi-F,TaPP2C.D1-R)鉴定小麦转基因事件。引物如下:
Ubi-F:5’-GCCCTGCCTTCATACGCT-3’
TaPP2C.D1-R:5’-TCAGTAGGTGCTGCTCACCAT-3’
(8)提取小麦Total RNA并用TaPP2C.D1引物(TaPP2C.D1-RT-F、TaPP2C.D1-RT-R)鉴定转基因小麦植株表达量变化。引物如下:
TaPP2C.D1-RT-F:5’-CTGAACCTGCGATTGCTG-3’
TaPP2C.D1-RT-R:5’-GATCTTCTTCAGGTCCGAG-3’
(9)将转基因小麦种子与对照YM20种子泡发过夜,平铺于浸湿的滤纸上,待种子萌发后转移到锥形瓶或花盆中。
(10)待部分锥形瓶中的TaPP2C.D1-OE、TaPP2C.D1-RNAi及YM20生长至一叶一心时施加100mM NaHCO3:Na2CO3=9:1,处理10天,剩余TaPP2C.D1-OE、TaPP2C.D1-RNAi及YM20正常培养用作对照。
(11)将鲜重作为表型标准,正常生长时TaPP2C.D1-OE鲜重低于YM20,TaPP2C.D1-RNAi鲜重高于YM20,而在100mM NaHCO3:Na2CO3=9:1处理时TaPP2C.D1-OE鲜重低于YM20,而TaPP2C.D1-RNAi鲜重较YM20更高,通过比较处理与未处理的相对鲜重表明TaPP2C.D1过表达不利于小麦对碱胁迫下生存,而TaPP2C.D1干涉促进小麦对碱胁迫的抗性。
结果见图2、图3。
Claims (5)
1.一种小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1,其特征在于:所述基因cDNA的核苷酸序列如SEQ ID No.1所示。
2.一种含有权利要求1所述基因TaPP2C.D1的植物表达载体,其特征在于:所述植物表达载体是pGA3426-TaPP2C.D1或pTCK303-TaPP2C.D1。
3.权利要求1所述小麦耐碱D型蛋白磷酸酶基因TaPP2C.D1在培育抗碱植物中的应用。
4.权利要求2所述植物表达载体pGA3426-TaPP2C.D1或pTCK303-TaPP2C.D1在培育抗碱植物中的应用。
5.根据权利要求3或4所述的应用,其特征在于:所述植物是普通小麦。
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Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2008201714A1 (en) * | 2001-09-05 | 2008-05-08 | Basf Plant Science Gmbh | Protein phosphatase stress-related polypeptides and methods of use in plants |
CN104498508A (zh) * | 2015-01-13 | 2015-04-08 | 山东大学 | 小麦渐渗系应答非生物胁迫调控基因TaGBF及其应用 |
CN107475270A (zh) * | 2017-09-08 | 2017-12-15 | 云南农业大学 | 甘蔗细茎野生种中干旱胁迫表达的2C型蛋白磷酸酶基因ScPP2C |
CN107746851A (zh) * | 2017-10-30 | 2018-03-02 | 齐齐哈尔大学 | 砂藓蛋白磷酸酶2C基因RcPP2C及其编码蛋白和应用 |
CN114574509A (zh) * | 2022-03-11 | 2022-06-03 | 河北省农林科学院粮油作物研究所 | 一种提高小麦抗旱性的基因TaPP2C59.2及其应用 |
-
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- 2022-12-12 CN CN202211590406.0A patent/CN116200412B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
AU2008201714A1 (en) * | 2001-09-05 | 2008-05-08 | Basf Plant Science Gmbh | Protein phosphatase stress-related polypeptides and methods of use in plants |
CN104498508A (zh) * | 2015-01-13 | 2015-04-08 | 山东大学 | 小麦渐渗系应答非生物胁迫调控基因TaGBF及其应用 |
CN107475270A (zh) * | 2017-09-08 | 2017-12-15 | 云南农业大学 | 甘蔗细茎野生种中干旱胁迫表达的2C型蛋白磷酸酶基因ScPP2C |
CN107746851A (zh) * | 2017-10-30 | 2018-03-02 | 齐齐哈尔大学 | 砂藓蛋白磷酸酶2C基因RcPP2C及其编码蛋白和应用 |
CN114574509A (zh) * | 2022-03-11 | 2022-06-03 | 河北省农林科学院粮油作物研究所 | 一种提高小麦抗旱性的基因TaPP2C59.2及其应用 |
Non-Patent Citations (1)
Title |
---|
NCBI: "PREDICTED: Triticum aestivum probable protein phosphatase 2C 28 (LOC123105866), transcript variant X2, mRNA", NCBI, 25 October 2021 (2021-10-25), pages 044528031 * |
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