CN113930440B - 一种通过抑制OsSDP基因表达提高水稻耐盐性的方法 - Google Patents
一种通过抑制OsSDP基因表达提高水稻耐盐性的方法 Download PDFInfo
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Abstract
本发明公开了一种通过抑制OsSDP基因表达提高水稻耐盐性的方法。本发明提供了OsSDP蛋白或其相关生物材料在调控植物耐盐性中的应用;所述相关生物材料为能够表达所述OsSDP蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。本发明实验显示敲除水稻日本晴中OsSDP蛋白的编码基因后植株的耐盐性提高,证明OsSDP蛋白及其编码基因可以调控植物的耐盐性,对于培育植物耐盐新品种具有重要意义。
Description
技术领域
本发明涉及基因工程领域,具体涉及一种通过抑制OsSDP基因表达提高水稻耐盐性的方法。
背景技术
在水稻的生长发育过程中会受到各种各样的非生物胁迫,如高盐、高温、干旱及冷害等。水稻最早起源于淡水沼泽环境,是一种对盐胁迫中度敏感的作物,土壤盐化自然也成为了影响水稻产量的主要不利因素之一。
盐胁迫是由土壤或溶液中高浓度的盐离子引发而产生的对作物的危害作用,包括直接的离子毒害作用和间接的渗透胁迫、离子失衡和营养缺乏等。盐胁迫几乎会影响水稻所有的生长代谢过程。在盐胁迫下,水稻会产生各种生理生化变化,根据前人的研究,盐胁迫对水稻的伤害机制主要有以下几方面:(1)过多的盐离子破坏水稻细胞质膜的完整性,导致其选择透过性能下降直至消失,从而使细胞内Na+、Cl-等离子大量积累,K+、Ca2+等营养元素则大量外渗,引起细胞内离子浓度的动态平衡失调,破坏细胞膜和细胞器的结构,引起叶片的气孔导度、叶绿素含量以及核酸含量等下降,引发一系列的代谢紊乱,使细胞的功能下降,加速衰老或死亡;(2)高盐降低土壤溶液的水势,形成水分胁迫,使水稻根部吸水困难,细胞内盐分浓度提高,从而生理代谢失调,进而影响水稻的出苗和生长发育;(3)盐分胁迫促使活性氧O2 -、H2O2、OH等的产量增加,破坏或减弱细胞内抗氧化系统如超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POX)、谷胱甘肽(GSH)、抗坏血酸(ASC)等的活性或含量,从而影响体内活性氧代谢系统的平衡;活性氧含量的增加能加剧膜质过氧化,破坏膜的完整性,选择透过性丧失,电解质及某些小分子有机物大量渗入,物质交换平衡被破坏,使水稻收到伤害;(4)盐胁迫降低了光合速率,同化物和能量的供给减少,抑制蛋白质的合成,此外水稻为了适应高盐环境和维持其生长,需要消耗更多的能量来进行离子的主动吸收和运输、区域化分配以及渗透调节物质的合成,从而限制水稻的生长发育,大大影响其生产量和品质。
水稻不同品种、不同时期和不同器官受盐胁迫伤害的程度不同,耐盐性也不同。在水稻种子萌发阶段,由于吸水受到高盐抑制,种子发芽不齐,发芽率降低,甚至根本不能发芽而在土内变质腐烂。种子萌发后,对盐胁迫极为敏感,会表现出芽尖枯黄、弯曲,迟迟不能绿化,直至出苗后的秧苗死亡。出苗后,水稻从异养阶段过渡到自养阶段,此时若遭遇盐害,则会发生卷叶和枯萎,叶片伸长和新生叶的形成都会受到抑制。水稻分蘖期、幼穗形成期和抽穗开花期遭受盐胁迫,其分蘖和伸长将受到抑制,无效分蘖增多,抽穗推迟,穗短粒少,籽粒不饱满,最终导致产量下降。
水稻对盐胁迫的应答适应和耐盐性是近年来研究的一个重点和难点。耐盐性是水稻能在高盐基质上生长,并忍耐或抵抗盐胁迫并完成整个生命周期的能力。水稻的耐盐性是多个生理过程综合作用的表现,深入开展水稻对盐胁迫的适应和耐盐性的研究,对培育耐盐水稻品种,进一步扩大水稻的可种植面积,保证粮食的安全生产和品质具有重要意义。
发明内容
本发明的目的是提供一种通过抑制OsSDP基因表达提高水稻耐盐性的方法。
第一方面,本发明要求保护OsSDP蛋白或其相关生物材料在调控植物耐盐性中的应用。
所述相关生物材料为能够表达所述OsSDP蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系。
所述OsSDP蛋白可为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)将SEQ ID No.1所示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/或添加且具有相同功能的蛋白质;
(A3)与(A1)-(A2)中任一所限定的氨基酸序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且具有相同功能的蛋白质;
(A4)在(A1)-(A3)中任一所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白。
上述蛋白质中,所述标签是指利用DNA体外重组技术,与目的蛋白一起融合表达的一种多肽或者蛋白,以便于目的蛋白的表达、检测、示踪和/或纯化。所述标签可为Flag标签、His标签、MBP标签、HA标签、myc标签、GST标签和/或SUMO标签等。
上述蛋白质中,同源性是指氨基酸序列的同一性。可使用国际互联网上的同源性检索站点测定氨基酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对氨基酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述蛋白质中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
在所述应用中,所述OsSDP蛋白在所述植物中的表达量和/或活性降低,所述植物的耐盐性提高。所述OsSDP蛋白在所述植物中的表达量和/或活性提高,所述植物的耐盐性降低。
第二方面,本发明要求保护一种培育耐盐性改变的植物品种的方法。
本发明所要求保护的培育耐盐性改变的植物品种的方法,可为如下方法A1或方法A2:
方法A1:一种培育耐盐性提高的植物品种的方法(或称为“提高植物耐盐性的方法”),可包括使受体植物中OsSDP蛋白的表达量和/或活性降低的步骤。
方法B:一种培育耐盐性降低的植物品种的方法(或称为“降低植物耐盐性的方法”),可包括使受体植物中OsSDP蛋白的表达量和/或活性提高的步骤。
其中,所述OsSDP蛋白为前文(A1)-(A4)中任一所示蛋白。
第三方面,本发明要求保护一种培育耐盐性改变的转基因植物的方法。
本发明所要求保护的培育耐盐性改变的转基因植物的方法,可为如下方法B1或方法B2:
方法B1:一种培育耐盐性提高的转基因植物的方法,包括如下步骤:对受体植物中能够表达OsSDP蛋白的核酸分子进行抑制表达,得到转基因植物;所述转基因植物与所述受体植物相比耐盐性提高。
方法B2:一种培育耐盐性降低的转基因植物的方法,包括如下步骤:向受体植物中导入能够表达OsSDP蛋白的核酸分子,得到转基因植物;所述转基因植物与所述受体植物相比耐盐性降低。
其中,所述OsSDP蛋白为前文(A1)-(A4)中任一所示蛋白。
在所述方法B1中,对所述受体植物中能够表达所述OsSDP蛋白的核酸分子进行抑制表达可通过任何能够实现这一目的的技术手段实现,如通过序列特异核酸酶(如CRISPR/Cas9核酸酶)对所述核酸分子进行特异性剪切,从而降低其在所述受体植株中的表达。
在本发明中,具体是通过CRISPER/Cas9技术实现的;以SEQ ID No.3所示DNA片段中符合5’-NX-NGG-3’或5’-CCN-NX-3’序列排列规则的片段为靶序列;N表示A、G、C和T中的任一种,14≤X≤30,且X为整数,NX表示X个连续的脱氧核糖核苷酸。更加具体的,在本发明的一个具体实施例中,所述靶序列具体为SEQ ID No.4。
在上述方法B2中,所述核酸分子可通过重组载体的形式导入所述受体植物中。
在所述方法中,将携带有所述核酸分子的所述重组载体或者用于对所述受体植物中所述核酸分子进行敲除或抑制表达时采用的基因编辑工具导入所述受体植物,具体可为: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。
在上述各方面中,能够表达所述OsSDP蛋白的核酸分子可为如下任一所述的DNA分子:
(a1)SEQ ID No.2所示的DNA分子(cDNA);
(a2)SEQ ID No.3所示的DNA分子(基因组DNA);
(a3)在严格条件下与(a1)-(a2)中任一限定的DNA分子杂交且编码所述OsSDP蛋白的DNA分子;
(a4)与(a1)-(a3)中任一限定的DNA序列具有99%以上、95%以上、90%以上、85%以上或者80%以上同源性且编码所述OsSDP蛋白的DNA分子。
上述核酸分子中,所述严格条件可为如下:50℃,在7%十二烷基硫酸钠(SDS)、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,2×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.5×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在50℃,0.1×SSC,0.1%SDS中漂洗;还可为:50℃,在7%SDS、0.5M Na3PO4和1mM EDTA的混合溶液中杂交,在65℃,0.1×SSC,0.1%SDS中漂洗;也可为:在6×SSC,0.5%SDS的溶液中,在65℃下杂交,然后用2×SSC,0.1%SDS和1×SSC,0.1%SDS各洗膜一次。
上述核酸分子中,同源性是指核苷酸序列的同一性。可使用国际互联网上的同源性检索站点测定核苷酸序列的同一性,如NCBI主页网站的BLAST网页。例如,可在高级BLAST2.1中,通过使用blastp作为程序,将Expect值设置为10,将所有Filter设置为OFF,使用BLOSUM62作为Matrix,将Gap existence cost,Per residue gap cost和Lambda ratio分别设置为11,1和0.85(缺省值)并进行检索一对核苷酸序列的同一性进行计算,然后即可获得同一性的值(%)。
上述核酸分子中,所述95%以上的同源性可为至少96%、97%、98%的同一性。所述90%以上的同源性可为至少91%、92%、93%、94%的同一性。所述85%以上的同源性可为至少86%、87%、88%、89%的同一性。所述80%以上的同源性可为至少81%、82%、83%、84%的同一性。
在上述各方面中,所述植物可为单子叶植物。
进一步地,所述单子叶植物可为禾本科植物。
更进一步地,所述禾本科植物可为水稻。
在本发明的具体实施方式中,所述水稻具体为水稻品种日本晴。
实验证明,敲除了水稻日本晴中OsSDP蛋白的编码基因后植株的耐盐性提高,本发明证明OsSDP蛋白及其编码基因可以调控植物的耐盐性,对于培育植物耐盐新品种具有重要意义。
附图说明
图1为CRISPR/Cas载体BGK03的元件示意图。
图2为pCRISPR-OsSDP转基因植株鉴定测序结果。
图3为ossdp突变体耐盐性检测。A为NIP和ossdp-1植株在盐处理前后的生长状态,bar=5cm;B为NIP和ossdp-1盐处理前后的存活率统计。显示的值为平均值±标准差,n=3。*,差异显著,P<0.05;**,差异极显著,P<0.01,统计分析方法为单因素方差分析。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南,并不以任何方式构成对本发明的限制。
下述实施例中的实验方法,如无特殊说明,均为常规方法,按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
CRISPR/Cas载体BGK03:杭州百格生物技术有限公司,产品目录号BGK03。
N6D2培养基:含300mg/L水解酪蛋白、500mg/L脯氨酸、500mg/L谷氨酰胺、30g/L蔗糖和2mg/L 2,4-D的固体MS培养基。
N6D2S1培养基:含25mg/L潮霉素和600mg/L头孢霉素的N6D2培养基。
N6D2S2培养基:含50mg/L潮霉素和300mg/L头孢霉素的N6D2培养基。
分化培养基A:含300mg/L水解酪蛋白、50mg/L潮霉素、1mg/L 6-BA、0.5mg/L KT、0.2mg/L ZT、0.25mg/L NAA、30g/L蔗糖和30g/L山梨醇的N6D2培养基。
分化培养基B:含300mg/L水解酪蛋白、50mg/L潮霉素、1mg/L 6-BA、0.5mg/L KT、0.2mg/L ZT、0.5mg/L NAA、30/L蔗糖和20g/L山梨醇的N6D2培养基。
生根壮苗培养基:含1mg/L多效唑和0.5mg/L NAA的固体1/2MS培养基。
木村B培养液的配方见表1,pH值为5.8,溶剂为水。
表1木村B培养液的配方
水稻日本晴中的OsSDP蛋白如SEQ ID No.1所示。水稻日本晴的cDNA中编码OsSDP蛋白的开放阅读框如SEQ ID No.2所示。水稻日本晴的基因组DNA中编码OsSDP蛋白的基因如SEQ ID No.3所示。
实施例1、通过CRISPER/Cas9技术抑制OsSDP基因从而增强水稻耐盐性
一、重组质粒的构建
靶序列为:5’-GTCTCCAGAGACCGCGCGG-3’(SEQ ID No.4)
1、合成单链DNA分子Ⅰ和单链DNA分子Ⅱ。
单链DNA分子Ⅰ:5’-TGTGTGGTCTCCAGAGACCGCGCGG-3’;
单链DNA分子Ⅱ:5’-AAACCCGCGCGGTCTCTGGAGACCA-3’。
2、将单链DNA分子Ⅰ和单链DNA分子Ⅱ进行退火,得到具有粘末端的双链DNA分子。
3、取步骤2得到的双链DNA分子,与CRISPR/Cas载体BGK03(元件示意图如图1所示)连接,得到重组质粒pCRISPR-OsSDP。
根据测序结果,对重组质粒pCRISPR-OsSDP进行结构描述如下:具有SEQ ID No.4所示的DNA分子,表达SEQ ID No.5所示的sgRNA。
二、制备转基因植株
1、将重组质粒pCRISPR-OsSDP导入根癌农杆菌EHA105,得到重组农杆菌。
2、取步骤1得到的重组农杆菌的菌液,对日本晴的愈伤组织进行浸染,然后用含300mg/L头孢霉素的无菌水洗涤4-5遍,然后无菌滤纸吸干,然后转移至N6D2S1培养基上,培养2周。
3、完成步骤2后,取愈伤组织,转移到N6D2S2培养基上培养2周,然后转移到新的N6D2S2培养基上培养2周。
4、完成步骤3后,取生长旺盛的愈伤组织,转移到分化培养基A上培养7d,然后转移到分化培养基B上培养至长出再生苗。培养条件:12小时光照/12小时黑暗;光照强度为8000lux;光照时温度为28℃,黑暗时温度为25℃。
5、完成步骤4后,将再生苗转移到生根壮苗培养基上进行培养,待小苗长至10cm左右时,打开容器封口膜,炼苗2-3d,然后将小苗移入人工气候室栽培。
6、步骤5得到的再生植株,取叶片,进行OsSDP基因突变的检测。
(1)提取叶片的基因组DNA。
(2)以步骤(1)得到的基因组DNA为模板,采用F1和R1组成的引物对进行PCR和测序鉴定。
F1:5’-CTGGACGACCTCATCAAG-3’;
R1:5’-TTGGCAATTCTGGGATTCA-3’。
最终得到1个OsSDP基因发生突变的转基因单株,将其命名为ossdp-1,如图2所示。ossdp-1两个等位基因都发生了碱基的插入,最终导致移码突变,OsSDP完全缺失。
实验同时设置向根癌农杆菌EHA105中导入CRISPR/Cas载体BGK03后侵染日本晴的空载对照。
三、ossdp突变体的耐盐性鉴定
光暗交替培养的参数如下:光照强度为120μmol·m-2·s-1,温度为28℃/25℃(白天/黑暗),光周期10h光照/14h黑暗。
待测水稻种子为ossdp-1突变体T2代纯合种子及其背景材料日本晴(NIP)和空载对照。
实验重复3次取平均值,每次重复的步骤如下:
1、针对各材料,分别取待测水稻种子32粒,分装种子于牛皮纸袋中,放于28℃~30℃水中浸种48h。
2、完成步骤1后,将种子在28℃~30℃条件下催芽24h(催芽过程中需保持种子湿润),获得已催芽的种子。
3、完成步骤2后,取96孔板,每孔剪去部分下缘,然后每孔放入1粒已催芽的种子(胚芽向上、胚根向下)。
4、完成步骤3后,将所述96孔板(其上有已催芽的种子)置于盛有木村B培养液的塑料盒上并使已催芽的种子浸于木村B培养液,光暗交替培养3周,得到生长至三叶期阶段的水稻幼苗。光暗交替培养期间,每隔7d需更换木村B培养液。
5、完成步骤4后,将所述96孔板(其上有生长至三叶期阶段的水稻幼苗)置于分别盛有含180mM NaCl和200mM NaCl的木村B培养液的塑料盒上并使根部完全浸于含180mMNaCl和200mM NaCl的木村B培养液,光暗交替培养下高盐胁迫8d(高盐胁迫期间,每隔2d更换一次含180mM NaCl和200mM NaCl的木村B培养液)。
6、完成步骤5后,将所述96孔板(其上有水稻幼苗)置于盛有木村B培养液的塑料盒上并使根部完全浸于木村B培养液,光暗交替培养下恢复15d。
观察水稻幼苗的生长状态并统计存活率。存活率=存活的水稻幼苗数/32×100%。
水稻幼苗的生长状态见图3中A。存活率统计结果见图3中B。
结果表明,盐处理前,ossdp-1与NIP相比略矮;180mM NaCl盐溶液处理后,NIP的存活率为26.5%,而ossdp-1的存活率为75%;200mM NaCl盐溶液处理后,NIP的存活率为11.2%,而ossdp-1的存活率为56%,统计学分析结果表明ossdp-1的存活率显著高于NIP,说明ossdp-1突变体的耐盐性得到了显著的提高。空载对照的表型和存活率与背景材料日本晴(NIP)基本一致,无统计学差异。
以上对本发明进行了详述。对于本领域技术人员来说,在不脱离本发明的宗旨和范围,以及无需进行不必要的实验情况下,可在等同参数、浓度和条件下,在较宽范围内实施本发明。虽然本发明给出了特殊的实施例,应该理解为,可以对本发明作进一步的改进。总之,按本发明的原理,本申请欲包括任何变更、用途或对本发明的改进,包括脱离了本申请中已公开范围,而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围,可以进行一些基本特征的应用。
<110> 中国科学院植物研究所
<120> 一种通过抑制OsSDP基因表达提高水稻耐盐性的方法
<130> GNCLN201474
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 277
<212> PRT
<213> Oryza sativa L.
<400> 1
Met Ser Ser Gly Leu Asp Met Ser Leu Asp Asp Leu Ile Lys Gln Ser
1 5 10 15
Lys Thr Lys Pro Lys Gly Gly Ala Pro Ser Ser Ser Gly Pro Thr Arg
20 25 30
Arg Ala Ala Pro Pro Ala Ala Arg Ala Ala Pro Tyr Pro Pro Ala Gly
35 40 45
Pro Lys Ala Ala Gly Gly Ala Ser Pro Tyr Gly Val Tyr Ser Glu His
50 55 60
Val Ala Ala Met Ala Gly Val Val Pro Arg Pro Arg Pro Pro Pro Ala
65 70 75 80
Ala Ala Ala Ala Ala Ala Arg Ser Leu Glu Thr Gly Thr Lys Leu His
85 90 95
Ile Ser Asn Leu Asp Pro Gly Val Thr Val Asp Asp Val Gln Glu Leu
100 105 110
Phe Ser Glu Ile Gly Glu Leu Lys Arg Tyr Ser Val Asn Tyr Asp Lys
115 120 125
Asp Gly Lys Ser Gln Gly Thr Ala Glu Val Val Phe Ala Arg Lys Val
130 135 140
Asp Ala Leu Glu Ala Ile Lys Arg Tyr Asp Gly Val Ile Leu Asp Gly
145 150 155 160
Asn Pro Met Lys Ile Asp Leu Ile Gly Asn Asn Ser Glu Thr Ser Pro
165 170 175
Met Pro Pro Thr Ala Pro Leu Leu Tyr Asn Pro Pro Phe Pro Asn Tyr
180 185 190
Pro Asn Ser Val Pro Arg Arg Gly Gly Gln Arg Gly Gln Phe His Gln
195 200 205
Gly Asn Gly Arg Pro Gly Asn Ser Gln Gly Ile Gly Gly Gly Pro Arg
210 215 220
Gly Phe Gln Gly Ser Gly Arg Pro Gly Ser Gly Ser Gln Gly Gly Gly
225 230 235 240
Gly Cys Ser Gln Gly Lys Thr Arg Gly Asn Glu Arg Ser Arg Ile Gln
245 250 255
Lys Ser Ala Ala Asp Leu Asp Ala Glu Leu Asp Gln Tyr His Ala Glu
260 265 270
Ala Val Lys Glu Lys
275
<210> 2
<211> 834
<212> DNA
<213> Oryza sativa L.
<400> 2
atgtcgagtg gcctggacat gtccctggac gacctcatca agcagtccaa gaccaagccc 60
aagggcgggg ccccctcctc gtcggggccc acccgccgcg cggcgccgcc ggcggcgcgc 120
gcggcgccgt acccaccggc cggccctaag gccgccggcg gcgcttcgcc gtacggggtc 180
tactccgagc acgtggccgc catggcgggg gtcgtgccgc ggccgcggcc gccgccagcc 240
gccgccgccg ccgccgcgcg gtctctggag accgggacga agctgcacat ctccaacctc 300
gaccccggcg tcaccgtcga cgacgtccag gagctcttct cggagattgg tgagctcaaa 360
cgctattctg ttaactatga taaggatgga aaatcacagg gaacggcaga agttgtgttt 420
gcaagaaaag tggatgcttt ggaggctatt aagagatatg atggtgtaat acttgatggt 480
aaccccatga agatagatct catcggaaat aattctgaga catccccaat gcccccaaca 540
gcacctttgt tgtacaatcc accttttccg aactacccta acagtgtacc acggagaggt 600
ggccaaagag gacaatttca tcaaggtaat ggtcgccctg gaaacagtca gggcattgga 660
ggtgggccaa gaggatttca aggtagcggt cgtcctggaa gcggtagtca gggcggtggt 720
ggctgcagcc aggggaaaac ccgtggaaat gaacggagtc gcatacaaaa atcagctgca 780
gatcttgatg ctgaattgga ccagtatcac gcagaagcag tgaaggagaa atga 834
<210> 3
<211> 7684
<212> DNA
<213> Oryza sativa L.
<400> 3
agaaactata acagagacat cattaaacca gacagctact tataggtgga tttaaaccat 60
catgaggata aatatagtac taacgtggca acataactta tattataatg gacattaccc 120
attttggaga tataacgcaa gggagattat aggaggatac caggtggtaa tcatagagcg 180
actgtgagcg tggtttggtg ttaaaagtga tagaaagata aagacggctg cccaccagat 240
cttgatctga cggtcgcccg tggaatacca aaaccggaaa aaaaatccag aggaaacgca 300
tcctcagctc tcacctcact ctctcagccc accgccgccg ccgccgccgc cgtcaccgac 360
gtagcgctgg ttccccttcc ccgcagccgg cgaccatgtc gagtggcctg gacatgtccc 420
tggacgacct catcaagcag tccaagacca agcccaaggg cggggccccc tcctcgtcgg 480
ggcccacccg ccgcgcggcg ccgccggcgg cgcgcgcggc gccgtaccca ccggccggcc 540
ctaaggtacg ccgcgcgcgc cctcccactc acctgtggcc tcctcgggct ctcctcccct 600
cctccgcaag aaaaccctaa ctccgccggc tgcttgtcca ggccgccggc ggcgcttcgc 660
cgtacggggt ctactccgag cacgtggccg ccatggcggg ggtcgtgccg cggccgcggc 720
cgccgccagc cgccgccgcc gccgccgcgc ggtctctgga gaccgggacg aagctgcaca 780
tctccaacct cgaccccggc gtcaccgtcg acgacgtcca ggtgctgttt gttcttgtcc 840
tcgctgctgg tttcgccatt tttttgccgt acttcgcttg tccattttca aatccctgga 900
gctgaatccc agaattgcca aggaaatttg tccactccca ctagcgcgca tctatgtttt 960
cctttcttgt gatgaaaaat taccatgtga aatgttgcaa ttaccttacc cacctgaatt 1020
ctcaacctca tgaatggggg gatgagttta gtgttgctca tgtttctcta ctagcacctc 1080
ctgttgtttt gcttgttggg ccgcgaacat tgacaagggt ataatacata tctcaaacaa 1140
accgtaagac gccacacaca tgcatcctta ttggcaccta gctgctttaa ggtcaccact 1200
tgaggatatg aagatgtagg taatcaagat tggctcattt cttgattgga tggtcagttt 1260
atggggaagg aaggataaca ttacggggac tggaaattcg catggcaggt tgtcactctc 1320
ggtggtaggg agtggtgaga tgtgacctct gaggggaggc ggtgctaggt ctatgagcaa 1380
tggcggtatc tgtgattaac tggttctcat ttaagattgg taatagatat gctgaggtat 1440
taaatagcgc cgggtgtgcg tgtttatatg tgtaataggg caggtatgac atattgggaa 1500
tggaaagtaa tcttttggtg gtttacatgt gttgtaggat gaatgacatg taactgaaat 1560
cgatgtctca gtgccattag aacccaattt tccatgattt gtattatact agttgttggt 1620
aaaacatgat tccagttatt tctttgcata atatactccc tctattgcat aataactgac 1680
aatgggcact attcacttta aaactttgac cactcgtctt tttcgttgaa tatctacaaa 1740
tactagaaat gatgccatgc tataaagtga tgtatgccct aaatataagt ataaaacttt 1800
tgttcatttg atctaacaat tcaagggcaa ttaatggtca aagtttaaat gggaagtcaa 1860
aattgtcagt tattaagaaa tgggtggagt agtttgcaat ctccaaattc taagtgttca 1920
agtaagtaaa agtgcagaga ccgttcttga taacatggaa atcaggttaa taaactcatc 1980
ttcacataca ctttgaagaa gcacagtgtt gtctttcctt gctatgtgat taactgatac 2040
attgatattg tacccttttg tgaaagattt aaattctcat cccaaggcaa gcatatgatg 2100
atgatacttt tttctgtact ctgaccaatg acattttaaa ggcatagatt cgttgcacag 2160
agagattcgt cataaatata tttccagtgt ttattcttaa gttttactaa gctatcatga 2220
attcatcata ttgttataaa aactacttgg ttaagcatgt catggagatt gtgttaaaag 2280
caccaacact ttgtgattga agggaataat atgaaacttt tagtttttgt ttaagaatat 2340
aagcacagaa tctcaattat tctgtgattg tagctgatgg gctatattat tttctttcct 2400
caggagctct tctcggagat tggtgagctc aaacgctatt ctgttaacta tgataaggat 2460
ggaaaatcac aggtagacca tggagtattg tcttgccttt ttctgtatat tgctcagatc 2520
tactctctgt tctcattttt ctgacatttt ttcttaggga acggcagaag ttgtgtttgc 2580
aagaaaagtg gatgctttgg aggctattaa gagatatgat ggtgtaatac ttgatggtaa 2640
ccccatgaag atagatctca tcggaaataa ttctgagaca tccccaatgc ccccaacagc 2700
acctttgttg tacaatccac cttttccgaa ctaccctaac aggtctgttt caactgttcc 2760
ttgaattcat atattggttt taaaatcaat aaaaatacta ttagtgtgga gcatcctttt 2820
ctcgtttttc actcatttag agacattatt ttgctagcaa ttaactgcca ctaagaatat 2880
tataacaaac agacttgagg tgttctctaa tttcatggag gatttcgtca atttgatatc 2940
tccttaggtt ttgatacatc aaactgtaat tgcgtactga acccaattgg caaaaatgag 3000
gcgcataatc cttcagggaa atagttttca agtctcacga attgattttt cgaaatgcca 3060
cttaagtagc tcacaaatca catttcagac tagttttctc tgtactatta tactcaatga 3120
atttgtgccc cttcaagttc agaagctgtt tgggctgtgc tgatactgct gcaatctagc 3180
cttgcacaga gtgatgttat ccacacaatt tatttactgc ttaatttctt tcatgtttgt 3240
gcacatgata ctgttctgca cggttcagtt ggatgcttct atttacagta aaatgtgaaa 3300
aattgagaat gttgtgaaat atcttagatt tgttgggcag atggagagag agtaaatcaa 3360
cacaggagag agttctgtta tgtttgaagg tcaatgaata aaaactttgg atgataaact 3420
ggagagcttt tagagctaac tagtaattaa actactatgt agtctatgtt gttaaacatg 3480
ttgctttaag gtttgtgctc gacagttgaa actattgtcc atgttaccat catatcatac 3540
ctaatgcttg aattagtggg ttttgttgcc tgttcaggag ttatgtgcaa ctgaaagata 3600
aatttagatg ttcaatagct acagtgtgct atgcttgtgt aacatagtat tgtcgaagca 3660
gtataggaca tgatgagttc tctggttgca ataggctttg ttgttgtaga ctaatagtca 3720
ccatgtctgg caccagcact tgccttgtta gggcgttcac ggtgtgtcga catgtcgttc 3780
gccctcaccc ttccacgtgg gagattcgtc tccattcttg ctcctatcgt gaccaaacca 3840
agaccgtgca gccaaaatga gccacggatt cttcgcacgg atcgaggggc tgtggcgtga 3900
ggatagatcg ggcgcgaatg catagctcgt agcctggcct cgcgcggctt cgcgcgcgct 3960
caccctaaat cgccggcgga cagggggaga tgagggctgg agaagtgagg agaaagcctt 4020
gtcacccact gccagagaag aaaggatgga aagttggcac ggagcatgct ggccggcggc 4080
gagccggtgc agaacctcat cgctgcggcc agatccacgc tgccgccgag attagccaga 4140
tcagccgctc ctcagggtcc tccgccgcac cgccggccat cagtccgcct cggagtcctc 4200
cgccgctgag gcccaccgct agatccgcac tgttccactc catgctgcca cggttagcca 4260
gatctgcgcc gtcagcccca tcgccgcccg ccatagaaag aagaggatgg aaggtggagc 4320
tcgacggagt ggcgttggtg gccgctttga cccgtctgca gcagcagctc gacagcagcc 4380
gcttcaacgt gtctgcggct tccttgccgc accgccggag ctgccctcag ccttgtcgct 4440
gtttcgatct actctacggc cgcacggata gaggcggtgc cagggagagg gtgtaggcgg 4500
cagcacctag gagagagggg agaaacggcg ccaccgggaa gcggataggg gatcgggggt 4560
ggagggaatt aggtggggag gggagagatt ttatattact ttcttgattt tgggtgttag 4620
agtgcaaatt tcggagtact atgtaaattg aaaactaaaa gagtgcaaaa tgtctgtagg 4680
tcccaccata gatttttcta aaccacacct tgcactgtgg gtgtagtgtc ctttgaagtt 4740
cttaagccta catggcagct gaggcctagc aaattgtttg gacgcatttg ctattgccct 4800
tagaaaacct tgttgaacac caaagatggg catgtttcaa ttatgtcact ggagtcacct 4860
acactgagag atgcagtacg atttcctaaa ccatattctt ttctctaaaa aaggatcaac 4920
actgtaggtc cggacgtcta cgatatcgga attattagag cagaagcatg cggtatcaat 4980
tggaaatcaa taattcatgt tggaaacacc gaccataaca ccgtatgttc atgcgcttga 5040
ccacaactag gtaaaataaa ctttcatagg cactatacta atgatttatt caacaccaat 5100
ggcatattca tagtgaactg aagttaccat tttgctgagc tgcaaagcta cgtgtactgt 5160
tagatgttac gtagttatat cgattttatt tgcttgtgaa acaactgaca tagactatga 5220
tatttaagaa aactattgag cttaggcatt gacccgaaca aggactcagg ttaccaaatc 5280
ggaccgcatg taaatgtggt gtccatacag cgctattaat gttcttacaa tttatgcttc 5340
acaatggaaa atgagtgttg gtttgcatgt tagtcgggga ttggaggtac ttgattcgat 5400
tctcaggctc cccacactag cacaattttt tgaattgctc atgcaggaag tattgggcca 5460
ggcctcagag tggtaaccac atggaaaaca tgggctgtga ggctgtgacg gcgtggctat 5520
ggcacatgaa tgcatggcca ctgggataca ttaggcctag gctgttgggg tagcagaatt 5580
ctgaaattac agacggatga atatagtaaa ccaaggaaac ggtcagcata aaagtggaat 5640
cgtttccgct gctgtttctg gcttgttcta ccatttccat ttatgcttcg ggcaattact 5700
gtttccagtt gatttggtca atagaaatca gaaatgagca tcgggcggtc ccgaatttcc 5760
atgactattt tcaccctagt tccatgtatg agaaggtgcc agggacagta agcaatgttc 5820
aataaggtgc ttgttgggca gcatggttct gcctaggaac taggggcacc catgtggtgc 5880
cctgggacta gctagcccac actgttttgg gagtagcaca gcgatggaga agaggttaga 5940
tcaaatggta atacctgcag ggttggatag cttcgtgatg gcatagagga tggagaatgt 6000
gccagggagg ttgcattgca ctggcatgga agatggattt ggtcggatat gagacagacc 6060
cgccaggagg aaggtgactt gggccagcaa catgatggat ttagtggagg agttggttcg 6120
gctgatgaca agatggattt gatgatgtgc ttgtggtgtt atatatattt ggctgtagac 6180
gtgccatcag atgcagtcaa cttcgccatt tagtgaggtt gtgtgtcctt gtcctgctat 6240
gtggtctgta ggttcaacca taaatttgtc agcgtttgtc cactcttcct tgatagtttt 6300
tatgacttcc taattcctat aagttccttg tcagctgggg tattacaaac caaaacatgc 6360
atctcgccat ccatgatgta ccatagtttt tattacttcc tataagttac agggtaagat 6420
tgctgcgaca gctttcattc attccttgtg cacaacattg aaaaacaaaa caacgtatac 6480
cacttttgtc tgtttctgtt ttcacaatgt ggaatcctgt gggcaagatc taacagatcc 6540
gactcaggaa tcctgtgggc aaccactaaa tgtctaattc taagaaatat ttaatgaaaa 6600
tttggcgtcc atactgcttt ataagatata atccaaacac ctgttgtctc atctacaatg 6660
gaaggacggc caagaaagga aaatattgta attgcacttg atgcatcaat ggacttacat 6720
tatggaatga ctacatccaa gtgcaccttt gtgtgtaata gagatagtag tccaagtgta 6780
cctgttgtcc atggtttgat tttgaaggga gcagatcaca ctgctgcagc atttttgctc 6840
ttttttgcac tgttctagat tgtccagtcc ttattagaca acatttcagg atatacaaac 6900
tgcatcatgt actcccacta ttttaatagc tagtttctga gagtctaatg aggtggcttt 6960
tcattaagag taggacatcg tttgcagaca catacatcta cagaatttat attcacatgc 7020
acagccaata ttgttatggg acactctttt gtttgtggga gatgatgacc acattctgta 7080
acctctcctt caaactggta attcctctgt caggaagcat ttcaaatagc gaatatatcc 7140
aaatcttagg ttcatcatgt ccttctaagg aactatcttc tattactaaa tactcgtagc 7200
attttggtta ctttgcctgt agtgtaccac ggagaggtgg ccaaagagga caatttcatc 7260
aaggtaatgg tcgccctgga aacagtcagg gcattggagg tgggccaaga ggatttcaag 7320
gtagcggtcg tcctggaagc ggtagtcagg gcggtggtgg ctgcagccag gggaaaaccc 7380
gtggaaatga acggagtcgc atacaaaaat cagctgcaga tcttgatgct gaattggacc 7440
agtatcacgc agaagcagtg aaggagaaat gaggttctct tctattttta ctagtgggaa 7500
tgtgggatga cactgaaact acctggcatt gatttaaaga tgtgaaatta tcttgctagt 7560
gttttcatgt tttgtgctag aatttttgtt gttagtgaca accattcagg ataacatgct 7620
taatcatgtg tttggacttt gtaatgtgca gttaaagtta ttttctgttt gatcaaatgc 7680
ttgg 7684
<210> 4
<211> 19
<212> DNA
<213> Artificial sequence
<400> 4
gtctccagag accgcgcgg 19
<210> 5
<211> 95
<212> RNA
<213> Artificial sequence
<400> 5
gucuccagag accgcgcggg uuuuagagcu agaaauagca aguuaaaaua aggcuagucc 60
guuaucaacu ugaaaaagug gcaccgaguc ggugc 95
Claims (7)
1.OsSDP蛋白或其相关生物材料在调控植物耐盐性中的应用;
所述相关生物材料为能够表达所述OsSDP蛋白的核酸分子或含有所述核酸分子的表达盒、重组载体、重组菌或转基因细胞系;
所述OsSDP蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
在所述植物中敲除OsSDP基因,所述植物的耐盐性提高;所述OsSDP基因编码所述OsSDP蛋白;
所述植物为水稻。
2.根据权利要求1所述的应用,其特征在于:能够表达所述OsSDP蛋白的核酸分子为如下任一所述的DNA分子:
(a1)SEQ ID No.2所示的DNA分子;
(a2)SEQ ID No.3所示的DNA分子。
3.一种培育耐盐性提高的植物品种的方法,包括敲除受体植物中OsSDP基因的步骤;所述OsSDP基因编码OsSDP蛋白;
所述OsSDP蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻。
4.一种培育耐盐性提高的转基因植物的方法,包括如下步骤:敲除受体植物中OsSDP基因,得到转基因植物;所述转基因植物与所述受体植物相比耐盐性提高;
所述OsSDP基因编码OsSDP蛋白;
所述OsSDP蛋白为如下任一所示蛋白质:
(A1)氨基酸序列为SEQ ID No.1的蛋白质;
(A2)在(A1)所限定的蛋白质的N端和/或C端连接标签后得到的融合蛋白;
所述植物为水稻。
5.根据权利要求4所述的方法,其特征在于:所述敲除受体植物中OsSDP基因通过CRISPR/Cas9技术实现。
6.根据权利要求5所述的方法,其特征在于:通过CRISPR/Cas9技术实现敲除受体植物中OsSDP基因时,靶序列为所述受体植物基因组中SEQ ID No.4所示的DNA分子。
7.根据权利要求3-6中任一所述的方法,其特征在于所述OsSDP基因为如下任一所述的DNA分子:
(a1)SEQ ID No.2所示的DNA分子;
(a2)SEQ ID No.3所示的DNA分子。
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