CN104762305A - 与小麦抗旱相关的基因TaUreG及其应用 - Google Patents
与小麦抗旱相关的基因TaUreG及其应用 Download PDFInfo
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- CN104762305A CN104762305A CN201510207339.3A CN201510207339A CN104762305A CN 104762305 A CN104762305 A CN 104762305A CN 201510207339 A CN201510207339 A CN 201510207339A CN 104762305 A CN104762305 A CN 104762305A
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Abstract
本发明公开了一种与小麦抗旱相关的基因TaUreG,其具有SEQ ID NO:1所示的cDNA核苷酸序列或SEQ ID NO:2所示的gDNA核苷酸序列。本发明应用了植物基因工程技术,首次从小麦中克隆了与小麦抗旱关联的基因TaUreG,通过实时荧光定量PCR检测,结果表明,所述基因TaUreG的表达受到干旱胁迫的调节,能够在小麦适应干旱胁迫的早期就起到重要作用;而且所述基因TaUreG的表达同样受ABA和H2O2的调节,说明TaUreG应该通过ABA依赖的信号转导途径参与了小麦对干旱胁迫的响应,并且参与了干旱胁迫引起的ROS水平升高的信号转导通路。因此,在培育小麦品种时,含有该基因的小麦植株具有较强的抗旱能力,这对筛选抗旱小麦品种以及培育具有高抗旱性的转基因小麦,提高小麦在干旱环境下的种植产量具有重大的意义。
Description
技术领域
本发明涉及生物基因工程技术领域,具体地说是一种与小麦抗旱相关基因TaUreG及其应用。
背景技术
目前,全球性气候变暖导致土壤干旱程度越来越严重,这对粮食生产构成直接威胁。小麦是世界上最主要的粮食作物之一,也是我国第二大粮食作物。植物在应对干旱胁迫时会产生一系列的保护机制,研究小麦抗旱的分子机制,培养抗旱小麦新品种是提高小麦产量的一个主要途径,对于保障国家粮食安全和水资源安全具有重要意义。
植物在干旱胁迫下产生一系列的基础反应,包括生长和代谢水平的抑制、基因表达水平的变化、体内激素水平的变化、诱导和抑制信号转导途径的产生、可溶性渗透保护剂的积累、活性氧簇(ROS)水平升高引起的质膜氧化等。干旱信号主要通过脱落酸(ABA)依赖的以及ABA不依赖的DREB转录因子介导的信号转导途径调节下游基因的表达(Yoshida et al. ABA-dependent
and ABA-independent signaling in response to osmotic stress in plants. 2014)。研究表明,植物在应对干旱胁迫时会产生一系列的保护机制;干旱胁迫能诱导许多植物基因的表达,有些基因的表达产物可直接增强胁迫耐性,有些可以感应和转导胁迫信号调控基因表达。近年来,随着基因组学和分子生物学的发展,中外学者从小麦中克隆了一批抗旱相关的基因,如2011年Liu et al.报道的Expression of a wheat MYB gene in transgenic tobacco enhances
resistance to Ralstonia solanacearum,
and to drought and salt stresses;2012年Niu et al.报道的Wheat WRKY
genes TaWRKY2 and TaWRKY19 regulate abiotic
stress tolerance in transgenic Arabidopsis plants;2012年Qin et al.报道的Over-expression
of TaMYB33 encoding a novel wheat MYB
transcription factor increases salt and drought tolerance in Arabidopsis;2012年Tang et al.公开的Molecular characterization of novel TaNAC genes in wheat and overexpression of TaNAC2a
confers drought tolerance in tobacco;这些均为筛选抗旱小麦品种、提高在干旱环境下小麦种植产量奠定了坚实的基础。
发明内容
本发明的目的是提供一种与小麦抗旱相关基因TaUreG及其应用。
一种与小麦抗旱相关的基因TaUreG,其具有SEQ ID NO : 1所示的cDNA核苷酸序列或SEQ ID NO : 2所示的gDNA核苷酸序列。
本发明所述gDNA包含7个外显子和6个内含子。
一种与小麦抗旱相关的基因TaUreG,该基因的编码蛋白如SEQ ID NO : 3所示。
本发明克隆所述基因TaUreG的上游引物TaUreG-F1的核苷酸序列如SEQ ID NO : 4所示、下游引物TaUreG-R1的核苷酸序列如SEQ ID NO : 5所示。
本发明所述基因位于小麦1A染色体上。
本发明提供的基因来源于小麦科农9204,根据序列结构特征,命名为TaUreG,其cDNA序列全长945 bp,包含一个长为855 bp的开放阅读框,编码284个氨基酸;gDAN序列全长2546 bp,由7个外显子和6个内含子组成。从5'端开始,外显子的长度依次为77 bp、204 bp、61 bp、202 bp、122 bp、105 bp、84 bp,内含子的长度依次为86 bp、600 bp、336 bp、345 bp、129 bp、105 bp,基因结构见图1。
本发明公开的抗旱相关的基因TaUreG通过实时荧光PCR检测分析时,其上游引物TaUreG-F2的核苷酸序列如SEQ ID NO :8所示、下游引物TaUreG-R2的核苷酸序列如SEQ ID NO : 9所示;内参上游引物TaActin-F2的核苷酸序列如SEQ ID NO :10所示、内参下游引物TaActin-R2的核苷酸序列如SEQ ID NO : 11所示,其扩增体系为:SYBR® Premix Ex
Taq II(2×)10 μl,Forward Primer(10 μM)0.8 μl,Reverse Primer(10 μM)0.8 μl,ROX Reference Dye II(50×)0.4 μl,cDNA模板2 μl,ddH2O 6 μl;共计20 μl;其PCR反应程序:95℃预变性30 s;95℃变性5 s,60℃退火并延伸34 s,40个循环。
本发明所用科农9204为审定品种,于2002年通过河北省农作物品种审定委员会审定;2003年通过全国品种审定,品种审定编号为国审麦2003037。
由于本发明公开的基因TaUreG具有抗旱性,故可将所述核苷酸序列如SEQ ID NO : 1或其编码蛋白质如SEQ ID NO.3所示的基因TaUreG转入小麦组织,从而培育成耐旱性小麦。
本发明应用了植物基因工程技术,首次从小麦中克隆了与小麦抗旱相关的基因TaUreG,通过实时荧光定量PCR检测,其结果表明,所述基因TaUreG的表达受到干旱胁迫的调节,能够在小麦适应干旱胁迫的早期就起到重要作用;而且所述基因TaUreG的表达同样受ABA和H2O2的调节,说明TaUreG是通过ABA依赖的信号转导途径参与了小麦对干旱胁迫的响应,并且参与了干旱胁迫引起的ROS水平升高的信号转导通路;TaUreG参与小麦抗旱机制的挖掘为该基因在小麦抗旱育种中的利用提供了更为可靠的证据。由此表明,含有TaUreG基因的小麦植株具有较强的抗旱能力,与小麦抗旱相关的基因TaUreG在选育抗旱小麦品种中可以得到应用;尤其是可以将所述核苷酸序列如SEQ ID NO:1所示的基因TaUreG转入小麦组织培育出具有高抗旱性的转基因小麦。
附图说明
图1为TaUreG基因结构图。方框代表外显子,线条代表内含子。ATG和TAG分别为起始密码子和终止密码子。
图2为利用中国春缺体-四体系对TaUreG进行染色体定位的结果。CS表示中国春;M表示marker;以TaActin(可扩增A,B,D组)为内参证明DNA的完整性。
图3为实时荧光定量PCR对TaUreG在ABA处理不同时间下表达特征的分析结果。以TaActin为内参。
图4为实时荧光定量PCR对TaUreG在H2O2处理不同时间下表达特征的分析结果。以TaActin为内参。
图5为实时荧光定量PCR对TaUreG在PEG-6000人工模拟干旱处理不同时间下表达特征的分析结果。以TaActin为内参。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。实施例中所用的试验材料、试剂等,如无特殊说明,均可从商业途径得到。以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
实施例1 小麦抗旱相关基因TaUreG的克隆
1、小麦TaUreG的cDNA序列的电子克隆
(1)以NCBI提交的水稻OsUreG(HM369059)基因序列为探针,搜索小麦EST数据库,将得到的序列用DNAMAN软件拼接,对开放阅读框(ORF)进行预测,以获取完整的小麦TaUreG cDNA序列;
(2)根据得到的全长cDNA序列用Primer5软件设计并筛选出一对特异引物TaUreG-F1和TaUreG-R1,分别用于小麦TaUreG cDNA和gDNA基因克隆。其上、下游引物分别为:
上游引物TaUreG-F1:5’-CCTTCCACTTCCAAGTCTGG-3’(SEQ ID NO:4)
下游引物TaUreG-R1:5’-CATACGTGAACACATGCCCG-3’(SEQ ID NO:5)。
2、利用Trizol(Invitrogen)提取总RNA,具体步骤如下:
(1)液氮研磨50-100 mg小麦叶片,研碎后转移到含1 ml Trizol试剂的EP管中,充分混匀,室温静置5 min;(2)加入0.2 ml氯仿,充分混匀,室温静置2-3 min;在4℃下,12000 × g离心15 min;(3)把上层无色的水相转移到一个洁净的1.5 ml EP管中,加入0.5 ml异丙醇,混匀后室温放置10 min;在4℃下,12000 × g离心10 min;(4)去上清,将沉淀用1 ml 75%乙醇清洗,在4℃下,7500 × g离心5 min,之后将沉淀风干;(5)将沉淀溶于适量RNase-free的去离子水中,60℃促溶10 min;即得所需总RNA;可微量分光光度计NanoDrop ND-2000 Spectrotometer定量检测,电泳分析RNA的完整性。
3、利用植物基因组DNA提取试剂盒(天根,DP305)提取DNA,具体步骤如下:
(1)取小麦叶片约100 mg,加入液氮充分碾磨;(2)将研磨好的粉末迅速转移到预先装有700 µl 65℃预热缓冲液GP1的离心管中(实验前在预热的GP1中加入巯基乙醇,使其终浓度为0.1%),迅速颠倒混匀后,将离心管放在65℃水浴20 min,水浴过程中颠倒离心管以混合样品数次;(3)加入700 µl氯仿,充分混匀,12000 rpm离心5 min;(4)小心地将上一步所得上层水相转入一个新的离心管中,加入700 µl缓冲液GP2,充分混匀;(5)将混匀的液体转入吸附柱CB3中,12000 rpm离心30 s,弃掉废液;(6)向吸附柱CB3中加入500 µl缓冲液GD,12000 rpm离心30 s,倒掉废液,将吸附柱CB3放入收集管中;(7)向吸附柱CB3中加入600 µl漂洗液PW,12000 rpm离心30 s,倒掉废液,将吸附柱CB3放入收集管中;(8)重复操作步骤(7);(9)将吸附柱CB3放回收集管中,12000
rpm离心2 min,倒掉废液,将吸附柱CB3置于室温放置数分钟,以彻底晾干吸附材料中残余的漂洗液;(10)将吸附柱CB3转入一个干净的离心管中,向吸附膜的中间部位悬空滴加50-200 μl洗脱缓冲液TE,室温放置2-5 min,12000 rpm离心2 min,将溶液收集到离心管中,即为所需的DNA。
4、利用GoldScript cDNA合成试剂盒(Invitrogen,c81401190)进行第一链cDNA合成,具体步骤如下:
(1)在0.2 ml离心管中依次加入步骤2提取的总RNA 5 µl(200 ng/µl)、10 mM dNTP mix 1 µl、Oligo dT Primer 1 µl、DEPC水3 µl;(2)将上述RNA/引物混合物放置在65℃孵育5 min,然后放置在冰上1-2
min;(3)在另一管中,配制2×反应混合液,按顺序加入下列组分:10×RT缓冲液 2 µl,25 mM
MgCl2 4 µl,0.1 M DTT 2 µl,重组RNase抑制剂(40U/ µl)1 µl;(4)向上述第(2)步的每个RNA/引物预混液中加入9 µl 2×反应预混液,轻柔混匀,7500 rpm离心30 s;(5)42℃孵育2 min;在每个管中加入1µl GoldScript RT;42℃孵育50 min;(6)70℃孵育15 min,终止反应,冰上冷却;(8)7500 rpm离心30 s,向每个管中加入1 µl RNaseH,37℃孵育20 min,-20℃保存。
5、聚合酶链式反应(PCR)反应进行序列扩增
用TaUreG-F1和TaUreG-R1引物分别对小麦cDNA和gDNA进行PCR扩增;
其PCR扩增体系为50 μl,包括:5×PrimeSTAR GXL Buffer 10 µl,dNTP Mixture 4 µl,上游引物TaUreG-F1(10 μM)2.5 μl,下游引物TaUreG-R1(10 μM)2.5 µl,模板cDNA或gDNA 2 µl,PrimeSTAR GXL DNA Polymerase(TaKaRa,R050Q)0.5 µl,ddH2O 28.5 µl。
其PCR扩增反应程序:98℃预变性2 min;98℃变性10 s,58℃退火15 s,68℃延伸3 min,30个循环;68℃延伸10 min;4℃保存。
6、将PCR扩增产物纯化回收,使用琼脂糖凝胶回收试剂盒(天根,DP209),具体步骤如下:
(1)柱平衡步骤:向吸附柱CA2中(吸附柱放入收集管中)加入500 μl平衡液BL,12000 rpm 离心1 min,倒掉收集管中的废液,将吸附柱重新放回收集管中;(2)将单一的目的DNA条带从琼脂糖凝胶中切下放入干净的离心管中,称取重量;(3)向胶块中加入等倍体积溶液PN,50℃水浴放置,其间不断温和地上下翻转离心管,以确保胶块充分溶解;(4)将上一步所得溶液加入一个吸附柱CA2中(吸附柱放入收集管中),室温放置2 min,12000 rpm离心30-60 s,倒掉收集管中的废液,将吸附柱CA2放入收集管中;(5)向吸附柱CA2中加入600 μl漂洗液PW,12000 rpm离心30-60 s,倒掉收集管中的废液,将吸附柱CA2放入收集管中;重复操作该步骤;(6)将吸附柱CA2放回收集管中,12000 rpm离心2 min,尽量除尽漂洗液;将吸附柱CA2置于室温放置数分钟,彻底地晾干;(7)将吸附柱CA2放到一个干净离心管中,向吸附膜中间位置悬空滴加适量洗脱缓冲液EB,室温放置2 min;12000 rpm离心2 min收集DNA溶液。
7、基因克隆
取4 μl PCR纯化回收后的产物,加入1 μl pEASY-Blunt
Cloning载体(全式金,CB101-01),轻轻混合,在温度为20℃-37℃下反应5 min,完成连接;将连接产物转化大肠杆菌DH5α菌株,在表面涂有8 μl IPTG(500 mM),40 μl X-gal(20 mg/ml)的卡那霉素(50 μg/ml)LB 平板37℃生长过夜;挑选白色菌落,通过快速PCR挑选阳性克隆测序。
8、小麦TaUreG序列分析
运用DNAMAN软件对测序结果进行分析,以TaUreG-F1和TaUreG-R1引物扩增的小麦TaUreG的cDNA序列如SEQ ID NO:1 所示,其cDNA 序列全长945 bp,包含一个长为855 bp的开放阅读框,编码284个氨基酸,氨基酸序列如SEQ
ID NO : 3所示。以TaUreG-F1和TaUreG-R1引物扩增的小麦TaUreG的gDNA序列如SEQ ID NO:2 所示,其gDNA序列全长2546 bp,包含7个外显子和6个内含子。从5’端开始,外显子的长度依次为77 bp、204 bp、61 bp、202 bp、122 bp、105 bp、84 bp,内含子的长度依次为86 bp、600 bp、336 bp、345 bp、129 bp、105 bp,基因结构参见图1。
实施例2 小麦抗旱基因TaUreG的染色体定位
以一套普通小麦品种中国春的缺体-四体系材料的基因组DNA为模板,以TaActin为内参(引物TaActin-F1:5’-GTTCCAATCTATGAGGGATACACGC-3’(见SEQ ID NO:6),TaActin-R1:5’-GAACTTCCACTGAGAACAACATTACC-3’ (见SEQ ID NO:7)。用TaUreG特异引物TaUreG-F1和TaUreG-R1进行PCR扩增。参看图2,结果显示,TaUreG在缺失染色体1A的缺体-四体系中2.5 Kb特异扩增该条带缺失,证明该基因定位在小麦1A染色体上。
实施例3 实时荧光定量PCR(Real-time PCR)分析验证TaUreG在小麦干旱相关的ABA信号通路中的作用
实验方法:
(1)材料培养及ABA处理:科农9204小麦种子播种于铺有2层滤纸的培养皿中,dH2O浸湿,萌发4天,去胚乳,Hogland培养至第10天,挑选长势一致的幼苗进行100
μM ABA 处理,0 h、1 h、3 h、6 h分别取样,液氮冷冻后-80℃保存;
(2)利用Trizol提取总RNA,具体步骤参见实施例1的第2步;
(3)利用PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa,RR047A)进行第一链cDNA合成;
a、去除基因组DNA,反应体系:5×gDNA
Eraser Buffer 2 µl,gDNA Eraser 1 µl,Total RNA 5 µl(200 ng/µl),RNase Free dH2O 2 µl;反应程序:42℃孵育2 min,4℃保存。
b、反转录,反应体系:步骤a的反应液10 µl,PrimeScript RT Enzyme Mix I 1µl,RT Primer Mix 1 µl,5×PrimeScript Buffer 2 4 µl,RNase Free dH2O
4 µl;反应程序:37℃孵育15 min,85℃ 5 s终止反应,4℃保存。
(4)使用SYBR® Premix Ex Taq II(TaKaRa,RR820A)试剂盒进行实时荧光定量PCR分析,具体步骤:
混匀如下反应体系,然后分装于96孔光学版中,并覆盖上光学膜,扩增使用的仪器为ABI PRISM 7500
real-time PCR仪;
PCR反应程序:95℃预变性30 s;95℃变性5 s,60℃退火并延伸34 s,40个循环;
20 μl PCR反应体系配制如下:SYBR® Premix Ex Taq II(2×)10 μl,Forward Primer(10 μM)0.8 μl,Reverse Primer(10 μM)0.8 μl,ROX Reference Dye II(50×)0.4 μl,cDNA模板2 μl,ddH2O 6 μl;
引物序列为:
上游引物TaUreG-F2:5’-GCCGATTTGCTGCTCTGT GA-3’(SEQ ID NO:8),
下游引物TaUreG-R2:5’-GCCTGTTCTTGGTATCTTGTCCC-3’(SEQ ID NO:9);
内参TaActin引物TaActin-F2:5’-TGCTATCCTTCGTTTG GACCTT-3’ (SEQ ID NO:10),
内参TaActin引物TaActin-R2:5’-AGCGGTTGTTGTGAGGGAGT-3’(SEQ ID NO:11)。
(5)实验结果:如图3所示,随着处理时间的推移,TaUreG在小麦叶片和根中的表达均受ABA调节,呈现先上升再下降的趋势。在叶片中1小时达到最高峰,在根中3小时到达最高峰,之后表达量下降,说明TaUreG参与了ABA信号转导通路;TaUreG应该是通过ABA依赖的信号转导途径参与了小麦对干旱胁迫的响应。
实施例4 实时荧光定量PCR(Real-time PCR)分析验证TaUreG在小麦干旱相关的ROS信号通路中的作用
实验方法:
(1)材料培养及H2O2处理:科农9204小麦种子播种于铺有2层滤纸的培养皿中,dH2O浸湿,萌发4天,去胚乳,Hogland培养至第10天,挑选长势一致的幼苗进行10
mM H2O2处理,0 h、1 h、3 h、6 h分别取样,液氮冷冻后-80℃保存;
(2)利用Trizol提取总RNA,具体步骤参见实施例1的第2步;
(3)利用PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa,RR047A)进行第一链cDNA合成,具体步骤参见实施例3的第3步;
(4)使用SYBR® Premix Ex Taq II(TaKaRa,RR820A)试剂盒进行实时荧光定量PCR分析,具体步骤参见实施例3的第4步;
(5)实验结果:如图4所示,随着处理时间的推移,TaUreG在小麦叶片中的表达受H2O2调节,呈现先下降,后上升然后再下降的趋势。但是,TaUreG的表达在小麦根中则不受H2O2调节。实时荧光定量PCR结果说明TaUreG参与了干旱胁迫引起的ROS水平升高的信号转导通路。TaUreG参与小麦抗旱机制的挖掘为该基因在小麦抗旱育种中的利用提供了更为可靠的证据。在小麦中过表达TaUreG,或抑制TaUreG的表达,将影响小麦对干旱胁迫的适应能力,培育出具有高抗旱性的转基因小麦,对提高小麦产量具有重大的意义。
实施例5
实验方法:
(1)材料培养及PEG-6000人工模拟干旱处理:科农9204小麦种子播种于铺有2层滤纸的培养皿中,dH2O浸湿,萌发4天,去胚乳,Hogland培养至第10天,挑选长势一致的幼苗进行质量百分比浓度为20%的PEG-6000处理,0 h、1 h、3 h、6 h、12 h、24 h、48 h分别取样,液氮冷冻后-80℃保存;
(2)利用Trizol提取总RNA,具体步骤参见实施例1的第2步;
(3)利用PrimeScriptTM RT reagent Kit with gDNA Eraser(TaKaRa,RR047A)进行第一链cDNA合成,具体步骤参见实施例3的第3步;
(4)使用SYBR® Premix Ex Taq II(TaKaRa,RR820A)试剂盒进行实时荧光定量PCR分析,具体步骤参见实施例3的第4步;
(5)实验结果:如图5所示,随着处理时间的推移,TaUreG在小麦叶片和根中的表达均受干旱诱导明显上调,在叶片中12小时达到最高峰,在根中6小时到达最高峰,之后表达量下降,说明TaUreG在小麦适应干旱胁迫机制中起重要作用。在小麦中过表达TaUreG,或抑制TaUreG的表达,将影响小麦对干旱胁迫的适应能力,培育出具有高抗旱性的转基因小麦,对提高小麦产量具有重大的意义。由此表明,含有TaUreG基因的小麦植株具有较强的抗旱能力,与小麦抗旱相关的基因TaUreG在选育抗旱小麦品种中可以得到应用;可以采用所述方法筛选和选育含有该基因的抗旱小麦品种,当然,也可以将所述核苷酸序列如SEQ ID NO:1所示的基因TaUreG转入小麦组织培育出具有高抗旱性的转基因小麦。
上述描述仅作为本发明可实施的技术方案提出,不作为对其技术方案本身的单一限制条件。
SEQUENCE LISTING
<110> 中国科学院遗传与发育生物学研究所
<120> 与小麦抗旱相关的基因TaUreG及其应用
<130>
<160> 11
<170> PatentIn version
3.3
<210> 1
<211> 945
<212> DNA
<213> 小麦TaUreG基因cDNA
<400> 1
ccttccactt ccaagtctgg attccgtcca tggcgtccca ggatcaccac caccaccacg 60
gcggccactc ccacgacgac gaccaccatc accgccacca tcacggggat gccgccggga 120
agggggcggg ggcggggtcg tgggtcggcg aggacgggcg cgtgtggcac tcccacgacg 180
gcctggcgcc gcactcccac gagcccatct actccgccgg ggacttctcc aagcgcgcgc 240
cgccgctcga ctcccgcagc ttcgccgacc gcgccttcac cgtcggcatc ggcggccccg 300
tcggcaccgg gaagactgcc ctgatgttag cactctgcac ttgcctccgt gacaaatata 360
gtcttgcagc ggttacaaat gatatattca caaaagagga tggagaattc ttggtcaagc 420
atggagctct gcctgaagag cgcatacgtg ctgtcgaaac tggaggctgc cctcatgccg 480
ctatacgtga ggacatcagc ataaatctgg gccctctgga ggagctatcc aacttgtaca 540
aggccgattt gctgctctgt gaatctggag gagataacct ggcagccaac ttcagcaggg 600
agctagcaga ctacataatc tacatcatcg acgtgtccgg tggggacaag ataccaagaa 660
caggcggccc tgggataacc caagcagatc tcttggtcat aaacaagaca gaccttgcct 720
ccgcggttgg agccgaccta gccgtgatgg agcgagacgc ccttcggatg cgggaaggag 780
ggcccttcgt gttcgcccag gtgaaacacg gggttggcgt ggaggagatt gtggaccacg 840
tgctgcgggc ctgggagatc gccaccggca acaggcgccg atagagaggc tctctcacgc 900
gaccgaaacg ccggcgagta attttcgggc atgtgttcac gtatg 945
<210> 2
<211> 2546
<212> DNA
<213> 小麦TaUreG 基因gDNA
<400> 2
ccttccactt ccaagtctgg attccgtcca tggcgtccca ggatcaccac caccaccacg 60
gcggccactc ccacgacgac gaccaccatc accgccacca tcacgggtga gtctacccgc 120
ctccgcctcg ccatccttct cgccgtccgc gcagccgact caaccagcac ctgccctgct 180
ttgcgcttgc agggatgccg ccgggaaggg ggcgggggcg gggtcgtggg tcggcgagga 240
cgggcgcgtg tggcactccc acgacggcct ggcgccgcac tcccacgagc ccatctactc 300
cgccggggac ttctccaagc gcgcgccgcc gctcgactcc cgcagcttcg ccgaccgcgc 360
cttcaccgtc ggcatcggcg gccccgtcgg caccgggtac gcccagcttt tctcctgcgg 420
ttgcttaggt gccggactgg ccgttatgct gtgttgtccg gcgagatagg agccgtctct 480
gctgcatttt ccctctgaat ctgcctgtgt ttcccgcgta gtgtcggtgc agatgtcagc 540
tatagccttt tgatctttgg agataactgt ggatgcatgt actacacaca ttttagccgc 600
tgaaaaatgt tgctttgacc acaaatctgt tgtgaaatat tggagttatt ctcgagcaga 660
atagacatta tatgcactgt gattactgat tagtgaacac tgtcatatgc ttagggattg 720
tctgcctccc ttgctgtgct tacagccata cactattttg gtgttcttgc tgttgcggtt 780
atgtttccct gccacagtat aacatcctag cattttggaa ttcagaagcg agttctgggg 840
gtctgggcaa ttcgaagaaa tttgtgcaca gcctttttgt gttgacgaaa taacctcatg 900
tggtgaagca agggcttcta atttcatatc ataacaacta tggaatgacg aagtgagatg 960
acccccacca tcttctctct gttttgcctt atgcaggaag actgccctga tgttagcact 1020
ctgcacttgc ctccgtgaca aatatagtct tgcagcggta tgtggcatgg ttctatcctg 1080
tgcttctctt aagattgctt aagataccat ctacttctcc tgattaaact acaccgcttg 1140
ctgcatcttt gggcattgga ctttcatcat aacatttgaa gatatatttg gttggtcctg 1200
tttctgaact tcaaaacagt tgattcatat aacaggtcaa tttttgtctg tgggggccag 1260
cattgaaatc ttactacttc taatattctt gtgatatttt cttaaacaca aatacctaat 1320
agtctccacc ctgtatgctt gaattatatt acacccttag gatttctctt tctttatttt 1380
ttattatgtg caggttacaa atgatatatt cacaaaagag gatggagaat tcttggtcaa 1440
gcatggagct ctgcctgaag agcgcatacg tgctgtcgaa actggaggct gccctcatgc 1500
cgctatacgt gaggacatca gcataaatct gggccctctg gaggagctat ccaacttgta 1560
caaggccgat ttgctgctct gtgaatctgg aggaggtatt gtccctgttg aatcccacat 1620
tggaaggttt atcatcgtgt atgtttagac cggcttagtg ttgtcagttg actgtttcga 1680
aaatgtcctg tagctttttg ttttgcgatg aaacctgtat ccttttccgt aatattgttt 1740
aggaatatgc agtgtgcact actggttttt ctgaagctgt tttgttgttt cttgggcatt 1800
agttgaacat ctcgattgtc cctctgcttc tggataatgg gtcactgaag agcatcatca 1860
cggccccagc ttaagccatt ttgtgattaa gtccatgacc aggcactaag aaagctactt 1920
tttgtgcatc ttgtgtgcag ataacctggc agccaacttc agcagggagc tagcagacta 1980
cataatctac atcatcgacg tgtccggtgg ggacaagata ccaagaacag gcggccctgg 2040
gataacccaa gcagatctct tggtgcgtca ccgcaccttc taagccattc aactagactg 2100
caaacctgta tcacgatgct gatttatctt ctcaccttgc tgagacaagt tcaccgtttt 2160
cactaactcg gaccatccgt cccaaactca ggtcataaac aagacagacc ttgcctccgc 2220
ggttggagcc gacctagccg tgatggagcg agacgccctt cggatgcggg aaggagggcc 2280
cttcgtgttc gcccaggttc aagatcaaac catgcacaca catgcacaca tgcgcagttc 2340
tctattcctt gtaataatcg taatgccatg aactcattct gctctgacat cgctggtgca 2400
ggtgaaacac ggggttggcg tggaggagat tgtggaccac gtgctgcggg cctgggagat 2460
cgccaccggc aacaggcgcc gatagagagg ctctctcacg cgaccgaaac gccggcgagt 2520
aattttcggg catgtgttca cgtatg 2546
<210> 3
<211> 284
<212> PRT
<213> 小麦TaUreG
<400> 3
Met Ala Ser Gln Asp His His His His His
Gly Gly His Ser His Asp
1 5 10 15
Asp Asp His His His
Arg His His His Gly Asp Ala Ala Gly Lys
Gly
20 25 30
Ala Gly Ala Gly Ser Trp Val Gly Glu
Asp Gly Arg Val Trp His Ser
35 40 45
His Asp Gly Leu Ala Pro His Ser His Glu Pro Ile Tyr
Ser Ala Gly
50 55 60
Asp Phe Ser Lys Arg
Ala Pro Pro Leu Asp Ser Arg Ser Phe Ala Asp
65 70 75 80
Arg Ala Phe Thr Val Gly
Ile Gly Gly
Pro Val Gly Thr Gly Lys Thr
85 90 95
Ala Leu Met Leu Ala Leu Cys Thr
Cys Leu Arg
Asp Lys Tyr Ser Leu
100 105 110
Ala Ala Val Thr Asn
Asp Ile Phe Thr Lys Glu
Asp Gly Glu Phe Leu
115 120 125
Val Lys His Gly Ala Leu Pro Glu Glu
Arg Ile Arg
Ala Val Glu Thr
130 135 140
Gly Gly Cys Pro His Ala Ala Ile Arg
Glu Asp Ile Ser Ile Asn Leu
145 150 155 160
Gly Pro Leu Glu Glu Leu Ser Asn Leu Tyr Lys Ala Asp Leu Leu Leu
165 170 175
Cys Glu Ser Gly Gly
Asp Asn Leu Ala Ala Asn Phe
Ser Arg Glu Leu
180 185 190
Ala Asp Tyr Ile Ile
Tyr Ile Ile
Asp Val Ser Gly Gly Asp Lys Ile
195 200 205
Pro Arg Thr Gly
Gly Pro Gly Ile Thr Gln
Ala Asp Leu Leu Val Ile
210 215 220
Asn Lys Thr Asp Leu
Ala Ser Ala Val Gly Ala Asp Leu
Ala Val Met
225 230 235 240
Glu Arg Asp Ala Leu Arg Met Arg Glu
Gly Gly Pro Phe Val Phe Ala
245 250 255
Gln Val Lys His Gly Val Gly Val Glu Glu
Ile Val Asp His Val Leu
260 265 270
Arg Ala Trp Glu Ile
Ala Thr Gly Asn Arg Arg
Arg
275 280
<210> 4
<211> 20
<212> DNA
<213> TaUreG-F1
<400> 4
ccttccactt ccaagtctgg 20
<210> 5
<211> 20
<212> DNA
<213> TaUreG-R1
<400> 5
catacgtgaa cacatgcccg
20
<210> 6
<211> 25
<212> DNA
<213> TaActin-F1
<400> 6
gttccaatct atgagggata cacgc 25
<210> 7
<211> 26
<212> DNA
<213> TaActin-R1
<400> 7
gaacttccac tgagaacaac attacc 26
<210> 8
<211> 20
<212> DNA
<213> TaUreG-F2
<400> 8
gccgatttgc tgctctgtga
20
<210> 9
<211> 23
<212> DNA
<213> TaUreG-R2
<400> 9
gcctgttctt ggtatcttgt ccc 23
<210> 10
<211> 22
<212> DNA
<213> TaActin-F2
<400> 10
tgctatcctt cgtttggacc tt
22
<210> 11
<211> 20
<212> DNA
<213> TaActin-R2
<400> 11
agcggttgtt gtgagggagt
20
Claims (6)
1.一种与小麦抗旱相关的基因TaUreG,其特征在于,其具有SEQ ID NO : 1所示的cDNA核苷酸序列或SEQ ID NO : 2所示的gDNA核苷酸序列。
2.根据权利要求1所述的与小麦抗旱相关的基因TaUreG,其特征在于,所述的gDNA含有7个外显子和6个内含子。
3.一种与小麦抗旱相关的基因TaUreG,其特征在于,该基因的编码蛋白如SEQ ID NO :3所示。
4.根据权利要求1所述的与小麦抗旱相关的基因TaUreG ,其特征在于,克隆所述基因TaUreG的上游引物TaUreG-F1的核苷酸序列如SEQ ID NO : 4所示、下游引物TaUreG-R1的核苷酸序列如SEQ ID NO : 5所示。
5.根据权利要求1所述的与小麦抗旱相关的基因TaUreG ,其特征在于,所述基因位于小麦1A染色体上。
6.根据权利要求1所述的与小麦抗旱相关的基因TaUreG ,其特征在于,采用实时荧光PCR检测时,其上游引物TaUreG-F2的核苷酸序列如SEQ ID NO :8所示、下游引物TaUreG-R2的核苷酸序列如SEQ ID NO : 9所示;内参上游引物TaActin-F2的核苷酸序列如SEQ ID NO :10所示、内参下游引物TaActin-R2的核苷酸序列如SEQ ID NO : 11所示。
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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 |
CN114703199A (zh) * | 2022-04-15 | 2022-07-05 | 河北省农林科学院生物技术与食品科学研究所 | 一种植物抗旱性相关的基因TaCML46及应用 |
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CN103060340A (zh) * | 2013-01-30 | 2013-04-24 | 山东省花生研究所 | 一种花生中逆境胁迫AhMYBL6基因的克隆及功能表达方法 |
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
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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 |
CN114703199A (zh) * | 2022-04-15 | 2022-07-05 | 河北省农林科学院生物技术与食品科学研究所 | 一种植物抗旱性相关的基因TaCML46及应用 |
CN114703199B (zh) * | 2022-04-15 | 2023-02-28 | 河北省农林科学院生物技术与食品科学研究所 | 一种植物抗旱性相关的基因TaCML46及应用 |
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