CN116240219A - 一种小麦rth-1基因及其应用 - Google Patents
一种小麦rth-1基因及其应用 Download PDFInfo
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Abstract
本发明公开了一种小麦RTH‑1基因及其应用。所述小麦RTH‑1基因的核苷酸序列如SEQ ID No.1所示。本发明经过实验证实,小麦基因RTH‑1与小麦乙烯敏感性和耐盐性相关,并利用小麦幼胚遗传转化体系进一步敲除小麦基因RTH‑1得到基因编辑突变体。小麦RTH‑1基因编辑成功地大幅降低RTH‑1的基因表达水平,引起小麦乙烯敏感性和耐盐性发生变化。与野生型小麦相比,小麦RTH‑1基因编辑突变体的乙烯敏感性降低,且耐盐性提高。本发明对于利用小麦基因编辑技术提高植株耐盐性具有重要意义。
Description
技术领域
本发明属于分子生物学技术领域,具体涉及一种小麦RTH-1基因及其应用。
背景技术
乙烯是一类植物自身合成的气体植物激素,广泛分布于植物组织和细胞中。研究已经证实,乙烯在调控植物生长发育的各个方面都起到了很大作用,如种子萌发、果实成熟、花蕾发育、叶片发育、衰老和脱落等。乙烯还参与植物对生物和非生物胁迫反应中,包括干旱、低温和盐胁迫条件下幼苗生长的抑制等。
在模式植物拟南芥中,RTE1作为乙烯受体ETR1的活化因子,在乙烯信号转导中发挥重要作用。拟南芥RTE1编码一种由250个氨基酸组成的完整膜蛋白。RTE1与乙烯受体ETR1共同定位于细胞内质网和高尔基体,且发生蛋白直接相互作用,共同参与由乙烯受体介导的乙烯信号转导,调控植物的乙烯反应。在粮食作物小麦中,乙烯受体活化因子的同源基因RTH(RTE1 Homolog)及其功能并不清楚。
小麦是世界上种植最广泛的作物之一,也是人类饮食的主要组成部分。小麦是异源六倍体,由A、B和D三个不同的基因组构成,小麦的全基因组测序尚未全部完成。小麦基因组DNA重复序列比例高(>80%),不利于定向诱变育种。近年来,CRISPR/Cas9系统被广泛用于精确编辑植物基因组,通过可编程的sgRNA(single guide RNA)引导核酸酶在基因组中找到一个特定的目标位置,结合Cas蛋白质的“剪刀”功能,进而对基因进行定点的精确编辑。在CRISPR/Cas9基因编辑系统中,sgRNA起定位功能,Cas9蛋白能够切割目的基因,sgRNA能够决定Cas9蛋白特异性切割基因组DNA的部位,从而造成DNA双链的断裂,进而引发DNA的修复,通过同源重组或非同源重组产生基因突变。CRISPR/Cas9基因编辑无疑为小麦目标基因的精准改良提供了强有力的技术支撑。
在作物分子育种方面,基因编辑技术已广泛应用于各类农作物中,例如水稻、玉米、大豆、棉花、番茄等。同时,科学家也在积极探索并把CRISPR/Cas9基因编辑技术应用于小麦品质改良。目前为止,利用基因编辑技术创制小麦RTH-1基因突变体的研究尚未报道。
发明内容
本发明提供了一种小麦RTH-1基因及其应用。本发明获得了一种小麦基因RTH-1的编码序列,并利用农杆菌介导小麦基因编辑载体构建了小麦幼胚遗传转化体系,同时通过实验证实,小麦RTH-1基因能够调控小麦乙烯敏感性和提高其耐盐性。
为实现上述发明目的,本发明采用以下技术方案予以实现:
本发明提供了一种小麦RTH-1基因,其核苷酸序列如SEQ ID No.1所示。
进一步的:所述小麦RTH-1基因与小麦乙烯敏感性和耐盐性相关,RTH-1基因编辑突变体的根系明显变长。
本发明还提供了用于扩增所述小麦RTH-1基因的引物,其序列如下:
RTH-1-For:5’-CTGCAACATTCCTCTGGAGCAGTTC-3’;
RTH-1-Rev:5’-TCAAGCATCTCTACGCAAGACATG-3’。
本发明还提供了含所述小麦RTH-1基因的编辑载体,所述载体为pLGYE-002。
本发明还提供了含所述的小麦RTH-1基因的重组菌株,所述菌株为农杆菌。
本发明还提供了所述的小麦RTH-1基因在调节小麦乙烯敏感性和/或耐盐性中的应用。
进一步的:其特征在于,通过敲除所述小麦RTH-1基因,或者下调其表达,以降低小麦乙烯敏感性和提高小麦耐盐性。
进一步的:其特征在于,所述敲除所述小麦RTH-1基因的方法包括CRISPR/Cas9介导的基因编辑技术。
本发明还提供了所述的小麦RTH-1基因在筛选耐盐小麦品种中的应用。
本发明还提供了一种培育耐盐小麦的方法,其步骤为:利用基因编辑,获取敲除所述的小麦RTH-1基因或者下调其表达的载体,转入农杆菌,获得重组菌株,侵染小麦幼胚后,经培养、分化、生根、炼苗,获得耐盐小麦。
本发明还提供了一种试剂盒,其用于筛选和/或预测耐盐小麦品种,其中包含所述的引物。
与现有技术相比,本发明的优点和有益效果是:
本发明利用基因编辑技术,成功构建获得了小麦RTH-1的基因编辑载体,并通过农杆菌介导的小麦幼胚遗传转化方法获得小麦基因编辑突变体株系。通过对小麦基因编辑突变体的植株表型分析,并将其与相同条件下生长的野生型植株比较,证明小麦基因RTH-1具有调控小麦乙烯敏感性和参与小麦耐盐性的功能。小麦RTH-1被编辑后导致植株中乙烯反应变化而调控小麦的乙烯敏感性,与野生型植株相比表现为根的长度增加,RTH-1基因编辑突变体的耐盐性提高。本发明的技术方案对于利用基因编辑调控植物乙烯敏感性和获得耐盐小麦新种质具有重要意义。
附图说明
图1为小麦幼胚遗传转化;其中A:清洗后的小麦幼胚;B:共培养2d;C:诱导培养14d;D:分化培养14d;E:土壤培养30d;F:土壤培养90d。
图2为小麦基因编辑突变体中RTH-1表达分析。
图3为小麦基因编辑突变体耐盐性分析;其中A:100mM NaCl光照培养箱内培养72小时的小麦幼苗;B:不同浓度NaCl光照培养箱内处理72小时的小麦幼苗的最长根长统计分析。
图4为小麦基因编辑突变体的乙烯敏感性分析,其中A:300mM ACC黑暗处理72小时的小麦黄化小麦幼苗;B:不同浓度ACC黑暗处理72小时后的小麦黄化幼苗的最长根长分析。
具体实施方式
下面结合附图和具体实施例来对本发明的技术方案做进一步详细的说明。
本发明实验中用到的试剂等购自TAKARA、Promega、Sigma等公司。
实验中用到的LB培养基配方:1g胰蛋白胨(Tryptone)、0.5g酵母提取物(YeastExtract)、1g NaCl,加ddH2O定容至100mL;调节pH 5.7-5.8,固体培养基加入0.8g琼脂粉,121℃高压灭菌20min;
实验中用到的1/2MS液体培养基配方:0.22g MS、0.05g MES、1.0g蔗糖,加ddH2O定容至100mL;调节pH 5.7-5.8,固体培养基加入0.8g琼脂粉,121℃高压灭菌20min;
实验中用到的悬浮培养基配方:0.05g MES、0.22g MS、4g蔗糖,加ddH2O定容至100mL。
实验中用到的共培培养基配方:1.329g M519、7.2g麦芽糖、2.4g葡萄糖、0.585gMES、0.9g植物凝胶;
实验中用到的诱导培养基配方:1.29g M524、9g麦芽糖、1.05g植物凝胶;
实验中用到的分化培养基配方:1.329g M519、9g蔗糖、1.05g植物凝胶;
实验中用到的生根培养基配方:0.1g MES、0.44g MS、2g蔗糖、1.5g琼脂。
其它试剂配方:见《分子克隆》第三版。
实施例1、小麦RTH-1基因克隆
1、小麦籽粒的清洗和催种
挑选颗粒饱满、完整无损的小麦籽粒放于50mL离心管中,分别加入40mL的去离子水和10mL的次氯酸钠溶液(去离子水与次氯酸钠的比例为4:1),再加入15μL Triton X-100,平放于28℃的摇床里(200-250rpm/min),震荡15min,重复此操作两次;倒掉水后,只加高温高压灭菌后的去离子水,平放于摇床中震荡10min,重复此操作三次;清洗完的种子换上适量去离子水,于4℃冰箱黑暗放置过夜,后取出置于25℃光照培养箱中培养5d左右;将幼苗移植于营养土中,每3d浇一次水。
2、植物总RNA提取
(1)取新鲜的2-3片叶片于1.5mL离心管(无RNA酶)中,于液氮中速冻,-80℃冰箱储存备用;
(2)取1mL无RNA酶离心管,酒精灯外焰烧封枪头,作简易研磨棒使用,将样品置于液氮中,使用研磨棒迅速研磨样品,期间不断加入液氮冷冻;
(3)研磨样品至粉末后,加入1mL TRNzol Universal总RNA提取试剂,弹起底部样品与试剂充分混合,室温静置5min;
(4)4℃低温离心机中12000rpm,离心11min,吸取800μL上清液于新离心管,加入200μL氯仿溶液,震荡混匀,室温静置溶液至分层;
(5)4℃低温离心机中12000rpm,离心11min,吸取400-500μL上清液于新离心管,加入等体积异丙醇,轻轻混匀;
(6)放置于-20℃的冰箱,冷冻30min以上,有利于RNA沉淀;
(7)4℃低温离心机中12000rpm,离心11min,弃上清,于滤纸上扣干;
(8)加入1mL 75%乙醇溶液,弹起沉淀,4℃低温离心机12000rpm,离心5min后,弃上清;
(9)重复步骤(8),滤纸上扣干;
(10)空管短暂离心,吸走乙醇,通风橱中吹5min;
(11)加入30μL RNase-Free ddH2O,60℃的水浴锅中加热10min,充分溶解RNA,涡旋震荡混匀,-80℃冰箱保存。
3、RNA的反转录
详细方法见PrimeScriptTMII 1st Strand cDNA Synthesis Kit TAKARA cDNA反转录试剂盒和PrimeScriptTMRT reagent Kit with gDNA Eraser(Perfect Real Time)TAKARA去除基因组Real Time RT-PCR专用试剂盒说明书。
4、PCR扩增
以上述反转录合成的cDNA为模板,PCR法扩增基因片段。
(1)PCR引物由北京擎科生物科技有限公司青岛分公司合成:
RTH-1-For:5’-CTGCAACATTCCTCTGGAGCAGTTC-3’(SEQ ID No.2);
RTH-1-Rev:5’-TCAAGCATCTCTACGCAAGACATG-3’(SEQ ID No.3)。
(2)PCR反应体系
反应程序如下:预变性:95℃,3min;循环反应(循环数35):95℃,15sec;55℃,15sec,72℃,1min/kb;彻底延伸:72℃,10min。
由此得到基因RTH-1,其核苷酸序列如SEQ ID No.1所示。
实施例2、CRISPR/Cas9基因编辑载体的构建
1、按照实验例1克隆得到正确的目的基因RTH-1,在目的基因RTH-1序列上寻找合适的PAM序列,记录所选PAM序列前端19bp碱基序列,并将其作为靶序列,合成引物。依据PAM序列的所在位置,设计前、中、后三个靶标sgRNA:
sgRNA1:5’-CTCCCATACCCTTCATCACA-3’(SEQ ID No.4);
sgRNA2:5’-ACACCGGCGCAGAGACGACA-3’(SEQ ID No.5);
sgRNA3:5’-TCTACGCCGGCCACGACAAC-3’(SEQ ID No.6)。
2、制备Oligo二聚体,
将前引物(RTH-1-For)和后引物(RTH-1-Rev)等量混合后,在PCR仪上将引物退火为双链,其反应体系和程序如下:
PCR反应程序为:95℃,30sec;72℃,2min;37℃,2min;25℃,2min。
3、基因编辑载体的构建
(1)载体酶切:对pLGYE-002进行酶切(pLGYE-002载体由山东省农业科学院作物研究所李根英研究员惠赠)。
其反应体系如下:
37℃恒温水浴锅,酶切1-2h。
(2)基因编辑载体连接:将Oligo二聚体与酶切后的pLGYE-002进行连接,其反应体系如下:
混匀后,在低温恒温水浴锅中,16℃反应过夜连接。
4、大肠杆菌DH5α感受态细胞转化
(1)取制备好的大肠杆菌感受态细胞100μL于冰上解冻,在超净工作台内加入10μL连接产物或5μL待转化质粒,轻轻吸打混匀后冰上放置30min;
(2)放置于42℃水浴锅中热激90sec,立即冰上静置5min;
(3)加入900μL的LB(-琼脂粉)液体培养基,37℃摇床中200-250rpm/min振荡培养2h;
(4)室温4000g离心5min,弃上清,留约100μL上清轻轻吸打混匀菌体,将悬浮液全部涂布于含有筛选抗生素的固体培养基上,使用封口膜密封培养皿,至37℃恒温培养箱中培养过夜培养。
将连接产物转化到大肠杆菌感受态细胞中,将在抗性培养基中生长的大肠杆菌单菌落进行PCR反应和琼脂糖凝胶电泳验证,将条带正确且明亮的菌液提质粒后进行测序。将测序成功的质粒转化到EHA105农杆菌中,用于后续实验。至此,成功得到了含有前、中、后3个靶点的小麦基因编辑载体pLGYE-002-RTH-1。
实验例3、农杆菌介导的小麦幼胚遗传转化体系
1、活化菌体:取已灭菌的试管,加入5mL含有Rif+Kan的LB液体培养基,加入10μL已经冻融的农杆菌EHA105,放置于28℃摇床中,200-250rpm/min培养2d;
2、转接菌液:取新的已灭菌的试管,加入10mL含有Rif+Kan的LB液体培养基,加入50μL上述步骤活化后的农杆菌,放置于28℃的摇床中,200-250rpm/min培养12h;
3、大量培养菌液:取已灭菌的250mL锥形瓶,加入50mL含有Rif+Kan的LB液体培养,加入500μL上述步骤转接后的农杆菌,放置于28℃的摇床中,200-250rpm/min培养OD600至0.6-0.8;
4、配制侵染液:将上述步骤大量培养后的菌液倒入50mL已灭菌的圆底离心管,配平,4℃低温离心机,5000rpm/min,离心10min。倒掉上清液,加入适量悬浮培养液,将沉淀轻轻吸打混匀;
5、清洗小麦幼胚,取花后14d的小麦幼胚种子清洗(使用高温高压灭菌后去离子水);
6、刀切和侵染小麦幼胚:首先在超净工作台中将清洗后的幼胚放于已灭菌的滤纸吸干,使用已灭菌的手术刀将幼胚损伤并用已灭菌的镊子取下放入已加入侵染液的50mL锥形瓶中,放置于28℃摇床中,120rpm/min,黑暗侵染30min;
7、共培小麦幼胚:将侵染后的小麦幼胚放于滤纸上,吸干多余菌液,使用镊子转移到共培培养基中(切面贴合培养基),放置于25℃的光照培养箱中黑暗培养2d;
8、诱导培养小麦幼胚:使用镊子将共培后的小麦幼胚转移到诱导培养基上,25℃黑暗培养15d;
9、继代培养小麦幼胚:将愈伤组织用镊子小心取出,用尖头镊小心将胚与愈伤组织剥离,移到新的诱导培养基上,25℃黑暗培养15d;
10、分化筛选培养:将愈伤组织小心地转移到分化筛选培养基上,25℃光照培养30d;
11、生根培养:待愈伤组织上分化出绿芽后,使用镊子将其转移到生根培养基中,25℃光照培养7d;
12、炼苗培养:当绿苗生长到较大时,打开培养瓶瓶盖,加入蒸馏水,25℃光照培养3d;
13、土壤培养:将炼好的苗移入营养土中,继续培养,每3d浇一次水。
实验结果如图1所示,成功获得小麦RTH-1基因编辑转化苗。
实验例4、小麦基因编辑突变体的分子鉴定
将农杆菌侵染获得的第一代小麦基因编辑产生的种子命名为T0代,取小麦幼嫩叶片提取DNA,方法如下:
1、小麦DNA提取
(1)取新鲜的2-3片叶片放于2.0mL离心管(已加入研磨珠),然后在高通量组织研磨机震荡研磨10min左右;
(2)于离心管中加入500μL的DNA提取液,混匀,65℃水浴加热30min;
(3)取出离心管,待样本冷却至室温,加入等体积的氯仿/异戊醇(24:1)混合液,再轻颠倒混匀,静置10min后12000rpm/min离心11min;
(4)吸取500μL上清液至新的已灭菌的1.5mL离心管中,加入等体积的异丙醇溶液,再轻颠倒混匀,-20℃冰箱中放置30min以上(可过夜),10000rpm/min离心11min;
(5)弃上清液,加入1mL 70%乙醇溶液洗涤沉淀物,重复两次,短暂离心后吸取多余液体,放置于洁净工作台中,自然风干;
(6)加20μL ddH2O溶解。
2、将提取的小麦DNA进行PCR反应。
(1)设计引物
PCR引物由北京擎科生物科技有限公司青岛分公司合成:
引物RTH-1-For1:5’-GAGCTCGGTTTCCTTGTTGC-3’(SEQ ID No.7);
引物RTH-1-For1:5’-CCCGCGCTCACCCAGCGGC-3’(SEQ ID No.8);
引物RTH-1-For2:5’-TTGCCTTTGGAGCTGTTACA-3’(SEQ ID No.9);
引物RTH-1-Rev2:5’-CCCGCGCTCACCCAGCGGC-3’(SEQ ID No.10);
引物RTH-1-For3:5’-TACAACCTGTTCACCTGCAA-3’(SEQ ID No.11);
引物RTH-1-Rev3:5’-AACGTCGTGCCGCCGAGGAG-3’(SEQ ID No.12);
引物RTH-1-For4:5’-GAGCCTGGCCGCGGTGATGT-3’(SEQ ID No.13);
引物RTH-1-Rev4:5’-GAACCACCCGGTCATGACG-3’(SEQ ID No.14);
PCR反应体系如下:
反应程序如下:预变性:95℃,3min;循环反应(循环数35):95℃,15sec;55℃,20sec;72℃,30sec;彻底延伸:72℃,10min。
将PCR反应后的产物进行凝胶电泳检测,条带有且亮的样品送样进行测序。
取小麦T0代部分植株进行测序,结果显示,株系4-c中的5A、5B、5D染色体上均出现了突变位点,株系5-e中的5A、5B染色体上出现了碱基替换,说明小麦基因编辑的效果较好。进一步利用小麦基因编辑T1代株系测序,结果如表1所示,T1代植株2B1-b1、5A-a2中的5A、5B染色体上均出现了突变位点,在靶点2上出现单个碱基缺失,突变率为18.95%,在靶点3上出现单个碱基的缺失,突变率为17.45%。株系10A2-a在靶点1上的5D染色体出现了3个碱基的缺失,突变率为10.59%。株系10A2-b1、10A2-b2、10A2-c在靶点2上的5D染色体上都出现同样的突变,即3个碱基的缺失,突变率分别为14.02%、21.59%。
表1:小麦基因编辑突变体T1代分子鉴定(·为缺失碱基)
实验例5、小麦基因编辑突变体的RTH-1基因表达分析
取小麦基因编辑T1代植株(10A2-a、10A2-c、10A2-b1)及野生型(FLD)幼嫩叶片提取细胞总RNA,反转录获得cDNA,将cDNA稀释5倍后做模板,经过荧光定量PCR技术分析,对基因编辑植株中的RTH-1基因进行表达水平分析。
结果如图2所示,三个基因编辑突变体的RTH-1表达量相对于野生型显著降低,说明利用基因编辑技术能成功地将RTH-1基因的表达水平大幅度降低。
实验例6、小麦基因编辑突变体的耐盐性分析
挑选颗粒饱满,完整无损的小麦基因编辑突变体T1代(10A2-a、10A2-c、10A2-b1)籽粒放于50mL离心管中,进行清洗及催种后,铺两层滤纸于干净的平板中,加入不同浓度的NaCl溶液浸透滤纸(NaCl浓度梯度为0,50,100,200,300mM),将小麦籽粒的腹沟朝下均匀摆于平板中,平放置于25℃光照培养箱中培养72h后,观察幼苗的根长变化,拍照并进行数据统计分析。
实验结果如图3所示,在不同NaCl浓度下,小麦幼苗根的长度随着NaCl浓度的增大而减小,基因编辑突变体的最长根长都长于野生型(FLD)对照。在100mM NaCl浓度处理时,基因编辑突变体的最长根长的长度比野生型更加明显(如图3B所示)。结果说明,基因编辑突变体比野生型更耐盐。
实验例7、小麦基因编辑突变体的乙烯敏感性分析
挑选颗粒饱满,完整无损的小麦基因编辑突变体T1代(10A2-a、10A2-c、10A2-b1)的籽粒放于50mL离心管中,清洗及催种后,铺两层滤纸于干净的平板中,加入不同浓度的ACC溶液浸透滤纸(ACC浓度梯度为0,100,200,300,400μM),将小麦籽粒的腹沟朝下均匀摆于平板中,进行遮光处理后,平放置于25℃光照培养箱中培养72h后,观察幼苗的根长变化,拍照并进行数据统计分析。
实验结果如图4所示,在不同ACC浓度下,发现小麦幼苗的根系长度随着ACC浓度的增大而减小,而基因编辑突变体幼苗的最长根长都长于野生性(FLD)对照。在300μM ACC浓度处理时,基因编辑突变体幼苗的最长根长度比野生型更加明显。结果说明,基因编辑突变体的幼苗的乙烯敏感性降低。
以上实施例仅用以说明本发明的技术方案,而非对其进行限制;尽管参照前述实施例对本发明进行了详细的说明,对于本领域的普通技术人员来说,依然可以对前述实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换;而这些修改或替换,并不使相应技术方案的本质脱离本发明所要求保护的技术方案的精神和范围。
Claims (10)
1.一种小麦RTH-1基因,其特征在于,所述小麦RTH-1基因的核苷酸序列如SEQ ID No.1所示。
2.用于扩增权利要求1所述的小麦RTH-1基因的引物,其特征在于,所述引物的序列如下:
RTH-1-For:5’-CTGCAACATTCCTCTGGAGCAGTTC-3’;
RTH-1-Rev:5’-TCAAGCATCTCTACGCAAGACATG-3’。
3.含权利要求1所述的小麦RTH-1基因的编辑载体,其特征在于,所述载体为pLGYE-002。
4.含权利要求1所述的小麦RTH-1基因的重组菌株,其特张在于,所述菌株为农杆菌。
5.权利要求1所述的小麦RTH-1基因在调节小麦乙烯敏感性和/或耐盐性中的应用。
6.根据权利要求5所述的应用,其特征在于,通过敲除所述小麦RTH-1基因,或者下调其表达,以降低小麦乙烯敏感性和提高小麦耐盐性。
7.根据权利要求6所述的应用,其特征在于,所述敲除小麦RTH-1基因的方法包括CRISPR/Cas9介导的基因编辑技术。
8.权利要求1所述的小麦RTH-1基因在筛选耐盐小麦品种中的应用。
9.一种培育耐盐小麦的方法,其特征在于,所述方法的步骤为:利用基因编辑,获取敲除权利要求1所述的小麦RTH-1基因或者下调其表达的载体,转入农杆菌,获得重组菌株,侵染小麦幼胚后,经培养、分化、生根、炼苗,获得耐盐小麦。
10.一种试剂盒,其特征在于,用于筛选和/或预测耐盐小麦品种,其中包含权利要求2所述的引物。
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104513825A (zh) * | 2014-12-25 | 2015-04-15 | 中国科学院遗传与发育生物学研究所 | 一种小麦耐盐基因TaNAS1及其应用 |
CN105063062A (zh) * | 2015-08-10 | 2015-11-18 | 济南大学 | 小麦耐盐、抗旱基因TaDHN3,表达载体及其应用 |
WO2020156406A1 (zh) * | 2019-01-31 | 2020-08-06 | 中国农业大学 | 小麦单倍体诱导基因及其应用 |
CN113481210A (zh) * | 2021-07-27 | 2021-10-08 | 中国农业科学院棉花研究所 | 棉花GhDof1.7基因在促进植物耐盐中的应用 |
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Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104513825A (zh) * | 2014-12-25 | 2015-04-15 | 中国科学院遗传与发育生物学研究所 | 一种小麦耐盐基因TaNAS1及其应用 |
CN105063062A (zh) * | 2015-08-10 | 2015-11-18 | 济南大学 | 小麦耐盐、抗旱基因TaDHN3,表达载体及其应用 |
WO2020156406A1 (zh) * | 2019-01-31 | 2020-08-06 | 中国农业大学 | 小麦单倍体诱导基因及其应用 |
CN113481210A (zh) * | 2021-07-27 | 2021-10-08 | 中国农业科学院棉花研究所 | 棉花GhDof1.7基因在促进植物耐盐中的应用 |
Non-Patent Citations (3)
Title |
---|
GENBANK: "登录号XM_048695529", GENBANK * |
GENBANK: "登录号XM_048695530", GENBANK * |
隋新莹等: "小麦耐盐、富铁突变体新品系的鉴定及分析", 山东农业科学, vol. 53, no. 7 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116891861A (zh) * | 2023-08-18 | 2023-10-17 | 青岛农业大学 | 植物AtRTH基因在铁吸收转运中的应用 |
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