CN112725357A - 一种培育耐低钾水稻的方法 - Google Patents
一种培育耐低钾水稻的方法 Download PDFInfo
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- CN112725357A CN112725357A CN202110197937.2A CN202110197937A CN112725357A CN 112725357 A CN112725357 A CN 112725357A CN 202110197937 A CN202110197937 A CN 202110197937A CN 112725357 A CN112725357 A CN 112725357A
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- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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Abstract
本申请提供了一种培育耐低钾水稻的方法,具体包括以下步骤:对水稻OsHAK7基因进行克隆;构建pHB‑OsHAK7过表达重组质粒;获得OsHAK7过表达阳性植株;获得耐低钾水稻。本申请提供了OsHAK7基因在提高水稻耐低钾能力的应用。OsHAK7基因在水稻中负责从土壤中吸收K+。OsHAK7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。OsHAK7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。本申请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新的基因资源。
Description
技术领域
本申请涉及基因工程技术领域,尤其涉及一种培育耐低钾水稻的方法。
背景技术
钾离子是植物细胞中十分丰富的两价阳离子,在光合作用,调节细胞膨压和渗透势等一系列生理生化过程中起着重要作用。钾是植物生长发育所必需的矿质元素,缺钾会严重影响作物的产量和质量。
水稻从土壤吸收钾离子,通过木质部导管将钾离子运输到地上各个部分以及钾离子再分配的生理过程由细胞膜上的转运蛋白负责,细胞膜上的各种钾转运蛋白和通道蛋白又受到转录因子以及蛋白激酶等调控因子的调控,这些膜蛋白的活性调控是植物钾营养效率调控的关键和基础。但是到目前为止,有关水稻耐低钾的分子机制还不清楚,参与水稻耐低钾的相关转运蛋白还报道较少。因此挖掘水稻耐低钾膜转运蛋白,解析它们在水稻钾高效利用中的活性调控机制是提高水稻钾高效利用率以及培育钾高效利用水稻品种的有效途径。
发明内容
本申请提供了一种培育耐低钾水稻的方法,以解决水稻耐低钾能力低以及耐钾水稻品种缺乏的问题。
本申请提供了一种培育耐低钾水稻的方法,具体包括以下步骤:
对水稻OsHAK7基因进行克隆;
构建pHB-OsHAK7过表达重组质粒;
获得OsHAK7过表达阳性植株;
获得耐低钾水稻。
可选的,所述对水稻OsHAK7基因进行克隆包括设计特异性的引物序列克隆OsHAK7的编码区序列;所述引物序列包括上游引物序列F1和下游引物序列R1;
F1:5'-ATGCCCAGCTACCAATATCT-3';
R1:5'-AGACATAGTAGATCATGCCGACT-3'。
可选的,所述构建pHB-OsHAK7过表达重组质粒包括构建引物组对日本晴cDNA模板进行PCR扩增,回收目的带后将OsHAK7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OsHAK7的目的片段连到pHB载体上。
可选的,所述获得OsHAK7过表达阳性植株包括将构建成功的pHB-OsHAK7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培养基上诱导培养愈伤组织,将愈伤组织进行筛选以及分化培养成幼苗。
可选的,所述引物组包括上游引物序列F2和下游引物序列R2;
F2:5'-ACCAGTCTCTCTCTCAAGCTTATGCCCAGCTACCAATATCTGC-3';
R2:5'-GATACGAACGAAAGCTCTAGATTAGACATAGTAGATCATGCCGACTTC-3'。
由以上技术方案可知,本申请提供了一种培育耐低钾水稻的方法,具体包括以下步骤:对水稻OsHAK7基因进行克隆;构建pHB-OsHAK7过表达重组质粒;获得OsHAK7过表达阳性植株;获得耐低钾水稻。以解决水稻耐低钾能力低以及耐钾水稻品种缺乏的问题。本申请提供了OsHAK7基因在提高水稻耐低钾能力的应用。OsHAK7基因在水稻中负责从土壤中吸收K+。OsHAK7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。OsHAK7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。本申请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新的基因资源。
附图说明
为了更清楚地说明本申请的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,显而易见地,对于本领域普通技术人员而言,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1为一种培育耐低钾水稻的方法的流程图;
图2为OsHAK7过表达株系的检测结果;
图3为OsHAK7敲除突变体的检测结果;
图4为突变植株(Oshak7-1、Oshak7-2)和过表达植株(OsHAK7-1、OsHAK7-2)在低钾条件下的表型观察结果;
图5为突变植株(Oshak7-1、Oshak7-2)和过表达植株(OsHAK7-1、OsHAK7-2)在低钾条件下的根长、地上部分长测定结果;
图6为突变植株(Oshak7)和过表达植株(OsHAK7)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。
具体实施方式
下面将详细地对实施例进行说明,其示例表示在附图中。下面的描述涉及附图时,除非另有表示,不同附图中的相同数字表示相同或相似的要素。以下实施例中描述的实施方式并不代表与本申请相一致的所有实施方式。仅是与权利要求书中所详述的、本申请的一些方面相一致的系统和方法的示例。
参见图1,为一种培育耐低钾水稻的方法的流程图。本申请提供了一种培育耐低钾水稻的方法,具体包括以下步骤:
对水稻OsHAK7基因进行克隆;构建pHB-OsHAK7过表达重组质粒;获得OsHAK7过表达阳性植株;获得耐低钾水稻。
进一步地,本申请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,在121℃高温高压下灭菌15min。
(2)AB培养基:NaH2PO4·2H2O 1300mg/L,K2HPO42950mg/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由表1所示:
表1
其中,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)水稻水培液配方由表2所示:
表2
本申请提供的一种培育耐低钾水稻的方法,具体实施例包括以下步骤:
首先,对水稻的OsHAK7基因进行克隆;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OsHAK7的编码区序列并测序;所述引物序列包括上游引物序列F1和下游引物序列R1;F1:5'-ATGCCCAGCTACCAATATCT-3';R1:5'-AGACATAGTAGATCATGCCGACT-3'。用上述引物进行PCR扩增,测序得到日本晴OsHAK7基因的编码区序列。具体地,OsHAK7基因编码区序列全长2436bp,见序列表。
进一步地,对pHB-OsHAK7过表达重组质粒进行构建;其中用于构建pHB-OsHAK7重组质粒的引物组包括上游引物序列F2和下游引物序列R2;上游引物序列F2为:
F2:5'-ACCAGTCTCTCTCTCAAGCTTATGCCCAGCTACCAATATCTGC-3';
下游引物序列为:
R2:5'-GATACGAACGAAAGCTCTAGATTAGACATAGTAGATCATGCCGACTTC-3'。
利用上述F2和R2引物进行PCR扩增日本晴cDNA模板,回收2436bp的目的带,使用HindIII+XbaI将OsHAK7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OsHAK7的目的片段连到pHB载体上。
进一步地,获得OsHAK7过表达的阳性植株;将构建成功的pHB-OsHAK7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。将含有重组载体的农杆菌EHA105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。使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仪进行OsHAK7表达量的检测,OsHAK7表达量上调的为OsHAK7的过表达阳性植株,共检测到23株阳性植株。
其中,检测引物序列为包括上游引物F3和下游引物R3;
F3:5'-TTTCCGAGACACGACAGT-3';
R3:5′-TGGTGACAAAGTGCGAGA-3′。
进一步地,获得耐低钾的水稻;将随机挑选的阳性植株OsHAK7-1和OsHAK7-2进行繁种,后代植株具有潮霉素抗性且OsHAK7表达量上调即为耐低钾水稻。
为了验证耐低钾水稻在低钾条件下对钾离子的吸收能力以及长势,接着利用OsHAK7基因获得低钾敏感水稻;
获得低钾敏感水稻的具体步骤包括:首先,利用CRISPER/Cas9敲除进而获取OsHAK7靶标序列;利用CRISPER/Cas9系统,根据OsHAK7编码区序列选择特异的使OsHAK7蛋白失活的靶标序列。其中,靶标序列:TGGAGAAGCACAGGAAGCTACGG。
进一步地,构建含上述靶标序列片段的pCRISPER/Cas9重组质粒;在靶序列设计接头引物后的完整靶序列如下:
F4:5'-TGGAGAAGCACAGGAAGCTAGTTTTAGAGCTAGAAAT-3';
R4:5'-TAGCTTCCTGTGCTTCTCCACGGCAGCCAAGCCAGCA-3'。
将F4引物、R4引物稀释成浓度为10μM的溶液备用。取pYLgRNA-OsU6/LacZ质粒1μg,在25μL反应体系,用10UBsaI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×T4 DNA ligase Buffer 1μL,pYLgRNA-OsU6/LacZ 0.5μL,接头0.5μL,T4 DNAligase(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R4(反应1)、接头正向F4引物/gR-R引物(反应2)各0.2μM,适量高保真PCR酶。其中U-F引物和gR-R引物如下:
UF引物:5'-CTCCGTTTTACCTGTGGAATCG-3';
gR-R引物:5'-CGGAGGAAAATTCCATCCAC-3'。
其中,PCR扩增26循环为:94℃10s,60℃15s,68℃20s。取4μL用2%琼脂糖胶电泳检查(产物长度约700bp)。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)。接着,PCR扩增22循环:95℃10s,56℃15s,68℃20s。取2μl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/μLpYLCRISPR/Cas9质粒为模板,组装sgRNA表达盒到pYLCRISPR/Cas9载体。双元载体与sgRNA表达盒的酶切-连接反应体系如下:
双元载体与sgRNA表达盒的酶切-连接反应体系
接着,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OsHAK7目标插入序列的LacZ-OsU6-gRNA表达盒的完整重组质粒。
进一步地,获得OsHAK7敲除的阳性植株;分别将上一步骤构建成功的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0.3。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25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(含靶标位点的1775bp以内的DNA片段)、扩增得到的PCR产物经纯化后测序,测序结果与野生型植株序列比对,T0代部分阳性植株靶位点变化如下表所示:
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) | ACAGGAAGCTACGGACTTGTT | 野生型 |
7-1 | ACAGGAAGCCTACGGACTTGTT | 纯合突变体 |
野生型(WT) | CTGGAGAAGCACCAGTGGTGATAGGTG | 野生型 |
7-2 | CTGGAGA…TAGGTG | 纯合突变体 |
其中,设计的靶标位点检测引物为F5:5'-TTGCTTTATACTCACTTCTCTGCAG-3',R5:5'-GGACTACTTTCTTAAAACAGCTTGC-3',扩增片段长度为1775bp。根据测序比对结果,选取OsHAK7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7-1和7-2,进行繁种。
进一步地,获得低钾敏感的水稻;将所述突变体植株进行繁种,于后代植株中分离OsHAK7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Oshak7-1突变体和Oshak7-2突变体。
由图2-图6所示,将获得低价耐受水稻OsHAK7-1以及低钾敏感水稻Oshak7-1突变体和Oshak7-2突变体进行表型鉴定;将野生型日本晴(Nip)、OsHAK7的敲除突变体(Oshak7-1、Oshak7-2)和其过表达植株(OsHAK7-1)的种子催芽3天后,分别在含有不同钾离子浓度(10mM、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。结果发现在钾离子充足条件下,野生型日本晴、OsHAK7的敲除突变体(Oshak7-1、Oshak7-2)的长势一致。而在低钾处理下,OsHAK7的敲除突变体(Oshak7-1、Oshak7-2)的植株矮小、根部变短,根部和地上部分钾离子含量、根部的钾离子吸收能力比野生型日本晴少88%左右。过表达植株(OsHAK7-1)在低钾条件下的长势明显强于野生型日本晴和敲除突变体(Oshak7-1、Oshak7-2)。其根部和地上部分钾离子含量、根部的钾离子吸收能力也明显高于野生型日本晴和敲除突变体(Oshak7-1)。
本申请提供了一种培育耐低钾水稻的方法,具体包括以下步骤:对水稻OsHAK7基因进行克隆;构建pHB-OsHAK7过表达重组质粒;获得OsHAK7过表达阳性植株;获得耐低钾水稻。以解决水稻耐低钾能力低以及耐钾水稻品种缺乏的问题。本申请提供了OsHAK7基因在提高水稻耐低钾能力的应用。OsHAK7基因在水稻中负责从土壤中吸收K+。OsHAK7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。OsHAK7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。本申请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新的基因资源。
本申请提供的实施例之间的相似部分相互参见即可,以上提供的具体实施方式只是本申请总的构思下的几个示例,并不构成本申请保护范围的限定。对于本领域的技术人员而言,在不付出创造性劳动的前提下依据本申请方案所扩展出的任何其他实施方式都属于本申请的保护范围。
序列表
<110> 湖南师范大学
<120> 一种培育耐低钾水稻的方法
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2436
<212> DNA
<213> 水稻(Oryza sativa)
<400> 1
atgcccagct accaatatct gctttctctc ttgttttaca tactggactg tactgatcgg 60
ttctcagtaa tagttactat tcacaatcac agggtcggcg tactgatgat tgtgcttttg 120
caggaccaat ggaaaagtta ttgcagaact atctctctgc tagcatttca gagctttggt 180
gtggtctatg gagacttgag tacatcaccc ctgtacgtct acaaaagtgc attttctggg 240
aggctgaata attaccgtga tgagacaacc atatttggat tgttttcgct gatattttgg 300
actttgacac ttcttccgtt gctgaagtat gtaataattg tattgaacgc agatgataat 360
ggggaaggtg gaacatttgc tttatactca cttctctgca ggcatgcaaa gttcagcctg 420
cttccaaacc agcaatcggc agacgaagag ctttcaacgt actatcagcc tggagttggt 480
ggcattattt cctctccact caaaagattc ctggagaagc acaggaagct acggacttgt 540
ttactacttt ttgttctgtt tggcgcatgc atggtgatag gtgatggtgt tttcacacct 600
gctatctctg tcttgtcagc catctctggt ttaaaagacc ctggcccagg aggaatacca 660
gatggttggg tggtgttcat tgcatgtatt gtgcttgttg gcctgtttgc actgcaacac 720
cgaggaactc atagggtagc atttatgttt gcgcccattg ttgtagtatg gctgttgagt 780
attggtgtta ttgggcttta taatatcatc cactggaacc acagaatatt tcttgctctt 840
tctccacatt atgttataaa gttcttcaag atgacaggaa aagatggttg gctttctctt 900
ggaggagtac ttcttgctat aacaggtact gaagctatgt ttgctgatct tggccacttc 960
actgctgcat ctattaggtt ggcttttgtt ggtgccatat atccctgtct tgtgctgcaa 1020
tacatggggc aggctgcatt tctttccaga aacatgtcag ctgtagaaga tagcttttac 1080
caatctgtac caagatccct gttttggcca gtgtttgtca ttgcaactct tgcagcggtt 1140
gtgggcagcc agtctatcat atctgctacc ttttcaattg ttaagcagtg tctttctttg 1200
ggatgttttc cacgggttaa ggtcgtgcat acatcaaggt ggatccatgg tcagatttac 1260
atacctgaga ttaattggat tctgatggtt ctttgtcttg ctgtgacact tggtttccga 1320
gacacgacag ttattggaaa tgcttacggt cttgcgtgta tcgttgtgat gtttgttacc 1380
acctggttga tggcattggt tatcatattt gtgtggcaaa aaaacatcct gctcgccttg 1440
ttatttgtcg tggcgtttgg gtcaatcgaa gtggtgtacc tgtcggcagc agtcactaaa 1500
gttcctcagg gaggatgggc accaatcgta tttgccttcg tgttcatgct cgtcatgtac 1560
gtatggcact acggctctcg gaggaagtac ctgtttgatc tccagaacaa ggtctcaatg 1620
aaatggatcc tgacacttgg tccgagcctt gggatcgttc gggtgcctgg aatcggtctc 1680
atctacacag agctggtcac cggggtgcct tccatcttct cgcactttgt caccaacctg 1740
cctgctttcc atcaggttct ggtcttcgtt tgcgtcaagt ccgtacctgt gccgttcgtt 1800
ccagaggacg aacggtacct cattggacgg atcggcccca gggaataccg tatgtaccgg 1860
tgcatcgtga ggtatggcta caaagatgtg cagaaagatg acgagaactt tgagaaccac 1920
ttggtgatga gcattgccaa gttcatccaa atggaagccg aggaggcagc ctcctctggg 1980
agctacgaat cttcggaagg gaggatggct gtcatacaca cagaggacac aaccggcacc 2040
ggtctggtga tgagggactc caataacgag gcttcaggca cgtccctgac aaggagcagc 2100
aggtcggaga cgctccggag cctccagtcc atctacgagc aggagtccgg tagtctcagc 2160
cggcgtcgca gagtccgttt tgagattgcg gaggaggagc ggatcgaccc ccaggtgcgg 2220
gacgagctgg ctgatctcct ggacgccaag gaggccggcg tgacttacat catcggccac 2280
tcctacgtga aggcaaggaa gaactccaac ttcctcaaga cgttcgccat cgactacgct 2340
tactcgttcc tccgcaagaa ctgcaggggt cccgcggtgg cgctgcacat accccacatt 2400
agcctcgtcg aagtcggcat gatctactat gtctaa 2436
<210> 2
<211> 498
<212> PRT
<213> 水稻(Oryza sativa)
<400> 2
Met Asp Ala Glu Thr Gly Pro Ala Ala Pro Gln Asp Gln Trp Lys Ser
1 5 10 15
Tyr Cys Arg Thr Ile Ser Leu Leu Ala Phe Gln Ser Phe Gly Val Val
20 25 30
Tyr Gly Asp Leu Ser Thr Ser Pro Leu Tyr Val Tyr Lys Ser Ala Phe
35 40 45
Ser Gly Arg Leu Asn Asn Tyr Arg Asp Glu Thr Thr Ile Phe Gly Leu
50 55 60
Phe Ser Leu Ile Phe Trp Thr Leu Thr Leu Leu Pro Leu Leu Lys Tyr
65 70 75 80
Val Ile Ile Val Leu Asn Ala Asp Asp Asn Gly Glu Gly Gly Thr Phe
85 90 95
Ala Leu Tyr Ser Leu Leu Cys Arg His Ala Lys Phe Ser Leu Leu Pro
100 105 110
Asn Gln Gln Ser Ala Asp Glu Glu Leu Ser Thr Tyr Tyr Gln Pro Gly
115 120 125
Val Gly Gly Ile Ile Ser Ser Pro Leu Lys Arg Phe Leu Glu Lys His
130 135 140
Arg Lys Leu Arg Thr Cys Leu Leu Leu Phe Val Leu Phe Gly Ala Cys
145 150 155 160
Met Val Ile Gly Asp Gly Val Phe Thr Pro Ala Ile Ser Val Leu Ser
165 170 175
Ala Ile Ser Gly Leu Lys Asp Pro Gly Pro Gly Gly Ile Pro Asp Gly
180 185 190
Trp Val Val Phe Ile Ala Cys Ile Val Leu Val Gly Leu Phe Ala Leu
195 200 205
Gln His Arg Gly Thr His Arg Val Ala Phe Met Phe Ala Pro Ile Val
210 215 220
Val Val Trp Leu Leu Ser Ile Gly Val Ile Gly Leu Tyr Asn Ile Ile
225 230 235 240
His Trp Asn His Arg Ile Phe Leu Ala Leu Ser Pro His Tyr Val Ile
245 250 255
Lys Phe Phe Lys Met Thr Gly Lys Asp Gly Trp Leu Ser Leu Gly Gly
260 265 270
Val Leu Leu Ala Ile Thr Gly Thr Glu Ala Met Phe Ala Asp Leu Gly
275 280 285
His Phe Thr Ala Ala Ser Ile Arg Leu Ala Phe Val Gly Ala Ile Tyr
290 295 300
Pro Cys Leu Val Leu Gln Tyr Met Gly Gln Ala Ala Phe Leu Ser Arg
305 310 315 320
Asn Met Ser Ala Val Glu Asp Ser Phe Tyr Gln Ser Val Pro Arg Ser
325 330 335
Leu Phe Trp Pro Val Phe Val Ile Ala Thr Leu Ala Ala Val Val Gly
340 345 350
Ser Gln Ser Ile Ile Ser Ala Thr Phe Ser Ile Val Lys Gln Cys Leu
355 360 365
Ser Leu Gly Cys Phe Pro Arg Val Lys Val Val His Thr Ser Arg Trp
370 375 380
Ile His Gly Gln Ile Tyr Ile Pro Glu Ile Asn Trp Ile Leu Met Val
385 390 395 400
Leu Cys Leu Ala Val Thr Leu Gly Phe Arg Asp Thr Thr Val Ile Gly
405 410 415
Asn Ala Tyr Gly Leu Ala Cys Ile Val Val Met Phe Val Thr Thr Trp
420 425 430
Leu Met Ala Leu Val Ile Ile Phe Val Trp Gln Lys Asn Ile Leu Leu
435 440 445
Ala Leu Leu Phe Val Val Ala Phe Gly Ser Ile Glu Val Val Tyr Leu
450 455 460
Ser Ala Ala Val Thr Lys Val Pro Gln Gly Gly Trp Ala Pro Ile Val
465 470 475 480
Phe Ala Phe Val Phe Met Leu Val Met Tyr Val Trp His Tyr Gly Ser
485 490 495
Arg Arg
Claims (5)
1.一种培育耐低钾水稻的方法,其特征在于,具体包括以下步骤:
对水稻OsHAK7基因进行克隆;
构建pHB-OsHAK7过表达重组质粒;
获得OsHAK7过表达阳性植株;
获得耐低钾水稻。
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,所述对水稻OsHAK7基因进行克隆包括设计特异性的引物序列克隆OsHAK7的编码区序列;所述引物序列包括上游引物序列F1和下游引物序列R1;
F1:5'-ATGCCCAGCTACCAATATCT-3';
R1:5'-AGACATAGTAGATCATGCCGACT-3'。
3.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,所述构建pHB-OsHAK7过表达重组质粒包括构建引物组对日本晴cDNA模板进行PCR扩增,回收目的带后将OsHAK7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OsHAK7的目的片段连到pHB载体上。
4.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,所述获得OsHAK7过表达阳性植株包括将构建成功的pHB-OsHAK7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培养基上诱导培养愈伤组织,将愈伤组织进行筛选以及分化培养成幼苗。
5.根据权利要求3所述的一种培育耐低钾水稻的方法,其特征在于,所述引物组包括上游引物序列F2和下游引物序列R2;
F2:5'-ACCAGTCTCTCTCTCAAGCTTATGCCCAGCTACCAATATCTGC-3';
R2:5'-GATACGAACGAAAGCTCTAGATTAGACATAGTAGATCATGCCGACTTC-3'。
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MARIA ANTONIA BANUELOS: "Inventory and Functional Characterization of the HAK Potassium Transporters of Rice", 《PLANT PHYSIOLOGY》 * |
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