CN114717241B - 一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用 - Google Patents
一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用 Download PDFInfo
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- CN114717241B CN114717241B CN202210427921.0A CN202210427921A CN114717241B CN 114717241 B CN114717241 B CN 114717241B CN 202210427921 A CN202210427921 A CN 202210427921A CN 114717241 B CN114717241 B CN 114717241B
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Abstract
本发明的目的在于公开一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用。所述基因OsMSRFP为如下1)或2)或3)所述的DNA分子:1)基因组序列如SEQ ID NO.1所示的DNA分子;2)CDS序列如SEQ ID NO.2所示的DNA分子;3)在严格条件下与1)或2)限定的DNA序列杂交且编码所述蛋白的DNA分子。本发明提供的基因OsMSRFP在调控水稻耐盐性中的基因工程应用,具体为敲除前述的基因OsMSRFP,提高水稻耐盐性;过表达前述的基因OsMSRFP,提高水稻盐敏感性。
Description
技术领域
本发明属于基因工程领域,具体涉及一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用。
背景技术
水稻是世界上最重要的粮食作物之一,全球一半以上的人口以稻米为主食,提高水稻产量对保障粮食安全意义重大。由于水稻种植常常依赖于灌溉,而不合理的灌溉促使地下水中的盐分沿土壤毛管孔隙上升并在地表积累,引起土壤次生盐渍化。在水稻种植地区盐渍化已成为普遍的土壤问题,极大限制了稻米生产。严重情况下,盐污染几乎完全阻碍了大面积土地上水稻的生长。因此,提高耐盐性已经成为水稻遗传改良的重要目标之一。
盐胁迫会对植物造成多个方面的伤害,包括渗透胁迫和离子毒害以及由此引起的次级胁迫如营养失衡和氧化胁迫等,严重危害植物的生长发育,使植物早衰甚至死亡,造成严重的产量损失。水稻属于甜土植物,耐盐性整体较差,不同生长发育时期受到盐胁迫伤害的程度也有所不同,其中幼苗期和生殖生长时期是对盐胁迫最为敏感的两个时期。水稻幼苗遭受盐胁迫,会导致根系生长迟缓、叶片卷曲枯萎,叶片伸长和新生叶的形成都会受到抑制。幼穗形成期和抽穗开花期遭受盐胁迫,会导致抽穗延迟、穗短粒少、育性降低、籽粒不饱满,最终导致产量下降。此外,在高盐胁迫下,稻米品质也会变劣,其加工品质、蒸煮食味品质和稻米淀粉黏滞特性明显降低,矿质元素含量、蛋白质含量等发生改变。
水稻对盐胁迫的应答由复杂的分子网络调控,耐盐性是多种生理生化反应的综合表现,采用传统育种方法进行水稻耐盐性改良难度较大。利用分子设计育种技术可以加快水稻耐盐新品种培育进程,但由于缺乏对耐盐分子基础的深入了解,相关工作进展缓慢。因此,挖掘优异耐盐新基因、解析其调控途径和分子机制尤为重要。
发明内容
为解决现有技术中的上述技术问题,本发明的目的在于公开一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用。
本发明的第一个目的是提供一种耐盐相关基因OsMSRFP,所述基因OsMSRFP为如下1)或2)或3)所述的DNA分子:
1)基因组序列如SEQ ID NO.1所示的DNA分子;
2)CDS序列如SEQ ID NO.2所示的DNA分子;
3)在严格条件下与1)或2)限定的DNA序列杂交且编码所述蛋白的DNA分子。
本发明的第二个目的是提供前述基因OsMSRFP编码的蛋白质。
进一步的,所述氨基酸序列如SEQ ID NO.3所示,或者,将SEQ ID NO.3的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且与耐盐性相关的由SEQ IDNO.3衍生的蛋白质。
本发明的第三个目的是提供一种包含前述基因OsMSRFP的重组表达载体、表达盒或重组菌。
进一步的,所述重组表达载体或表达盒是采用限制性内切酶BamH I和HindⅢ双酶切载体pCAMBIA1301的重组位点插入所述基因OsMSRFP得到;将所述重组表达载体或表达盒转入工程菌,得到所述重组菌。
含有以上任一所述基因的重组表达载体也属于本发明的保护范围。
可用现有的植物表达载体构建含有所述基因的重组表达载体。
所述植物表达载体包括双元农杆菌载体和可用于植物微弹轰击的载体等。所述植物表达载体还可包含外源基因的3’端非翻译区域,即包含聚腺苷酸信号和任何其它参与mRNA加工或基因表达的DNA片段。所述聚腺苷酸信号可引导聚腺苷酸加入到mRNA前体的3’端,如农杆菌冠瘿瘤诱导(Ti)质粒基因(如胭脂合成酶Nos基因)、植物基因(如大豆贮存蛋白基因)3’端转录的非翻译区均具有类似功能。
使用所述基因构建重组植物表达载体时,在其转录起始核苷酸前可加上任何一种增强型启动子或组成型启动子,如花椰菜花叶病毒(CAMV)35S启动子、玉米的泛素启动子(Ubiquitin),它们可单独使用或与其它的植物启动子结合使用;此外,使用本发明的基因构建植物表达载体时,还可使用增强子,包括翻译增强子或转录增强子,这些增强子区域可以是ATG起始密码子或邻接区域起始密码子等,但必需与编码序列的阅读框相同,以保证整个序列的正确翻译。所述翻译控制信号和起始密码子的来源是广泛的,可以是天然的,也可以是合成的。翻译起始区域可以来自转录起始区域或结构基因。
为了便于对转基因植物细胞或植物进行鉴定及筛选,可对所用植物表达载体进行加工,如加入可在植物中表达的编码可产生颜色变化的酶或发光化合物的基因(GUS基因、萤光素酶基因等)、具有抗性的抗生素标记物(庆大霉素标记物、卡那霉素标记物等)或是抗化学试剂标记基因(如抗除莠剂基因)等。从转基因植物的安全性考虑,可不加任何选择性标记基因,直接以逆境筛选转化植株。
所述重组表达载体可为在限制性内切酶BamH I和HindⅢ双酶切载体pCAMBIA1301的重组位点插入所述基因OsMSRFP得到的重组质粒。将含有OsMSRFP的pCAMBIA1301命名为pCAMBIA1301-OsMSRFP。
含有以上任一所述基因OsMSRFP的表达盒及重组菌均属于本发明的保护范围。
本发明的第四个目的是提供扩增前述基因OsMSRFP的引物。
扩增所述基因OsMSRFP全长或任一片段的引物对也属于本发明的保护范围;所述的引物对优选SEQ ID NO.4所示Primer1/SEQ ID NO.5所示Primer2、SEQ ID NO.9所示Primer5/SEQ ID NO.10所示Primer6。
本发明的第五个目的是提供前述的基因OsMSRFP、前述的蛋白质、前述的重组表达载体、表达盒或重组菌、或前述的引物在调控水稻耐盐性中的基因工程应用。
进一步的,敲除前述的基因OsMSRFP,提高水稻耐盐性。进一步的,过表达前述的基因OsMSRFP,提高水稻盐敏感性。
优选的,将前述的重组表达载体、表达盒或重组菌导入水稻,过表达前述的基因OsMSRFP。
本发明的第六个目的是提供一种调控水稻耐盐性的方法,所述方法为敲除水稻植株中前述基因OsMSRFP,从而提高水稻耐盐性;或过表达水稻植株中前述基因OsMSRFP,从而提高水稻盐敏感性。
具体地,可以利用CRISPR-Cas9技术将水稻中的所述基因进行编辑,敲除水稻植株中前述基因OsMSRFP,使该基因失去功能;
或利用前述的重组表达载体、表达盒或重组菌导入水稻,过表达水稻植株前述的基因OsMSRFP。
利用任何一种可以在植物中进行基因编辑的载体,将编码所述蛋白的基因进行敲除,可获得转基因细胞系及转基因植株。携带有所述基因的表达载体可通过使用Ti质粒、Ri质粒、植物病毒载体、直接DNA转化、显微注射、电导、农杆菌介导等常规生物学方法转化植物细胞或组织,并将转化的植物组织培育成植株。
有益效果:
本发明首次发现并克隆得到一个新的植物耐盐相关蛋白的基因OsMSRFP。本发明的植物耐盐相关蛋白影响植物的耐盐性。抑制该蛋白编码基因的表达可提高植物耐盐性,从而可以培育耐盐的转基因植物。将所述蛋白的编码基因导入植物中可导致植物耐盐性下降,从而可以培育盐敏感的转基因植物。所述蛋白及其编码基因可以应用于植物遗传改良。
附图说明
图1为野生型日本晴与突变体osmsrfp1和osmsrfp2中OsMSRFP基因的突变位点及其两侧序列。
图2为野生型日本晴与突变体osmsrfp1和osmsrfp2在盐胁迫下的表型(图A)和幼苗死亡率(图B)。
图3为野生型Dongjin与OsMSRFP过表达株系(OE-1、OE2和OE-3)中OsMSRFP基因的表达量。
图4为野生型Dongjin与OsMSRFP过表达株系(OE-1、OE2和OE-3)在盐胁迫下的表型(图A)和幼苗死亡率(图B)。
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。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
实施例1、水稻基因OsMSRFP的克隆
设计如下引物:
primer1:5'-ATGGGAGGGGCGCACTTCC-3'(SEQ ID NO.4);
primer2:5'-CTACGGATTGTTTTCGGACGAATCT-3'(SEQ ID NO.5)。
以primer1和primer2为引物,以日本晴幼苗叶片cDNA为模板,进行PCR扩增,获得目的基因OsMSRFP。
PCR扩增反应在Bio-rad T100 PCR仪中进行,反应体系(50μl)为:2×Phanta MaxBuffer 25μl,dNTP Mix(10mM)1μl,primer1(10uM)1.5μl,primer2(10uM)1.5μl,模板cDNA(50ng/μl)2μl,Phanta Max Super-Fidelity DNA Polymerase 1μl,ddH2O 18μl;程序为:95℃预变性3min;95℃变性15s、58℃退火15s、72℃延伸1min,循环35次;72℃延伸5min;15℃保存。
将PCR产物回收纯化后连接到pEASY-Blunt(北京全式金生物技术公司)上,转化大肠杆菌DH5α感受态细胞(北京Tiangen公司CB101),挑选阳性克隆后,进行测序。
测序结果表明,PCR反应获得的OsMSRFP基因片段具有SEQ ID NO.2所示的核苷酸序列,编码892个氨基酸残基组成的蛋白质(SEQ ID NO.3)。
实施例2、水稻OsMSRFP基因突变体转基因植株的获得和鉴定
一.OsMSRFP基因突变体转基因植株的获得
将OsMSRFP基因组序列(SEQ ID NO.1)提供给武汉伯远生物科技有限公司,由该商业公司设计CRISPR-Cas9 sgRNA靶点,构建OsMSRFP基因编辑载体,转化日本晴野生型愈伤组织,获得转基因T0代植株。
靶标序列为“ACGTGGGCAAGATGGAGCACGGG(SEQ ID NO.6)”,所用CRISPR-Cas9载体为pBWA(V)H_cas9i2。
二、OsMSRFP基因突变体植株的分子鉴定
将步骤一中获得的OsMSRFP基因突变体T0代植株提取基因组DNA作为模板,对SEQID NO.6所示编辑靶点及两侧的核苷酸片段进行PCR扩增,PCR引物序列如下:
Primer3:5'-CCAATTTCCATCGCAGCCAT-3'(SEQ ID NO.7);
Primer4:5'-AGGCTAAACTGGGGAAAAGGA-3'(SEQ ID NO.8)。
上述引物primer3和primer4均位于SEQ ID NO.1所示的OsMSRFP基因组序列中。
PCR产物用1%的琼脂糖电泳检测,送测序公司测序,结合测序峰图,筛选出OsMSRFP基因发生突变的两个突变体株系,即特异性敲除OsMSRFP的水稻株系(osmsrfp1和osmsrfp2)(图1)。
实施例3、水稻基因OsMSRFP过表达转基因植株的获得和鉴定
一.OsMSRFP基因过表达载体的构建
以日本晴幼苗叶片的基因组cDNA为模板,使用primer5和primer6进行PCR扩增,获得OsMSRFP基因全长CDS序列片段(SEQ ID NO.2)。
Primer5:5'-CGGGATCCATGGGAGGGGCGCACTTCC-3'(SEQ ID NO.9);
Primer6:5'-CCCAAGCTTCTACGGATTGTTTTCGGACGAATCT-3'(SEQ ID NO.10)。
扩增产物通过BamH I和HindⅢ双酶切位点连接至pCAMBIA1301载体,转化大肠杆菌DH5α,提取阳性质粒,进行测序。测序结果表明,得到了含有SEQ ID NO.2所示序列的重组表达载体,命名为pCAMBIA1301-OsMSRFP。
二.重组农杆菌的获得
用电击法将pCAMBIA1301-OsMSRFP转化农杆菌EHA105菌株,得到重组菌株,提取质粒进行PCR及酶切鉴定,将鉴定正确的重组菌株分别命名为EH-pCAMBIA1301-OsMSRF。
三.转基因植物的获得
将EH-pCAMBIA1301-OsMSRF菌株转化水稻品种Dongjin,具体方法为:
(1)28℃、200r过夜培养EH-pCAMBIA1301-OsMSRFP菌株,至OD600饱和;将菌液1:100接种到新的YEP液体培养基中,28℃、200r振荡培养至OD600=0.6-0.8,收集菌体;挑取农杆菌至30ml AAM感菌液中(含30ul 1000*AS),轻轻混匀,悬浮细胞,使OD600=0.05-0.1。
(2)将培养至一个月的日本晴野生型成熟胚胚性愈伤组织与步骤(1)的菌液混合侵染90s,滤纸吸干菌液后转入2N6-AS固体培养基上,28℃暗培养48-60h;
(3)将步骤(2)的愈伤取出,放入50ml的离心管中,加无菌水清洗8-10次,其间不断震荡,至水清澈为止;再用含有500mg/L Car的无菌水清洗2次,每次10-20分钟;去除无菌水,将离心管中的愈伤倒在无菌滤纸上沥干1-2小时;
(4)将步骤(3)的愈伤接种在含有50mg/L潮霉素的N6D-S固体培养基上,29.5℃培养,筛选3-4周;
(5)将筛选到的抗性愈伤转移到MS-NK固体培养基上,29.5℃培养3-4周,使愈伤分化产生不定芽;
(6)待不定芽生长至3-4cm时,将其转移到MS-HF生根培养基中生长1周;
(7)将分化出的转基因幼苗炼苗后转移至大田生长,获得的T0代植株。
四、转基因植株的鉴定
1、PCR分子鉴定
将步骤三获得的T0代植株提取基因组DNA作为模板,进行PCR扩增,PCR引物序列如下:
Primer7:5'-TGAGCGGATAACAATTTCACAC-3'(SEQ ID NO.11);
primer8:5'-GGATTGTTTTCGGACGAATCT-3'(SEQ ID NO.12)。
上述引物primer7位于pCAMBIA1301载体序列中,primer8位于SEQ ID NO.2所示的OsMSRFP基因的CDS序列中。
PCR产物用1%的琼脂糖电泳检测,阳性植株中可以检测到目标条带,而阴性植株中不能检测到。
2、OsMSRFP基因表达量的检测
将1中PCR分子鉴定筛选到的阳性OsMSRFP过表达转基因植株自交两代,获得纯合转基因株系,进行OsMSRFP基因过表达情况鉴定。两叶一心期幼苗取地上部,液氮冷冻匀样后利用TRIzol法提取RNA,取适量RNA利用反转录试剂盒获得cDNA作为荧光定量RT-PCR检测用的模板。取适量模板cDNA,以水稻OsUBQ5作为内参基因,利用HieffTM qPCRGreenMaster Mix(No Rox Plus)试剂盒,在Bio-Rad荧光定量PCR仪CFX96中检测OsMSRFP基因的表达量。所用OsUBQ5基因定量检测引物(primer9和primer10)和OsMSRFP基因定量检测引物(primer11和primer12)序列如下:
Primer9:5'-ACCACTTCGACCGCCACTACT-3'(SEQ ID NO.13);
Primer10:5'-ACGCCTAAGCCTGCTGGTT-3'(SEQ ID NO.14)。
Primer11:5'-ACACGAGTTCTCGACTGTGG-3'(SEQ ID NO.15);
primer12:5'-AACAATGCCCAGTGATGTGT-3'(SEQ ID NO.16)。
相对表达量采用2-△△CT法进行定量计算,所得结果如图3所示,以Dongjin野生型OsMSRFP相对OsUBQ5的表达量为1,3个过表达株系(OE-1、OE2和OE-3)中OsMSRFP基因的相对表达量均升高一倍多。
实施例4、OsMSRFP基因突变体和过表达转基因植株耐盐性鉴定
将实施例2筛选鉴定到的OsMSRFP基因突变体和实施例3筛选鉴定到的OsMSRFP基因过表达转基因植株分别自交两代,获得纯合OsMSRFP基因特异性敲除的突变株系和纯合OsMSRFP基因过表达转基因株系,用于幼苗期耐盐性鉴定。
幼苗期耐盐性鉴定具体方法为:
1.将突变体株系和过表达株系的水稻种子置于45℃烘箱处理一周打破休眠,28℃浸种48h,30℃催芽,直至萌发。
2.将萌发一致的种子播于去掉底部的96孔PCR板,放在黑色有机玻璃盒中,于人工气候室中培养。用纯水培养一周后,更换为用Yoshida营养液培养,每3天更换一次。培养条件为:14小时光照(28℃)/10小时黑暗(24℃),光照强度800μmol·m–2·s–1,相对湿度70%。
3.当幼苗生长至两叶一心时进行盐胁迫处理,在各组的Yoshida营养液中加入120Mm NaCl,每3天更换一次,以不添加NaCl的Yoshida营养液为对照(Control)。
4.盐处理7d后,将幼苗重新置于不添加NaCl的Yoshida营养液中,7d后统计各组幼苗存活率。
图2的实验结果显示,相较于其野生型日本晴(WT),两个OsMSRFP基因特异性敲除的突变体转基因株系在盐胁迫下均表现出较轻的叶片失绿和萎蔫症状,幼苗存活率显著提高,死亡率显著降低。
图4的实验结果显示,与其野生型Dongjin相比,三个OsMSRFP基因过表达转基因株系在盐胁迫下均表现出较重的叶片失绿和萎蔫症状,幼苗存活率显著降低,死亡率显著升高。
因此,根据这些实验结果可以明确OsMSRFP基因对水稻幼苗耐盐性具有重要的负向调控作用,敲除该蛋白编码基因可提高植物耐盐性,过表达该蛋白编码基因可提高水稻盐敏感性。
序列表
<110> 南京农业大学
<120> 一种水稻耐盐相关基因OsMSRFP及其编码蛋白质和应用
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 3658
<212> DNA
<213> 水稻(Oryza sativa)
<400> 1
gtgagtggcc tagcccggcc aatttccatc gcagccattt ttacccctag ccccctccgc 60
ttcctccttt ttacatccaa acccctcttc cactcccctc gccgccgccg cctccgccgt 120
cggggaggat gggaggggcg cacttccccg gtgagggcga ggtggtggcc ggggagggcg 180
acgccgtcgt ccccctccgc gacgtgggca agatggagca cgggtgagat ttcgcgggga 240
cctcctcctt ttttcccctc ttccccacgg gttcttgatt tgttctttcg cggtgaggtg 300
tgggatgatt ctgttcttcg tagggggtgc tgtgctgttt ccgctcgcta gctgtttcaa 360
tctttgtttc gcttgttctg ctggatttgc tgatctatgc ttttgctttt tggttgctga 420
atttcactga gtaaatctgc ccccctcccc gcgccatctc ttgttgtgtg caggtgcgag 480
cattacagga ggagatgcaa gatcgtggcg ccctgctgcg gcgaggtgtt cgcttgccgc 540
cattgccaca acgatgccac ggtaaccatg ctctgctgct gcggttgttt atcttgttca 600
tgtagaaggg gatttccatg ggatgccaag tgattagtat ttagttagtg cactgccatg 660
ctatctagta ctagtattgt ttggtagttc ttgcctggtt ggatatggaa ctagatcccc 720
caatcatcca aacatcaagt cttgaatggc aagctgacaa gtttgagctg attccttcat 780
atggaattgg tattttagtt cctataaatg cgctagtttt agtacttgac tccttttccc 840
cagtttagcc ttttgaacaa cttttcttgt ttgaacttat gcatgaaaag aaagttggtg 900
gtatagagac caatgaaatg caagtcgcag ggtgttgagt caatcagggt agaacataaa 960
aaaaagactt aacttgtcga tagttacttc caacatggag cttcaaaatc atttgagaat 1020
aagggcacat aatggagttc tgtaatccct tgcagaagtt aaaaagagaa actaaagaac 1080
aaaactgttt taactgttga gtttaactag gtttgatggt attgatattg ttctcattgt 1140
aggcttcagg tgatcggcat accatttgcc gccaggatgt tgaaaaagta cacctctcat 1200
gtcctttcta gtttggtttg tacaattgat actttattag gttcagaaat tccataaggt 1260
tgactttatt ctttcctgca ggtagtatgt ctactctgtg atactgaaca gccggtgcga 1320
cttaatttct agaagatact gtttacaata ccagtgtcat gcgatgagac catttataca 1380
tacactgact ttattttgtt tttcaggtgt cacaagtgtg cataaactgt ggtgtcaata 1440
tgggagagta cttttgtgat gtatgcaagt tttatgatga tgatgtaggt tcttaaattt 1500
attttctgtg gttatatgct ttttgggtgg ttcttatccg ttctcaactg atgcattcgt 1560
ctttttgcta cagacagaga aagggcagtt tcattgctac gattgtggca tatgcaggtg 1620
agcatgaaac aatagctcag caattatctt gtgaatcata gttctaattc tgtatccgct 1680
taatcaacca gggttggtgg caaggaaaac tacttccact gcgcaaagtg cggtatgctt 1740
tgtctgcact agtgtgatat gtcactttaa gataaatctt tccaatttgt gctgatcatt 1800
tttaactaac aagtgtggta tgtagatcat gtcaatatgc cataaatgca atcatagtct 1860
atttaggtga tactgaattt ggtaattaaa aaaaattgtt gcccagatga cttattttat 1920
tgcatagctt tctagtttct atcctaaaaa tgtcaatatg atgactattt ttttttctgg 1980
taatggttat atgtagtatc tattaattaa ttttgctgca acacgtaggg tcttgttatg 2040
ctgttgctct gcgtgataac catcagtgtg tggagaactc aatgaggcag aattgcccaa 2100
tttgttatga ggtaactttg agaacttgca ctctttcctt cctggttagc aacttagcaa 2160
gatgataaaa ctgaacttcc tgtgcagtat ctatttgatt cattaaaagg aacacgagtt 2220
ctcgactgtg gacacacgat gcacatggaa tgcttttctg agatggtgga acataacaag 2280
taaatccctc cacttgttgc cattattcat ttgcttaata cttctctgac actgtaacca 2340
gttcttgcct ttttttggag atataactag ttcctggcta tttggattat acagatacac 2400
ttgtccaata tgctccaaga cagctcttga tatgacacat cactgggcat tgttagatca 2460
agaggtatgt atttcttttc tacagattat aagtaaagtg tcacggtcaa acaaactcgg 2520
catattcaca ctgcagtgaa agaaacaatc tgaaacatta ttagaacaaa actagttgat 2580
gatcctcctg cccggaaatc aggaaatacg ttccattgga gatgagttgt gtccagtaaa 2640
actagtcagg catattggca ctccagtagc atctatcata cacttttatg atgatttgat 2700
ttgaacattt tagatatgat tggtgctttg ttaaataata acaagacgat gtgttctttt 2760
gtagattgaa gcaacaatca tgcctcccgt ctaccgttac aaggtattct ttttctgatt 2820
tagctacaca gttcaccttg ctttcattgc caaactgaag gttatgcttt gctaagaaat 2880
tcagaagtca gaactcacct cgaaaccttt ttctgctgcc tttttttttt tgataaatca 2940
tccaaaaata tgcagcaaaa gcagggacct tcattattat atatactgtt gacgatcact 3000
atcatgcaaa tcacacacaa tacccagatt taggctaaca aggtgttctt tccttgtgaa 3060
aattactagg tttgggtgct ctgtaatgac tgcaacaagg tctcagaagt ggacttccat 3120
gtgattggcc acaagtgcag ccactgcaac tcgtacaata ctcgatcgac atcacggcct 3180
gcggatttat caggaagcag ttctccctcg acgtcagatt cgtccgaaaa caatccgtag 3240
ataacatgcc atgttggtgc ccaacaagat ctacagttta gacaatcaag tcccagattc 3300
atacatatac aagtagtaga aggaacattg ttttgcggct agcctatcca agtgcctacc 3360
caattcttca agattcaatt gattccttga atcttcatgt gttggaactg ctactagaca 3420
aaagctattg tgatgatacc atatgttgtg ttgaatcttt aatcacagcc atggctgtta 3480
tcagtgtggg tgaaagtgaa acccatcatg ttatccagga gctgaatgtt gatgattgca 3540
atccatggaa aatgggtatg cgtactctac atgttgatga ttgtatgttg gttcagattt 3600
gtcttcgttt tacagtaagt gggcaaactt atcaacttaa gattcaataa gctaataa 3658
<210> 2
<211> 879
<212> DNA
<213> 水稻(Oryza sativa)
<400> 2
atgggagggg cgcacttccc cggtgagggc gaggtggtgg ccggggaggg cgacgccgtc 60
gtccccctcc gcgacgtggg caagatggag cacgggtgcg agcattacag gaggagatgc 120
aagatcgtgg cgccctgctg cggcgaggtg ttcgcttgcc gccattgcca caacgatgcc 180
acggcttcag gtgatcggca taccatttgc cgccaggatg ttgaaaaagt agtatgtcta 240
ctctgtgata ctgaacagcc ggtgtcacaa gtgtgcataa actgtggtgt caatatggga 300
gagtactttt gtgatgtatg caagttttat gatgatgata cagagaaagg gcagtttcat 360
tgctacgatt gtggcatatg cagggttggt ggcaaggaaa actacttcca ctgcgcaaag 420
tgcgggtctt gttatgctgt tgctctgcgt gataaccatc agtgtgtgga gaactcaatg 480
aggcagaatt gcccaatttg ttatgagtat ctatttgatt cattaaaagg aacacgagtt 540
ctcgactgtg gacacacgat gcacatggaa tgcttttctg agatggtgga acataacaaa 600
tacacttgtc caatatgctc caagacagct cttgatatga cacatcactg ggcattgtta 660
gatcaagaga ttgaagcaac aatcatgcct cccgtctacc gttacaaggt ttgggtgctc 720
tgtaatgact gcaacaaggt ctcagaagtg gacttccatg tgattggcca caagtgcagc 780
cactgcaact cgtacaatac tcgatcgaca tcacggcctg cggatttatc aggaagcagt 840
tctccctcga cgtcagattc gtccgaaaac aatccgtag 879
<210> 3
<211> 292
<212> PRT
<213> 水稻(Oryza sativa)
<400> 3
Met Gly Gly Ala His Pro Pro Gly Gly Gly Gly Val Val Ala Gly Gly
1 5 10 15
Gly Ala Ala Val Val Pro Leu Ala Ala Val Gly Leu Met Gly His Gly
20 25 30
Cys Gly His Thr Ala Ala Ala Cys Leu Ile Val Ala Pro Cys Cys Gly
35 40 45
Gly Val Pro Ala Cys Ala His Cys His Ala Ala Ala Thr Ala Ser Gly
50 55 60
Ala Ala His Thr Ile Cys Ala Gly Ala Val Gly Leu Val Val Cys Leu
65 70 75 80
Leu Cys Ala Thr Gly Gly Pro Val Ser Gly Val Cys Ile Ala Cys Gly
85 90 95
Val Ala Met Gly Gly Thr Pro Cys Ala Val Cys Leu Pro Thr Ala Ala
100 105 110
Ala Thr Gly Leu Gly Gly Pro His Cys Thr Ala Cys Gly Ile Cys Ala
115 120 125
Val Gly Gly Leu Gly Ala Thr Pro His Cys Ala Leu Cys Gly Ser Cys
130 135 140
Thr Ala Val Ala Leu Ala Ala Ala His Gly Cys Val Gly Ala Ser Met
145 150 155 160
Ala Gly Ala Cys Pro Ile Cys Thr Gly Thr Leu Pro Ala Ser Leu Leu
165 170 175
Gly Thr Ala Val Leu Ala Cys Gly His Thr Met His Met Gly Cys Pro
180 185 190
Ser Gly Met Val Gly His Ala Leu Thr Thr Cys Pro Ile Cys Ser Leu
195 200 205
Thr Ala Leu Ala Met Thr His His Thr Ala Leu Leu Ala Gly Gly Ile
210 215 220
Gly Ala Thr Ile Met Pro Pro Val Thr Ala Thr Leu Val Thr Val Leu
225 230 235 240
Cys Ala Ala Cys Ala Leu Val Ser Gly Val Ala Pro His Val Ile Gly
245 250 255
His Leu Cys Ser His Cys Ala Ser Thr Ala Thr Ala Ser Thr Ser Ala
260 265 270
Pro Ala Ala Leu Ser Gly Ser Ser Ser Pro Ser Thr Ser Ala Ser Ser
275 280 285
Gly Ala Ala Pro
290
<210> 5
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atgggagggg cgcacttcc 19
<210> 5
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
ctacggattg ttttcggacg aatct 25
<210> 6
<211> 23
<212> DNA
<213> 水稻(Oryza sativa)
<400> 6
acgtgggcaa gatggagcac ggg 23
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
ccaatttcca tcgcagccat 20
<210> 8
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aggctaaact ggggaaaagg a 21
<210> 9
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cgggatccat gggaggggcg cacttcc 27
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cccaagcttc tacggattgt tttcggacga atct 34
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tgagcggata acaatttcac ac 22
<210> 12
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
ggattgtttt cggacgaatc t 21
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
accacttcga ccgccactac t 21
<210> 14
<211> 19
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
acgcctaagc ctgctggtt 19
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
acacgagttc tcgactgtgg 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
aacaatgccc agtgatgtgt 20
Claims (3)
1.耐盐相关基因OsMSRFP,或基因OsMSRFP编码的蛋白质,或包含基因OsMSRFP的重组表达载体、表达盒或重组菌,或扩增基因OsMSRFP的引物在调控水稻耐盐性中的基因工程应用;
所述基因OsMSRFP为如下1)或2)所示的DNA分子:
1)SEQ ID NO. 1所示的DNA分子;
2)SEQ ID NO. 2所示的DNA分子。
2.根据权利要求1所述的应用,其特征在于,敲除权利要求1所述的基因OsMSRFP,提高水稻耐盐性。
3.一种调控水稻耐盐性的方法,其特征在于,所述方法为敲除水稻植株中权利要求1中所述基因OsMSRFP,从而提高水稻耐盐性。
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An apple MYB transcription factor regulates cold tolerance and anthocyanin accumulation and undergoes MIEL1-mediated degradation;Jian-Ping An等;Plant Biotechnology Journa;第18卷;第337–353页 * |
E3 ubiquitin-protein ligase MIEL1 [Oryza sativa Japonica Group] NCBI Reference Sequence: XP_015618332.1;NCBI;NCBI;第1-2页 * |
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