CN114686499A - 藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用 - Google Patents
藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用 Download PDFInfo
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Abstract
本发明公开了一种藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用,所述CqMSRA5.1基因的cDNA的核苷酸序列如SEQ ID No.1所示。该基因在培育抗逆作物中有潜在的应用价值。
Description
技术领域
本发明涉及生物基因工程技术领域,特别是,涉及一种藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用。
背景技术
干旱和高盐等逆境是限制植物产量的重要因素。因此,鉴定抗逆相关基因用于作物改良是未来抗逆育种的关键策略之一。MSR(甲硫氨酸亚砜还原酶)能被非生物胁迫诱导表达,而且MSR可以清除机体内过量的ROS,在保护植物的生长和产量中发挥重要作用。藜麦作为典型的杂粮作物之一,喜高海拔、耐寒、耐盐碱和贫瘠,是否可研究其内MSR及应用,是本领域值得关注的技术问题。
发明内容
为了克服现有技术的不足,本发明的目的在于提供一种藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用,该基因在培育抗逆作物中有潜在的应用价值。
为解决上述问题,本发明所采用的技术方案如下:
一种藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1,所述CqMSRA5.1基因的cDNA的核苷酸序列如SEQ ID No.1所示。
本发明还包括上述藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1的制备方法,包括步骤:
S101. 提取藜麦稼祺505总RNA并以总RNA反转录获得总cDNA;
S102. 以CqMSRA5.1-F、CqMSRA5.1-R为引物,以反转录获得的总cDNA为模板进行PCR扩增,得到目标片段;其中,CqMSRA5.1-F序列如SEQ ID No.2所示、CqMSRA5.1-R序列如SEQ ID No.3所示;
S103. PCR产物经凝胶回收、载体连接、大肠杆菌转化,测序得到藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1。
进一步的,所述步骤S103中,所述PCR的扩增体系为:
cDNA | 1 |
10×Buffer | 2 |
dNTP(2.5mM each) | 0.4 |
CqMSRA5.1-F引物F(10µM) | 0.5 |
CqMSRA5.1-R引物R(10µM) | 0.5 |
TransTaq HiFi DNA聚合酶 | 0.2 |
补充双蒸水至 | 20 。 |
进一步的,所述步骤S103中,扩增条件为:
95℃ | 5 min |
94℃ | 30 s |
55℃ | 30 s |
72℃ | 1.5 min |
2-4步骤 | 循环35次 |
72℃ | 10 min |
10℃ | 保存。 |
本发明还包括上述藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1在植株中表达以提高植株抗盐和耐旱性的应用。
优选的,所述植株为拟南芥。
相比现有技术,本发明的有益效果在于:利用基因工程技术,本发明首次克隆得到了藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1并进行了功能验证,通过不同诱导子处理显示该基因与抗盐和抗旱密切相关;对于该基因CqMSRA5.1的研究具有重要理论意义和应用前景。
附图说明
图1为CqMSRA5.1基因的cDNA电泳图,其中:Marker为DNA Marker,泳道2为CqMSRA5.1的cDNA;
图2为甲硫氨酸亚砜还原酶基因CqMSRA5.1在转基因拟南芥中RealTime-PCR表达分析,其中,Clo-0为野生型,AtOE1和AtOE 2为CqMSRA5.1基因在拟南芥中的过表达系,msrb5是MSRA5基因缺失系;
图3为转基因的拟南芥中NaCl处理分析,其中WT为野生型,AtOE1和AtOE 2为RealTime-PCR分析的过表达系,msrb5是MSRA5基因缺失系;
图4为转基因的拟南芥中控水试验的分析,其中WT为野生型,AtOE1和AtOE 2为RealTime-PCR分析的过表达系,msrb5是MSRA5基因缺失系。
具体实施方式
下面结合具体实施方式对本发明作进一步详细说明。
实施例1、CqMSRA5.1的克隆
1.1提取藜麦稼祺505总RNA
1)将稼祺505植物材料放入研钵中,加入液氮将其研磨成粉;
2)待液氮挥发后,取约100-200mg粉末转入到1.5ml离心管中,加入1ml Trizol提取液,涡旋震荡使样品充分溶入提取液中,室温静置5 min;
3)4℃,12000rpm,离心10min后,取0.9ml上清液转移到新的1.5ml离心管中,再加入0.2 ml氯仿剧烈振荡混匀15sec,室温静置2-5min;
4)4℃,12000rpm,离心10min后,取0.4ml上清液转移到新的1.5ml离心管中,加入0.4ml异丙醇后混匀,室温静置15mim;
5)4℃,12000rpm,离心10min,弃上清,用1ml 75%的乙醇洗涤沉淀两次后,于4℃,8000rpm,离心5 min;
6)弃上清,开盖于超净工作台中上干燥RNA约2-5min,加入40µl RNase-Free水,在60℃中充分溶解RNA 10min;
7)用紫外分光光度计测RNA样品的OD值和浓度,A260/A280达到1.7-2.0为佳;琼脂糖凝胶电泳检测的质量。
总cDNA的合成
1) 向离心管中依次加入下列物质(40µl 反应体系):
2) 轻轻混匀后,65℃变性5min,立即插到冰上,冰浴至少1min;
3) 向离心管中依次加入下列物质
4) 轻轻混匀后,42℃恒温水浴1h,65℃变性10min,-20℃保存备用。
反应
以CqMSRA5.1-F、CqMSRA5.1-R为引物,以反转录获得的总cDNA为模板进行PCR扩增,得到目标片段;引物序列如下:
CqMSRA5.1-F: -5’ ATGAATCGGAAGGCAAAAATAAGAA3’-
CqMSRA5.1-R: -5’ TTATATTTCCCTCAAAACAGGCCAG3’-
Trans HiFi Taq高保真酶扩增的反应体系如下(50µl体系):
总cDNA | 1 |
10×Buffer | 2 |
dNTP(2.5mM each) | 0.4 |
CqMSRA5.1-F引物F(10µM) | 0.5 |
CqMSRA5.1-R引物R(10µM) | 0.5 |
TransTaq HiFi DNA聚合酶 | 0.2 |
补充双蒸水至 | 20 |
扩增条件如下:
95℃ | 5 min |
94℃ | 30 s |
55℃ | 30 s |
72℃ | 1.5 min |
2-4步骤 | 循环35次 |
72℃ | 10 min |
10℃ | 保存 |
反应结束后,反应液于0.8%TAE琼脂糖凝胶电泳检测,电泳结果如图1所示。
凝胶回收并采用DP209试剂盒进行纯化
1) 将切下带有目的片段的凝胶回收放入1.5ml离心管并称凝胶重量,加入3倍体积的溶胶液,60 ℃溶胶10 min,溶胶期间不断的翻转;
2)凝胶完全融化后,全部吸取到回收柱中,放置片刻;
3) 室温,12000rpm,离心30Sec,弃溶液;
6) 空柱,12000rpm,离心2min;
7) 回收柱开盖晾干1-2min,放入新的干净的1.5ml离心管,加入60℃预热的40µl灭菌水或者EB缓冲液,放置2 min;
8) 12000rpm离心1min,所得溶液即为回收片段。
载体连接
用上述的回收目的片段与pEasy-T1 simple载体,25-30℃连接10-20 min。连接体系如下:
PCR回收产物 100~200 ng
混匀并短暂离心,25-30℃连接10-20 min,得到重组质粒pEasy-T1 simple,用于后面的反应。
大肠杆菌转化
1.61 首先用CaCl2法制备E. coli DH10B感受态细胞
a)挑取单菌落接种于5ml LB液体培养基中, 37℃振荡培养过夜;
b)按1:100-1:50的比例,取1ml菌液接种于100ml LB液体培养基中,37℃振荡培养至菌液OD600为0.3-0.6;
c)将菌液冰浴10min,4℃ 4000rpm离心10min,收集菌体;
d)沉淀加10ml预冷的0.1M CaCl2悬浮后,冰浴30min;
e)4℃4000rpm离心10min。沉淀重悬浮于1ml预冷的0.1M CaCl2,混匀并冰水浴中保存,备用或加入15%的甘油置于-70℃中保存;
1.62 热击法转化E. coli DH10B
b)在42℃恒温水浴中热激90sec;
c)冰浴3-5min;
d)加入800μl不含抗生素的LB液体培养基,混匀,37℃振荡培养45-60min;
f)将平板于37℃正向放置15-30min至液体被吸收,倒置平板,于37℃培养12-16h,观察平板会有蓝白斑,白斑用于后面的质粒提取。
阳性重组子的鉴定
1.71 首先利用碱裂解法提取含有重组子的大肠杆菌质粒
a)挑取白色单菌落,接入含抗生素Amp(50mg/L)的3ml LB液体培养基中,同时也要保存所挑取的白色单菌落于含抗生素Amp的固体LB平板中,37℃震荡培养12-16h;
b)12000rpm离心30sec,收集培养物中的菌体,尽量弃尽上清;
c)加入100μl冰预冷的溶液I,在涡旋器上使菌体充分重悬;
d)加入200μl溶液II,立即将离心管缓缓颠倒数次,冰浴5-10min;
e)加入150μl冰预冷的溶液Ⅲ,缓缓颠倒离心管数次直至白色沉淀充分形成,冰浴5-10min;
f)12000rpm离心3min,取上清转入另一微量离心管中,加入2倍体积 95%乙醇,混匀后,室温静置3 min;12000rpm离心3min,使质粒DNA沉淀;
h)加入等体积冰预冷的5mol/L LiCl溶液,冰浴5min,沉淀大量的RNA;
i)12000rpm,离心3min;
j)转移上清到另一离心管中,加入2倍体积的95%乙醇,混匀后于室温静置3min,12000rpm离心3min 使质粒沉淀;
k)弃上清后用1ml 70%乙醇洗涤沉淀,弃尽液体;
l)室温干燥后,取20μl含RNaseA(20μg/ml)的TE溶液溶解沉淀,37℃水浴30~60min消化RNA。
n)取上清,加入等体积的氯仿-异戊醇,重复以上操作;
o)取上清,加入1/10体积的3 mol/L醋酸钠(pH5.3)和2倍体积的无水乙醇,混匀后-20℃放置15min, 12000rpm离心3min 使质粒沉淀;
1.72 提出质粒后进行酶切验证,酶切反应体系如下表所示:
1.73 将上述反应体系混匀并5000rpm离心1min,37℃水浴反应过夜,然后进行1%的TAE琼脂糖凝胶电泳检测。
1.74 挑取含有重组质粒的阳性单菌落用含有Amp(50mg/L)的液体LB摇过夜,然后送上海博亚生物技术有限公司测序,得到测序结果:全长基因cDNA序列如序列表SEQ IDNo.1所示。经过NCBI blast分析,初步确定为甲硫氨酸亚砜还原酶基因,命名CqMSRA5.1。
实施例2、在拟南芥中对CqMSRA5.1进行功能验证
2.1 植物过表达载体的构建
2.11 设计引物
pSTART载体是常用的过表达双元载体,设计添加有合适酶切位点(正向引物添加EcoRⅠ,反向引物添加BamHI)的引物:
CqMSRA5.1-F-PSTART:CCGGAATTCATGAATCGGAAGGCAAAAATAAGAA
EcoRⅠ
CqMSRA5.1-R-PSTART:CGCGGATCCTTATATTTCCCTCAAAACAGGCCAG
BamHI
2.12 PCR反应:
以CqMSRA5.1-F-PSTART、CqMSRA5.1-R-PSTART为引物,以反转录获得的总cDNA为模板进行PCR扩增,得到目标片段;
Trans HiFi Taq高保真酶扩增的反应体系如下(50µl体系):
总cDNA | 1 |
10×Buffer | 2 |
dNTP(2.5mM each) | 0.4 |
CqMSRA5.1-F-PSTART (10µM) | 0.5 |
CqMSRA5.1-R-PSTART (10µM) | 0.5 |
TransTaq HiFi DNA聚合酶 | 0.2 |
补充双蒸水至 | 20 |
扩增条件如下:
95℃ | 5 min |
94℃ | 30 s |
55℃ | 30 s |
72℃ | 1.5 min |
2-4步骤 | 循环35次 |
72℃ | 10 min |
10℃ | 保存 |
反应结束后,反应液于0.8%TAE琼脂糖凝胶电泳检测。
凝胶回收并采用DP209试剂盒进行纯化
1)将切下带有目的片段的凝胶回收放入1.5ml离心管并称凝胶重量,加入3倍体积的溶胶液,60 ℃溶胶10 min,溶胶期间不断的翻转;
2)凝胶完全融化后,全部吸取到回收柱中,放置片刻;
3)室温,12000rpm,离心30Sec,弃溶液;
6)空柱,12000rpm,离心2min;
8)12000rpm离心1min,所得溶液即为回收片段。
载体连接
用上述的回收目的片段与pEasy-T1 simple载体,25-30℃连接10-20 min。连接体系如下:
PCR回收产物 100~200 ng
混匀并短暂离心,25-30℃连接10-20 min,得到重组质粒pEasy-T1 simple,用于后面的反应。
大肠杆菌转化
首先,用CaCl2法制备E. coli DH10B感受态细胞:
1)挑取单菌落接种于5ml LB液体培养基中, 37℃振荡培养过夜;
2)按1:100-1:50的比例,取1ml菌液接种于100ml LB液体培养基中,37℃振荡培养至菌液OD600为0.3-0.6;
3)将菌液冰浴10min,4℃ 4000rpm离心10min,收集菌体;
4)沉淀加10ml预冷的0.1M CaCl2悬浮后,冰浴30min;
5)4℃4000rpm离心10min。沉淀重悬浮于1ml预冷的0.1M CaCl2,混匀并冰水浴中保存,备用或加入15%的甘油置于-70℃中保存;
然后,热击法转化E. coli DH10B:
2)在42℃恒温水浴中热激90sec;
3)冰浴3-5min;
4)加入800μl不含抗生素的LB液体培养基,混匀,37℃振荡培养45-60min;
6)将平板于37℃正向放置15-30min至液体被吸收,倒置平板,于37℃培养12-16h,观察平板会有蓝白斑,白斑用于后面的质粒提取。
阳性重组子的鉴定
首先,利用碱裂解法提取含有重组子的大肠杆菌质粒:
1)挑取白色单菌落,接入含抗生素Amp(50mg/L)的3ml LB液体培养基中,同时也要保存所挑取的白色单菌落于含抗生素Amp的固体LB平板中,37℃震荡培养12-16h;
2)12000rpm离心30sec,收集培养物中的菌体,尽量弃尽上清;
3)加入100μl冰预冷的溶液I,在涡旋器上使菌体充分重悬;
4)加入200μl溶液II,立即将离心管缓缓颠倒数次,冰浴5-10min;
5)加入150μl冰预冷的溶液Ⅲ,缓缓颠倒离心管数次直至白色沉淀充分形成,冰浴5-10min;
6)12000rpm离心3min,取上清转入另一微量离心管中,加入2倍体积 95%乙醇,混匀后,室温静置3 min; 12000rpm离心3min,使质粒DNA沉淀;
8)加入等体积冰预冷的5mol/L LiCl溶液,冰浴5min,沉淀大量的RNA;
9)12000rpm,离心3min;
10)转移上清到另一离心管中,加入2倍体积的95%乙醇,混匀后于室温静置3min,12000rpm离心3min 使质粒沉淀;
11) 弃上清后用1ml 70%乙醇洗涤沉淀,弃尽液体;
12)室温干燥后,取20μl含RNaseA(20μg/ml)的TE溶液溶解沉淀,37℃水浴30~60min消化RNA;
14)取上清,加入等体积的氯仿-异戊醇,重复以上操作;
15)取上清,加入1/10体积的3 mol/L醋酸钠(pH5.3)和2倍体积的无水乙醇,混匀后-20℃放置15min, 12000rpm离心3min 使质粒沉淀;
然后,提出质粒后进行酶切验证,酶切反应体系如下表所示:
将上述反应体系混匀并5000rpm离心1min,37℃水浴反应过夜,然后进行1%的TAE琼脂糖凝胶电泳检测。挑取含有重组质粒的阳性单菌落用含有Amp(50mg/L)的液体LB摇过夜,然后送上海博亚生物技术有限公司测序。经电泳检测和测序发现,PCR后得到的基因序列为CqMSRA5.1基因。
过表达载体的连接、转化及鉴定
用步骤3)的回收目的片段与pSTART空载体,25-30℃连接10-20 min。连接体系如下:
PCR回收产物 100~200 ng
混匀并短暂离心,25-30℃连接10-20 min,得到过表达载体质粒,重复上述步骤5)和步骤6),将过表达载体质粒进行大肠杆菌转化,并进行鉴定,确定获得过表达载体质粒为CqMSRA5.1基因的过表达载体。
植物过表达载体转化农杆菌
2.2.1农杆菌感受态的制备(无菌操作)
1) 取农杆菌GV3101接种于10ml YEP液体培养基中,28℃摇床过夜;
2) 按1:50接种于50ml YEP液体培养基中,28℃振荡培养3-4小时,至OD600值为0.4-0.6;
3) 4℃,4200rpm,离心10min,收集菌体;
4) 弃上清,加入10ml预冷的0.15M的NaCl悬浮菌体;
5) 重复步骤3;
6) 弃上清,加入2ml预冷的20mM的CaCl2悬浮菌体,分装于1.5ml离心管中,现用或加入终体积7%DMSO经液氮速冻后-80℃保存备用。
农杆菌的转化(无菌操作)
1) 将10µl植物表达载体质粒DNA加入50µl的感受态细胞中,轻弹离心管混匀,冰浴30min;
2) 液氮速冻1min;然后37℃水浴5min,立即冰浴2-3min;
3) 加入1ml YEP培养基,28℃培养2-4h;
4) 室温,4000rpm,离心3min,收集菌体;
5) 将菌涂布于含有相应抗生素的YEP培养平板上,28℃倒置培养48h。
转化农杆菌的PCR验证
对阳性农杆菌菌落进一步鉴定,所用方法为菌落PCR技术,反应体系如下(20µl体系):
PCR扩增条件如下:
98℃ | 3min |
98℃ | 30 s |
55℃ | 30 s |
72℃ | 1.5 min |
2-4步骤 | 循环35次 |
72℃ | 10 min |
10℃ | 保存 |
反应结束后,反应液于0.8%TAE琼脂糖凝胶电泳检测。
花侵染法转化拟南芥
1)将适量待灭菌的野生型拟南芥(WT)种子放入1.5ml离心管中,加入1ml 75%的乙醇(含有0.03%体积比的TritonX-100)震荡消毒1min,再用70%的乙醇震荡消毒1min(两次),最后用吸头将种子吸到无菌滤纸上吹干,然后用无菌的牙签将其点入1/2MS培养基中。
2)生长至抽苔1cm时,将顶端减掉以诱导侧生花序的生成;
3)在转化前一天,取1ml活化过的含有表达载体质粒的农杆菌GV3101加到含相应抗生素及50μg/ml利福平的40ml YEP培养基中,28℃震荡培养至OD600约为1.0-1.2;
4)室温,4200rpm, 离心10min,收集菌体,用浸染液(5%蔗糖,0.05% Silwet L-77)重悬菌体,使OD600约为0.8;
5)用移液器将农杆菌滴到花序上进行浸染,待所有花序都被侵染后,将拟南芥放入真空干燥器中抽真空1min;
6)用保鲜袋覆盖花序,至于20-22℃避光培养一天剪开顶部露出花序,再培养一天后揭去保鲜袋,培养至种子成熟。
转基因纯系的获得
对收获的T0代种子进行表面消毒,然后均匀涂布于1/2MS平板上(含相应的抗生素卡那霉素)。春化处理3天后移入人工气候室生长。萌发约10天,子叶深绿色的植株为转基因植株,而子叶发浅绿甚至黄化的植株为非转基因植株。将转基因植株转入土中生长直至收获得到T1代转基因种子,T1代植株单株收种子,每株收取的种子继续筛选,将后代分离比为3׃1(阳性׃阴性)的阳性植株移栽后生长至收获T2代转基因种子,单株收种后,每株收取的种子经筛选可以得到纯系T2代转基因种子。
转基因植株的RT-PCR检测
1) 植株总RNA的提取和总cDNA合成方法参照1.1和1.2.
2)CqMSRA5.1基因RT引物序列:
CqMSRA5.1-RT-F:5’ ATGAATCGGAAGGCAAAAATAAGAA 3’
CqMSRA5.1-RT-R:5’ TTATATTTCCCTCAAAACAGGCCAG 3’
混匀体系并离心,放入PCR仪中进行PCR反应。
PCR反应程序:
之后利用琼脂糖电泳分析PCR结果,检测结果如图2A所示,表明成功获得了转基因株系,进一步对野生型(WT)、CqMSRA5.1基因过表达系(AtOE1和AtOE2)以及MSRA5基因的缺失系(Msrb5)植株总MSR酶活进行测定,结果如图2B所示,CqMSRA5.1基因过表达系(AtOE1和AtOE2)中MSR总酶活显著高于野生型(WT),而MSRA5基因的缺失系(Msrb5)中MSR总酶活显著低于野生型(WT)。
不同浓度盐和甘露醇处理拟南芥
1)将灭菌后的野生型拟南芥(WT)、CqMSRA5.1基因过表达系(AtOE1和AtOE2)以及MSRA5基因的缺失系(Msrb5)的种子分别点到1/2MS固体培养基上,4℃下无光培养3-4 d,后转移至到光照培养箱22℃竖直培养3 d;
2)分别制备加有100 mM/L浓度的NaCl、200 mM/L甘露醇的1/2MS固体培养基;
3)将根长0.8~1cm且生长一致的幼苗分别移入对照组A(1/2MS固体培养基),试验组B(加有100 mM/L浓度的NaCl的1/2MS固体培养基)和试验组C(加有200 mM/L甘露醇的1/2MS固体培养基)中竖直培养,培养10 d后拍照并统计根长,实验重复3次。
结果表明,CqMSRA5.1基因过表达系相较于野生型和MSRA5基因的缺失系相比,显著增强了对盐(图3B所示)和胁迫渗透(甘露醇模拟(图3C所述))的抗性。
控水实验
将野生型(WT)、CqMSRA5.1基因过表达系(AtOE1和AtOE2)以及MSRA5基因的缺失系(Msrb5)的种子点到土中,待长到5-7叶(大约五到六周)时,控水3周,之后再复水3 d。照相统计植株存活率(存活率=复水后叶片反绿的植株数/总植株数,实验重复3次。
当结束控水并复水一段时间后,CqMSRA5.1基因过表达系恢复了正常的生长状态,野生型和MSRA5基因的缺失系合仍然处于枯死状态(参见图4),表明CqMSRA5.1基因的过表达使拟南芥对干旱的耐受性增强。
对本领域的技术人员来说,可根据以上描述的技术方案以及构思,做出其它各种相应的改变以及形变,而所有的这些改变以及形变都应该属于本发明权利要求的保护范围之内。
序列表
<110> 山西农业大学
<120> 藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1及其制备方法和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 762
<212> DNA
<213> 藜麦(Chenopodium quinoa)
<400> 1
atgaatcgga aggcaaaaat aagaaccata actgcattaa tatctcattt tcttttactc 60
attctcgccg ggtttctttt tccggctccg gcaaagagca tccggctccc ggatcgaatg 120
ccggaattga cacctcccgc caaccagccc ttgaaaaccg ccgtgtttgc tctcggtagt 180
ttctggagat ctgatgctat ttttggttgc ttgaatggtg tggttcgcac caccgtcggc 240
tactccggtg gctctaagtc taatcctgag tatcgcagtt tgtccgatca cgctgaatct 300
gtacagattg aatatgatcc cagaggtata agcttccgag aactgttgga ggtcttctgg 360
tctagtcatg attcaaggca ggttttcggc cagggtcctg atgtcggtaa ccagtacaga 420
tctgtcatct ttaccaatgg tacgagtgaa tctagattgg cagccgccac taaggaaagg 480
gagcagacta aatcaaagag cagtatagtt acaacccaaa ttctacccct aggaaccttt 540
tatcctgctg aacctgagca tcagaaattt gagctcaaaa gaaatccttt tcttcttcac 600
ttgattggca atttgccaga ggaggagctc gagaagtcaa ccttggctgc aaaaatgaac 660
agttatgcag cagaactctg tcctcgaagg attcagaagc aaattgatgc caagattaat 720
gacattatta gtaaaggctg gcctgttttg agggaaatat aa 762
<210> 2
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
atgaatcgga aggcaaaaat aagaa 25
<210> 3
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ttatatttcc ctcaaaacag gccag 25
Claims (6)
1.一种藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1,其特征在于,所述CqMSRA5.1基因的cDNA的核苷酸序列如SEQ ID No.1所示。
2.权利要求1所述藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1的制备方法,包括步骤:
S101. 提取藜麦稼祺505总RNA并以总RNA反转录获得总cDNA;
S102. 以CqMSRA5.1-F、CqMSRA5.1-R为引物,以反转录获得的总cDNA为模板进行PCR扩增,得到目标片段;其中,CqMSRA5.1-F序列如SEQ ID No.2所示、CqMSRA5.1-R序列如SEQ IDNo.3所示;
S103. PCR产物经凝胶回收、载体连接、大肠杆菌转化,测序得到藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1。
3.如权利要求2所述的制备方法,其特征在于,所述步骤S103中,所述PCR的扩增体系为:
4.如权利要求2所述的制备方法,其特征在于,所述步骤S103中,扩增条件为:
5.权利要求1所述藜麦甲硫氨酸亚砜还原酶基因CqMSRA5.1在植株中表达以提高植株抗盐和耐旱性的应用。
6.如权利要求5所述的应用,其特征在于,所述植株为拟南芥。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
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CN104046639A (zh) * | 2014-07-02 | 2014-09-17 | 山东大学 | 小麦甲硫氨酸亚砜还原酶基因TaMsrB3.1及其应用 |
CN104087600A (zh) * | 2014-07-02 | 2014-10-08 | 山东大学 | 小麦甲硫氨酸亚砜还原酶基因TaMsrA4.1及其应用 |
CN113337526A (zh) * | 2021-06-08 | 2021-09-03 | 吉林大学 | 玉米甲硫氨酸亚砜还原酶基因ZmMSRB3及应用 |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104046639A (zh) * | 2014-07-02 | 2014-09-17 | 山东大学 | 小麦甲硫氨酸亚砜还原酶基因TaMsrB3.1及其应用 |
CN104087600A (zh) * | 2014-07-02 | 2014-10-08 | 山东大学 | 小麦甲硫氨酸亚砜还原酶基因TaMsrA4.1及其应用 |
CN113337526A (zh) * | 2021-06-08 | 2021-09-03 | 吉林大学 | 玉米甲硫氨酸亚砜还原酶基因ZmMSRB3及应用 |
Non-Patent Citations (2)
Title |
---|
"NCBI Reference Sequence: XM_021909570.1" * |
张映 等: "茄子甲硫氨酸亚砜还原酶(SmMsrA)基因cDNA全长的克隆和分析" * |
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