CN112094865A - 一种培育耐低钾水稻的方法 - Google Patents
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Abstract
本发明公开了一种培育耐低钾水稻的方法,包括如下步骤:1)对水稻OsHAK3基因进行克隆;2)构建pHB‑OsHAK3过表达重组质粒;3)获得OsHAK3过表达阳性植株;4)获得耐低钾水稻;5)获得CRISPER/Cas9敲除的OsHAK3靶标序列;6)构建含所述靶标序列片段的pCRISPER/Cas9重组质粒;7)获得OsHAK3敲除阳性植株;8)获得低钾敏感的水稻。本发明属于基因工程技术领域,具体是一种培育耐低钾水稻的方法,OsHAK3基因在水稻中负责从土壤中吸收K+,OsHAK3基因的敲除突变体对低钾敏感,水稻对K+的吸收显著减少,植株中钾离子含量明显下降,并出现明显的缺钾铁锈斑,OsHAK3基因过表达植株对K+的吸收能力明显增强,植株中钾离子含量明显增加,植株对低钾耐受能力明显增强,提高水稻耐低钾能力。
Description
技术领域
本发明属于基因工程技术领域,具体是指一种培育耐低钾水稻的方法。
背景技术
钾是植物生长发育所必需的矿质元素,对植物的生长发育、产量和品质等具有重要影响。钾离子作为多种酶的辅助因子,参与光合作用,调节细胞膨压和渗透势,维持细胞电荷平衡等一系列生理生化过程。植株长期缺钾会引起植株易倒伏、抗病虫害能力下降、结实率降低,导致作物减产。
水稻是我国的主要粮食作物,主要通过根部从土壤中吸收钾离子。逐年累月的耕作导致土壤中的钾离子逐渐匮乏,只有通过施加钾肥来缓解。目前世界钾资源稀缺,导致钾肥昂贵。另一方面水稻对钾肥利用率低,仅为40%左右,大量施用钾肥导致农田大量钾肥流失引起水体富营养化污染环境。我国约四分之一的农田缺钾,尤其南方稻区缺钾更为严重。我国钾肥资源十分短缺,90%的钾肥依赖进口。因此从经济上和长远的战略上考虑,充分挖掘耐低钾基因,深入了解和认识水稻对低钾胁迫的反应机制,对于培育耐低钾水稻品种、提高水稻产量和品质、减少环境污染具重要意义。
发明内容
针对上述情况,为克服现有技术的缺陷,本发明提供一种培育耐低钾水稻的方法,OsHAK3基因在水稻中负责从土壤中吸收K+,OsHAK3基因的敲除突变体对低钾敏感,水稻对K+的吸收显著减少,植株中钾离子含量明显下降,并出现明显的缺钾铁锈斑,OsHAK3基因过表达植株对K+的吸收能力明显增强,植株中钾离子含量明显增加,植株对低钾耐受能力明显增强,发明可为提高水稻耐低钾能力,为培育适用于钾缺乏土壤的水稻新品种提供保障。
本发明采取的技术方案如下:本发明一种培育耐低钾水稻的方法,包括如下步骤:
步骤一:对水稻OsHAK3基因进行克隆;
步骤二:构建pHB-OsHAK3过表达重组质粒;
步骤三:获得OsHAK3过表达阳性植株;
步骤四:获得耐低钾水稻;
步骤五:获得CRISPER/Cas9敲除的OsHAK3靶标序列;
步骤六:构建含所述靶标序列片段的pCRISPER/Cas9重组质粒;
步骤七:获得OsHAK3敲除阳性植株;
步骤八:获得低钾敏感的水稻。
进一步地,步骤3)所述的过表达阳性植株为HAK3-1和HAK3-2。
进一步地,步骤7)所述的过表达阳性植株为hak3-1和hak3-2。
采用上述结构本发明取得的有益效果如下:本方案一种培育耐低钾水稻的方法,OsHAK3基因在水稻中负责从土壤中吸收K+,OsHAK3基因的敲除突变体对低钾敏感,水稻对K+的吸收显著减少,植株中钾离子含量明显下降,并出现明显的缺钾铁锈斑,OsHAK3基因过表达植株对K+的吸收能力明显增强,植株中钾离子含量明显增加,植株对低钾耐受能力明显增强。本发明可为提高水稻耐低钾能力,为培育适用于钾缺乏土壤的水稻新品种提供保障。
附图说明
图1为本发明培育耐低钾水稻的方法OsHAK3过表达株系的检测结果;
图2为本发明培育耐低钾水稻的方法OsHAK3敲除突变体的检测结果;
图3为本发明培育耐低钾水稻的方法OsHAK3的敲除突变植株(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-1)在低钾条件下的表型观察结果;
图4为本发明培育耐低钾水稻的方法OsHAK3的敲除突变植株(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-1)在低钾条件下的根长、地上部分长及鲜重测定结果;
图5为本发明培育耐低钾水稻的方法OsHAK3的敲除突变植株(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-1)在低钾条件下的根部钾离子含量、地上部分钾离子含量及根部钾离子吸收速率测定结果;
图6为本发明培育耐低钾水稻的方法OsHAK3的敲除突变植株(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-1)在土壤中生长的表型。
附图用来提供对本发明的进一步理解,并且构成说明书的一部分,与本发明的实施例一起用于解释本发明,并不构成对本发明的限制。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例;基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如图1-6所示,本发明培育耐低钾水稻的方法,包括如下步骤:
步骤一:对水稻OsHAK3基因进行克隆;
步骤二:构建pHB-OsHAK3过表达重组质粒;
步骤三:获得OsHAK3过表达阳性植株;
步骤四:获得耐低钾水稻;
步骤五:获得CRISPER/Cas9敲除的OsHAK3靶标序列;
步骤六:构建含所述靶标序列片段的pCRISPER/Cas9重组质粒;
步骤七:获得OsHAK3敲除阳性植株;
步骤八:获得低钾敏感的水稻。
进一步地,步骤3)所述的过表达阳性植株为HAK3-1和HAK3-2。
进一步地,步骤7)所述的过表达阳性植株为hak3-1和hak3-2。
实施例中所用到的培养基的配方如下所示:
(1)LB液体培养基:10g/L胰蛋白胨,5g/L酵母提取物,10g/L NaCl,用NaOH调pH值到7.0,121℃高温高压灭菌15min。
(2)AB培养基:NaH2PO4·2H2O 1300mg/L,K2HPO4 2950mg/L,KCl 150mg/L,MgSO4·7H2O 296mg/L,CaCl2·2H2O 10mg/L,NH4Cl 1000mg/L。
(3)水稻愈伤组织培养基配方:
预培养培养基N6D(诱导愈伤组织分化),AAM培养液(浸染),共培养培养基2N6-AS(暗培养浸染后愈伤组织),筛选培养基(筛选抗性愈伤组织),分化培养基(RE-III),生根培养基(HF),CS培养基(阳性筛选)。筛选培养基需加Hygromycin B、Carbenicillindisodium;共培养培养基需加Acetosyringone;分化培养基需加KT、NAA;生根培养基需加NAA;CS培养基需加Hygromycin B、6-BA。
(4)水稻水培液配方:
实施例1
水稻OsHAK3基因的克隆:选取饱满一致的日本晴种子于37℃烘箱或Ms板催芽,选取长势一致的幼苗放入正常水培液培养7天,培养期间每2天更换一次水培液,提取所取材料的RNA,反转录成cDNA,设计特异性的引物序列克隆OsHAK3的编码区序列并测序;所用上游引物序列为:F1:5’-ATGCCGGTGGCCGACTGC-3’;下游引物序列为R1:5’-CTAGACGTAGTAGATCATGCCGACTT-3’。用上述引物进行PCR扩增,测序得到日本晴OsHAK3基因的编码区序列。
OsHAK3基因编码区序列全长2427bp,序列如下:
ATGCCGGTGG CCGACTGCGA AAGCGGTCTC TCGCCGGCAG ATGTCACCGG TGCAGGTGCCGCAAACGGGA ACCCGGGTCA CTGGAGGAGC TACTACAGGC ATGTGCTGCT GCTGGCGTAC CAGAGCTGCGGCGTGGTGTA CGGCGACCTG AGCACGTCGC CGCTGTACGT GTACAAGAGC ACCTTCATCA TCGGGTCGCTCCGGCGGTTC CAGGACGAGG AGATCGTGTT CGGCGTCTTC TCCCTAGTGT TCTGGACGCT GACCCTCATCCCGCTCCTCA AGTACGTCTT CATCGTGCTC GCCGCCGACG ACAACGGCGA GGGCGGCACG TTCGCGCTCTACTCGCTGCT GGTGCGCCAC GCCAAGTTCA GCCTCATGCC AAACCAGGAG GCCGCCGACG AGGAGCTCACCTCCTACTAC CGCCCCGGCT ACGCCCCCCA GGAGACCCCC ATCCTCACCG CGCTCCGTCG GTTCCTCGAGAACCACCGCA AGTCCCGCAC CTTCCTCCTC GTCACCGTCC TCTTCGGCGC CTCCCTCGTC ATCGGCGACGGCGTCCTCAC GCCGCCCATG TCCGTGCTGT CGTCCTTCTC TGGGCTGCAA GTGCACTCGA CCGCGCTGACGAGCGGGGAG GTGGAGATCC TGTCGTGCAC GGTGCTGGTG TGCCTGTTCA TGGTGCAGCA CTGGGGCACGCACAGGGTGG CGTTCCTGTT CGCGCCGGTG GTCATCGTCT GGCTCCTCCT CCTCGGGGCG CTCGGCGTCTACAACATCGT GGTGTGGAAC CCCAGGGTGC TGCGCGCCCT CTCCCCTTAC TACCTCGTCA GGTTCTTCCAGCACACCGGC AAGGACGGCT GGATCTCCCT CGGGGGAATT CTCCTCTCCA TGACAGGGAC CGAAGCCATGTATGCGGATC TTGGCCACTT CACAGCTGCA TCCATAAGGG TTGCGTTCGT GGGGCTCATC TACCCGTGCTTGGTGCTGCA GTACATGGGG CAGGCGGCCT TCCTGTCCAA ATCACCCCAC TGCGACATCC ACTTCGTATTCTTCGAGTCC ATTCCAACGG GCATCTTCTG GCCGGTGCTG GTGATCGCGA CGCTGGCGGC GATCGTGGGCAGCCAGGCGG TGATATCGGC GACCTTCTCC ATCGTGCGGC AGTGCACGGC GCTGGGCTGC TTCCCGCGCGTGAAGATCGT GCACACGTCG CGGCGGATCC ACGGGCAGAT CTACAGCCCG GAGATCAACT GGATCCTGATGCTGCTGTGC ATCGCCGTCA CCATGGGGCT CCGCGACACC ACCCTCATCG GCAACGCCTA CGGGATGGCGTGCGCGGGGG TGATGCTGGT CACCACGCTG CTCATGGCGC TCGTCATCGT CTTCGTCTGG CAGTACAGCTGCCTGGTGGC GGCGCTCTTC CTGGTGGCGT TCGGCGTCGT CGAGGCGGTG TACCTGTCGG CGGCGCTGATGAAGGTGCCC CAGGGCGGGT GGCTGCCGCT GGTGCTGTCG CTGGTGTTCG TCGCCGTCAT GTACGTGTGGCACTACGGCA CGCGGCGGAA GCACCAGTTC GACGTGCAGA ACAAGGTGTC GCTCAGGTGG ATCCACGCGCTCGGCCCCAG CCTGGGCATC GTGCGCGTCC CGGGGATCGG CATCATCTAC TCCGAGCTGG CCACCGGCGTGCCGGCCATC TTCTCGCACT TCGTCACCAA CCTGCCGGCG TTCCACCAGG TGCTCGTCTT CATCTGCGTCAAGGCCGTGC CGGTGCCGCA CGTCCGCGAC GAGGAGCGCC ACCTCGTCGG CCGCATCGGC CCGCGGGAGTTCCGCATGTA CCGCTGCGTC GTTCGGCACG GCTACAAGGA CGTGCTCGCC GAGGACACCG ACTTCGAGAACGACCTCGTG CTGCGGATCG CCGAGTTCGT GCAGATGGAG GCCGACTTCG ACCAGCGCTG CAGCATCAGCGACGATGGGG TCGTCGCCTC CGTGGAGGTG GAGGGCCGCA TGGCCGTGGT CCCGCGGCCC AGCGACCTTGCCAGGACGGG GCTCCTCATG CGGGAGCCCG GCGAGGAGGA GAGCGTGGTG GCGCGCGCCG CCGCGGCCGCCAAGCCGGAG TCGCTCATCC ACTCGATGCA CACGATGCAC GAGGCGGAGT CGCCGGGGTT CGCGAGCCGGCGGCGCGTGC GGTTCGAGGT GGCGAACCAG CACACGGACC CCAGGGTGAA GGAGGAGCTG AGCGCGCTGGTGGAGGCCAA GCACGCCGGC GTCGCCTACA TCATGGGCCA CTCGTACATC AAGGCGAGGA AGAGCTCGTCGGTGTTCAAG AAGTTCGCCG TCAACGTCGC CTACGCTTTC CTCCGCAAGA ATTGCAGAGG CCCCGGCCTCGTGCTCAACA TCCCGCACAT CAGCCTTATC GAAGTCGGCA TGATCTACTA CGTCTAG
pHB-OsHAK3过表达重组质粒的构建:用于构建pHB-OsHAK3重组质粒的上游引物序列为:F2:5’-CCCAAGCTTATGCCGGTGGCCGACTGC-3’,下游引物序列为:R2:5’-GCTCTAGACTAGACGTAGTAGATCATGCCGACTT-3’;利用上述引物PCR扩增日本晴cDNA模板,回收2427bp的目的带,用HindIII+XbaI将OsHAK3的目的片段和pHB空载体分别进行双酶切,回收酶切产物,通过同源重组的方法将OsHAK3的目的片段连到pHB载体上。
OsHAK3过表达阳性植株的获得:将构建成功的pHB-OsHAK3重组质粒用电转的方法转入农杆菌EHA105中,用日本晴种子在N6D培养基上诱导培养7天产生愈伤组织。将含有重组载体的EHA105菌在AB平板上划线,放置30℃生长3天后,用无菌的tip头将生长的菌转到AAM液体培养基中悬浮,调OD600到0.1。用无菌的镊子将愈伤组织转入菌液中轻摇90秒,放置N6-AS平板上25℃黑暗培养3天,清洗干净后放置含50mg/L潮霉素和400mg/L的羧苄青霉素的N6D培养基筛选两周。将筛选出的新鲜愈伤组织转到分化培养基上进行分化培养,两周后将分化出的幼苗转移至生根培养基上进行生根培养,生根一周后移栽至温室田中培养。
取上述移栽的T0代小苗的叶片放置含潮霉素B的初筛板进行初筛,叶片不褐化的初步判断为阳性植株。提取初筛为阳性植株的RNA,反转录成cDNA,利用PCR进行OsHAK3表达量的检测,OsHAK3表达量上调的OsHAK3的过表达阳性植株,为共检测到16株阳性植株。检测引物序列为:上游引物F3:5-AAGTCCCGCACCTTCCT-3′,下游引物R3:5′-AGCCAGACGATGACCAC-3′。
耐低钾水稻的获得:将所述阳性植株HAK3-1和HAK3-2进行繁种,后代植株具有潮霉素抗性且OsHAK3表达量上调即为耐低钾水稻。
进行CRISPER/Cas9敲除的OsHAK3靶标序列的获得:利用CRISPER/Cas9系统,根据OsHAK3编码区序列选择特异的使OsHAK3蛋白失活的靶标序列。靶标序列:CCTCCCTCGTCATCGGCGA(靶点1)和GGTGCTGGTGATCGCGACGC(靶点2)。
含所述靶标序列片段的pCRISPER/Cas9重组质粒的构建:靶点1序列设计接头引物后的完整靶序列如下:
F4:5’-CCTCCCTCGTCATCGGCGAgttttagagctagaaat-3’
R4:5’-TCGCCGATGACGAGGGAGGCcggcagccaagccagca-3’
将设计的靶点2序列添加pCRISPER/Cas9系统的特异粘性末端接头(F-GGCA,R-AAAC),合成完整的靶序列。完整靶序列如下:
F5:5’-GGCA-GGTGCTGGTGATCGCGACGC-3’
R5:5’-AAAC-GCGTCGCGATCACCAGCACC-3’
将F4引物、R4引物稀释成浓度为10μM的溶液,各取10μL混匀,在PCR仪中进行退火反应,使F4引物和R4引物互补形成双链小片段。用BsaⅠ酶切pOs-sgRNA原始载体,体系为:10×buffer BsaⅠ2μL,BsaⅠ酶1μL,pOs-sg RNA载体4μg,ddH2O补足20μL,37℃酶切12h,酶切产物用1%琼脂糖凝胶电泳检查条带大小后,回收酶切产物,加入灭菌的ddH2O溶解测定浓度后待用。用T4连接酶将上述双链小片段和酶切过的pOs-sgRNA载体连接,形成完整的包含针对OsHAK3蛋白的靶序列和sg-RNA的重组载体。连接体系为:10×T4ligation buffer 1.5μL,双链小片段4μL,酶切过的pOs-sgRNA载体3μL,T4 DNA ligase 1μL,于水浴锅中16℃连接12小时。将连接产物转化大肠杆菌TOP10,涂板到含卡那抗性的LB平板过夜培养,挑选阳性菌进行测序,得到正确的包含靶标序列和sg-RNA的重组载体。用LRmix将上述重组载体和包含Cas9的载体pH-Ubi-cas9-7进行LR反应重组,LR反应体系:包含靶标序列和sg-RNA的重组载体25-50ng,pH-Ubi-cas9-7载体75ng,5×LR ClonaseTM-buffer 1μL,TE Buffer(pH8.0)到4.5μL,LR ClonaseTM 0.51mL;于25℃下温育2h后,加入2μL 2μg/μL的Proteinase K于37℃下处理10min,取2μL该反应产物转入大肠杆菌TOP10,在含壮观霉素抗性LB平板培养过夜,挑选阳性菌进行测序,得到正确的包含OsHAK3蛋白靶标序列-sg-RNA+Cas9的完整的重组质粒。
将接头正向F5引物和接头反向R5引物稀释到10μM备用。取
pYLgRNA-OsU6/LacZ质粒1μg,在25μL反应体系,用10U BsaI酶切20min,冷冻保存。酶切过的pYLgRNA-OsU6/LacZ质粒与各所对应接头进行连接,连接反应体系如下:10×T4DNA ligase Buffer 1μL,pYLgRNA-OsU6/LacZ 0.5μL,接头0.5μL,T4 DNA ligase(Takara)0.05μL,ddH2O补足到10μL。室温连接10-15min。每个sgRNA表达盒分为2个PCR反应,每个反应各15μl反应体系:0.5μL连接产物,U-F引物/接头反向引物R5(反应1)、接头正向F5引物/gR-R引物(反应2)各0.2μM,适量高保真PCR酶。
UF引物:5’-CTCCGTTTTACCTGTGGAATCG-3’
gR-R引物:5’-CGGAGGAAAATTCCATCCAC-3’
PCR扩增26循环:94℃10s,60℃15s,68℃20s。取4μL用2%琼脂糖胶电泳检查(反应1产物长度约700bp,反应2产物长度约140bp)。取第一轮PCR产物1μL用H2O稀释10倍,各取1μL混合为模板。各表达盒40μl PCR,加入1/10量每种引物组合工作液(最终浓度0.15μM)。PCR扩增22循环:95℃10s,58℃15s,68℃20s。取2-3μl电泳检查产物长度是否符合,并估计样品的大致浓度。根据各样品产物估算的量,把所有产物大致等量混合,用胶回收试剂盒纯化PCR产物。取1μL纯化PCR产物,以4ng/μL pYLCRISPR/Cas9质粒为模板,组装sgRNA表达盒到pYLCRISPR/Cas9载体。反应体系如下:
双元载体与sgRNA表达盒的酶切-连接反应体系
进行连接转化,将其转入到大肠杆菌TOP10,37℃培养过夜。用卡那霉素(Kan+)筛选阳性克隆并进行PCR验证,最后分别进行测序以确保载体构建成功,获得包含正确OsHAK3目标插入序列的LacZ-OsU6-gRNA表达盒的完整重组质粒。
分别将上述构建成功的两种重组质粒电转转入农杆菌EHA105中,将日本晴种子在N6D培养基上诱导培养7天产生愈伤组织。将含有重组载体的EHA105菌在AB平板上划线30℃生长3天,挑取生长的菌转入AAM液体培养基中悬浮,调OD600到0.27。将愈伤组织转入菌液中轻摇90秒,于N6-AS平板上于25℃黑暗培养3天,清洗干净后在含50mg/L潮霉素和400mg/L的羧苄青霉素的N6D培养基筛选两周。将筛选出的新鲜愈伤组织挑到分化培养基上,进行分化培养两周后,将分化出的幼苗转移至生根培养基上进行生根培养,生根一周后移栽至温室田中培养。将移栽的小苗(T0代)提取DNA进行靶序列位点检测,共检测到22株阳性植株。将移栽的小苗提取DNA,设计特异性引物,扩增含靶标位点的DNA片段(含靶标位点1的650bp以内的DNA片段以及含靶标位点2的1000bp以内的DNA片段)、扩增得到的PCR产物经纯化后送公司测序,测序结果与野生型植株序列比对,部分突变分析结果如下表所示。
设计的靶标位点1检测引物为F6:
5’-GCGTCTTCTCCCTAGTGTTCT-3’,R6:5’-CCACACCACGATGTTGTAG-3’,扩增片段长度为650bp。靶标位点2的检测引物为F7:5’-TGGTGGTGAGGTGACATG-3’,R7:5’-TCCTGGCAAGGTCGCT-3’,扩增片段长度为1000bp。根据测序比对结果,选取OsHAK3开放阅读框发生移码突变提前终止或缺失起始密码子的T0代纯合突变株系3-1和3-2,繁种。
表1.T0代部分阳性植株靶位点变化
株系 | 靶位点序列 | 基因型 |
野生型(WT) | CCTCCCTCGTCATCGGCGA | 野生型 |
3-1 | CCTCCCTCGTCATCGGACGA | 纯合突变体 |
野生型(WT) | GGTGCTGGTGATCGCGACGC | 野生型 |
3-2 | GGTGCTGGTGATCGCGATACGC | 纯合突变体 |
将所述突变体植株进行繁种,于后代植株中分离OsHAK3基因功能缺失的突变体,即为对低钾敏感的水稻。分别命名为hak3-1突变体和hak3-2突变体。
苗期表型鉴定:将野生型日本晴(Nip)、OsHAK3的敲除突变体(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-2)的种子催芽5天后,分别在含有不同钾离子浓度(10mM、1mM、0.01mM、0mM K+)的水培液培养,培养14天后,观察各植株表型并拍照,检测钾离子含量。结果发现在正常钾离子条件下,野生型日本晴、OsHAK3的敲除突变体(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-2)的长势一致。而在低钾处理下,OsHAK3的敲除突变体(hak3-1、hak3-2)的植株矮小,根部变短,根部和地上部分钾离子含量明显低于野生型日本晴和其过表达植株(HAK3-1、HAK3-2),且突变体植株根部的钾离子吸收能力明显低于野生型日本晴和其过表达植株(HAK3-1、HAK3-2)。
成熟期表型鉴定:将野生型日本晴(Nip)、OsHAK3的敲除突变体(hak3-1、hak3-2)和其过表达植株(HAK3-1、HAK3-2)的种子催芽5天后,分别播种到土壤中进行土培,培养87天后,观察各植株农艺性状并拍照。结果发现OsHAK3的敲除突变体(hak3-1、hak3-2)的植株矮小,根部变短,叶片出现明显的缺钾铁锈斑,结实率和千粒重也明显低于野生型日本晴和其过表达植株(HAK3-1、HAK3-2)。
序列表
OsHAK3基因的外显子序列
OsHAK3的蛋白序列
人工序列
F1引物:5’-ATGCCGGTGGCCGACTGC-3’
R1引物:5’-CTAGACGTAGTAGATCATGCCGACTT-3’
F2引物:5’-CCCAAGCTTATGCCGGTGGCCGACTGC-3’
R2引物:5’-GCTCTAGACTAGACGTAGTAGATCATGCCGACTT-3’
F3引物:5′-AAGTCCCGCACCTTCCT-3′
R3引物:5′-AGCCAGACGATGACCAC-3′
F4引物:5’-CCTCCCTCGTCATCGGCGAgttttagagctagaaat-3’
R4引物:5’-TCGCCGATGACGAGGGAGGCcggcagccaagccagca-3’
F5引物:5’-GGCA-GGTGCTGGTGATCGCGACGC-3’
R5引物:5’-AAAC-GCGTCGCGATCACCAGCACC-3’
F6引物:5’-GCGTCTTCTCCCTAGTGTTCT-3’
R6引物:5’-CCACACCACGATGTTGTAG-3’
F7引物:5’-TGGTGGTGAGGTGACATG-3’
R7引物:5’-TCCTGGCAAGGTCGCT-3’
靶标序列:CCTCCCTCGTCATCGGCGA(靶点1)
GGTGCTGGTGATCGCGACGC(靶点2)
UF引物:5’-CTCCGTTTTACCTGTGGAATCG-3’
gR-R引物:5’-CGGAGGAAAATTCCATCCAC-3’
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
以上对本发明及其实施方式进行了描述,这种描述没有限制性,附图中所示的也只是本发明的实施方式之一,实际的结构并不局限于此。总而言之如果本领域的普通技术人员受其启示,在不脱离本发明创造宗旨的情况下,不经创造性的设计出与该技术方案相似的结构方式及实施例,均应属于本发明的保护范围。
Claims (3)
1.一种培育耐低钾水稻的方法,其特征在于:包括如下步骤:
步骤一:对水稻OsHAK3基因进行克隆;
步骤二:构建pHB-OsHAK3过表达重组质粒;
步骤三:获得OsHAK3过表达阳性植株;
步骤四:获得耐低钾水稻;
步骤五:获得CRISPER/Cas9敲除的OsHAK3靶标序列;
步骤六:构建含所述靶标序列片段的pCRISPER/Cas9重组质粒;
步骤七:获得OsHAK3敲除阳性植株;
步骤八:获得低钾敏感的水稻。
2.根据权利要求1所述的一种培育耐低钾水稻的方法,其特征在于:步骤3)所述的过表达阳性植株为HAK3-1和HAK3-2。
3.根据权利要求2所述的一种培育耐低钾水稻的方法,其特征在于:步骤7)所述的过表达阳性植株为hak3-1和hak3-2。
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Non-Patent Citations (3)
Title |
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L.ZHANG等: "A potassium (K)-transporter, OsHAK3, is required for K- homeostasis in rice under low-K and high-salinity conditions", 《AUTHOREA》 * |
T.OKADA 等: "Expression of OsHAK genes encoding potassium ion transporters in rice", 《PLANT BIOTECHNOLOGY》 * |
陈璇: "水稻耐低钾候选基因的初步研究", 《中国优秀博硕士学位论文全文数据库(硕士)基础科学辑》 * |
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