CN116574163A - OsKUA1基因在培育耐低温水稻中的应用及其培育耐低温水稻的方法 - Google Patents
OsKUA1基因在培育耐低温水稻中的应用及其培育耐低温水稻的方法 Download PDFInfo
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Abstract
本发明属于生物领域和作物育种领域,具体公开了OsKUA1基因在培育耐低温水稻中的应用和培育耐低温水稻的方法。利用CR1SPER/Cas9技术定向敲除水稻OsKUA1基因,发现OsKUA1基因敲除的转基因植株对低温敏感,在低温处理下植株叶片枯萎,植株存活率低。将OsKUA1基因在水稻中过表达,发现OsKUA1基因过表达的植株对低温耐受,在低温条件下长势较好,植株存活率高。上述结果表明,OsKUA1基因参与水稻耐低温信号途径,是一个水稻耐低温基因。本发明可以用于培育水稻耐低温水稻品种。
Description
技术领域
本发明属于基因工程领域,涉及OsKUA1基因在培育耐低温水稻中的应用。
背景技术
水稻是世界上的主要粮食作物之一,起源于温带气候区,对低温胁迫敏感。低温胁迫会引起水稻生理生化代谢紊乱,导致水稻在低温下出现生长缓慢,开花延迟等现象,严重影响水稻产量和品质,危及全球农业生产。因此,挖掘水稻优良耐低温基因并利用分子育种技术培育耐低温新品种具有重要意义。但迄今为止,可利用的优势耐低温基因资源有待进一步发掘。
MYB是植物中最大的转录因子家族之一,广泛参与植物生长发育和逆境胁迫的调控。根据MYB重复序列数目,将植物中的MYB转录因子分为4类:1R-MYB、R2R3-MYB、3R-MYB和4R-MYB。目前R2R3-MYB亚族被广泛研究,而有关1R-MYB类的报道较少。OsKUA1基因属于1R-MYB亚类,其在水稻中的生理功能还未见报道,其是否参与水稻对低温胁迫的响应也还不清楚。本发明发现OsKUA1基因的表达量受低温诱导,在此基础上,利用CRISPR/Cas9敲除技术和过表达技术分别创制出OsKUA1的敲除突变体及其过表达水稻植株,获得相关低温敏感和耐低温水稻材料。本发明利用OsKUA1基因提供了一种培育耐低温水稻中的方法。
发明内容
本发明在研究中发现OsKUA1基因具有参与脱落酸反应等逆境响应元件,而经实验发现OsKUA1基因的表达量受低温诱导,在此基础上,利用基因敲除技术和过表达技术分别创制出OsKUA1的敲除突变体及其过表达水稻植株,获得相关低温敏感和耐低温水稻材料。由此本发明利用OsKUA1基因提供了一种培育耐低温水稻中的方法。
因此,本发明提供OsKUA1基因在培育耐低温水稻或低温敏感水稻中的应用,所述OsKUA1基因编码的氨基酸序列如SEQ ID NO:2所示。
优选地,所述OsKUA1基因的核苷酸序列如SEQ ID NO:1所示。
具体地,将OsKUA1基因导入水稻,得到对低温逆境胁迫耐受能力增强的转基因植株;或者利用CRISPER/Cas9技术定向敲除水稻OsKUA1基因,得到对低温逆境胁迫敏感的转基因植株。
进一步地,所述水稻是粳稻。
本发明提供一种培育耐低温或低温敏感水稻的方法,其包括如下步骤:将OsKUA1基因导入水稻,得到对低温耐受能力增强的转基因植株;或者利用定向敲除水稻OsKUA1基因,得到对低温敏感的转基因植株。
具体地,培育耐低温水稻的方法是将OsKUA1基因导入水稻是通过导入含有OsKUA1基因的过表达重组载体,获得过表达阳性植株;任选地,进一步培育成耐低温水稻品种。更具体包括如下步骤:将OsKUA1过表达重组质粒转化水稻,获得OsKUA1过表达阳性植株,进而得到耐低温水稻植株进一步培育成耐低温水稻品种。
另外具体地,培育低温敏感水稻的方法通过CRISPER/Cas9敲除的OsKUA1基因,获得OsKUA1基因被敲除阳性植株;任选地,进一步培育成低温敏感的水稻品种。更具体地,通过设计敲除的OsKUA1靶标序列,构建含所述靶标序列片段的pCRISPER/Cas9重组质粒,转化水稻以得到OsKUA1敲除阳性植株,进而得到低温敏感水稻植株进一步培育成低温敏感水稻品种。
具体地,所述水稻是粳稻。
本发明公开了OsKUA1基因在提高水稻耐低温能力的应用。OsKUA1基因的敲除突变体对低温敏感,水稻存活率显著下降。OsKUA1基因过表达植株对低温耐受能力明显增强,水稻存活率显著明显提高。本发明可为提高水稻耐低温能力,为培育低温耐受水稻新品种提供保障。
附图说明
图1为OsKUA1基因在低温处理下的表达量结果。
图2为OsKUA1敲除突变体的检测结果。
图3为OsKUA1过表达株系的检测结果。
图4为OsKUA1的敲除突变植株(kua1-1、kua1-2)和其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)在低温条件下的表型观察结果。
图5为OsKUA1的敲除突变植株(kua1-1、kua1-2)和其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)在低温条件下的存活率结果。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白,以下结合具体实施例,并结合附图,对本发明进一步详细说明。
本发明提供了OsKUA1基因在培育耐低温水稻中的应用,包括如下步骤:水稻OsKUA1基因的克隆;pHB-OsKUA1过表达重组质粒的构建;OsKUA1过表达阳性植株的获得;耐低温水稻的获得。进行CR1SPER/Cas9敲除的OsKUA1靶标序列的获得;含所述靶标序列片段的pCR1SPER/Cas9重组质粒的构建;OsKUA1敲除阳性植株的获得;低温敏感的水稻的获得。
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15m1n。
(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-111),生根培养基(HF),CS培养基(阳性筛选)。筛选培养基需加Hygromyc1n B、Carben1c1ll1nd1sod1um;共培养培养基需加Acetosyr1ngone;分化培养基需加KT、NAA;生根培养基需加NAA;CS培养基需加Hygromyc1n B、6-BA。
(4)水稻水培液配方:
实施例1、水稻OsKUA1基因的克隆
通过定量PCR检测,发现OsKUA1基因的表达量受到低温诱导,在低温处理2小时,其表达量明显上调;当低温处理4小时,其表达量达到顶峰(如图1所示)。所用上游引物序列为:F1:5’-TCCTACTGTGGCAACTAC-3’;下游引物序列为R1:5’-CTTCTTCATCGGCAAACC-3’。用上述引物进行PCR扩增,测序得到OsKUA1基因在低温处理下的表达量结果(如图1所示)。因此,我们推测OsKUA1基因可能参与水稻对低温胁迫的响应。为了鉴定OsKUA1基因是否与水稻响应低温胁迫有关。首先,我们进行水稻OsKUA1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OsKUA1的编码区序列并测序;所用上游引物序列为:F2:5’-ATGGCGAGGAAGTGCTCCT-3’;下游引物序列为:R2:5’-:TCATGTGCCGATGGACG-3’。用上述引物进行PCR扩增,测序得到日本晴OsKUA1基因的编码区序列。
OsKUA1基因编码区序列全长906bp,序列如下:
1 ATGGCGAGGA AGTGCTCCTA CTGTGGCAAC TACGGCCACA ACTCAAGAAC CTGCAGCAGC
61 AGCGCCAGCG CTGGACACAG GGATACCACC ATGCTCTGCG ACGGCGGCGA CGGAGGTGGC
121 GGCAGTGGGC TGAGGCTGTT CGGAGTGCAG GTCCATGTCG CTGCCGGCGG CGGCGGTGGA
181 GGTGGAGGTG GAGGTTTGCC GATGAAGAAG AGCTACAGCA TGGACTGCCT GCAGCTGGCG
241 GCGGCGGGGG CGGCTCCGGG CTCGCTCGTG TCGCCGTCGT CGTCGTCCTC GTCGTCGATG
301 CTCCTGTCGA TCGACGAGGG GGGCTTGGAG AGGGCGTCCA ATGGGTACCT GTCTGATGGC
361 CCCCATGGCA GAATTGTCCA GGAGAGGAAG AAAGGAGTTC CGTGGAGCGA GGAGGAGCAC
421 CGGCTGTTCC TCGTCGGGCT CGAGAAGCTG GGCAAGGGCG ACTGGCGAGG CATCTCCCGG
481 AGCTACGTCA CGACGAGGAC CCCGACCCAG GTCGCCAGCC ACGCCCAGAA GTTCTTCCTC
541 AGGCAGAGCA GCATCGGCAA GAAGAAGCGC CGCTCCAGCC TCTTCGACAT GGTGCCGATT
601 TGCGAGAACG GTGCGCGCGT CTCGGAGCAG CTGAGCGGCG AAGGCGCGGC GGCGGCGGCG
661 GCGGCGTCGA CCTCACTGTC GCTGATGAAC ACGCACGAGA CCTCCTCGGA CAGAGTGGCG
721 GCAATTGATC TGAATTCCAC CGAGGAAGAT GACACGGTGG GCGCGTCAGG GAGGCCGTTT
781 TTCCCGGTGG TTCTGATGGA GCAGCAGCAG CAGGCTTCCC ATGGACATGG TCACCACCAC
841 CACTGCACGC CGCTCGACCT GGAGCTCGGC ATGTCCGTCT CGTCGACGCC GTCCATCGGC
901 ACATGA
实施例2、pHB-OsKUA1过表达重组质粒的构建
用于构建pHB-OsKUA1重组质粒的上游引物序列为:F3:5’-tctgcactaggtacctgcagATGGCGAGGAAGTGCTC-3’,下游引物序列为:R3:5’-atggatccgtcgacctgcagTACTGTGCCGATGGACG-3’。利用上述引物PCR扩增日本晴cDNA模板,回收906bp的目的带,用HindIII+XbaI将OsKUA1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OsKUA1的目的片段连到pHB载体上。
实施例3、OsKUA1过表达阳性植株的获得
将实施例2构建成功的pHB-OsKUA1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,用无菌的枪头将生长的菌转到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OsKUA1表达量的检测,OsKUA1表达量上调的OsKUA1过表达阳性植株,如图2所示,为共检测到21株阳性植株。检测引物序列为:上游引物F4:5′-ATGGCGAGGAAGTGCTC-3′,下游引物R4:5′-TCATGTGCCGATGGACG-3′。
将所述阳性植株KUA1-OE1、KUA1-OE2、KUA1-OE3进行繁种,后代植株具有潮霉素抗性且OsKUA1表达量上调即为耐低温水稻。
实施例4、用于CR1SPER/Cas9敲除的OsKUA1靶标序列和重组质粒的构建
利用CR1SPER/Cas9系统,根据OsKUA1编码区序列选择特异的使OsKUA1蛋白失活的靶标序列。靶标序列:GATACCACCATGCTCTGCGA(靶点1)和GTCGATCGACGAGGGGGGCT(靶点2)。
靶点1序列敲除的完整靶序列如下:
F5:5’-ATACCACCATGCTCTGCGAgttttagagctagaaat-3’
R5:5’-TCGCAGAGCATGGTGGTATCggcagccaagccagca-3’
靶点2序列敲除的完整靶序列如下:
F6:5’-TCGATCGACGAGGGGGGCTgttttagagctagaaat-3’
R6:5’-AGCCCCCCTCGTCGATCGACaacacaagcggcagc-3’
将F5引物、R5引物、F6引物、R6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F5引物和R5引物,F6引物和R6引物分别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OsKUA1蛋白的靶序列和sg-RNA的重组载体。连接体系为:10×T4连接buffer 1.5μL,双链小片段4μL,酶切过的pOs-sgRNA载体3μL,T4连接酶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LRm1x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的蛋白酶K于37℃下处理10m1n,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OsKUA1蛋白靶标序列-sg-RNA+Cas9的完整的重组质粒。
实施例5、OsKUA1敲除阳性植株的获得
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0.26。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38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以及含靶标位点2的500bp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和2的检测引物为F7 5’-AGGCACAGTACAAAGATTCAGC-3’,R7:5’-GCAAGGCCATTTCAACATTA-3’,扩增片段长度为483bp。根据测序比对结果,选取OsKUA1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1和2(如图3所示),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) | GATACCACCATGCTCTG.CGA | 野生型 |
1 | GATACCACCATGCTCTGTCGA | 纯合突变体 |
野生型(WT) | GTCGATCGACGAGGGGGGCT | 野生型 |
2 | GTCGATCGACGAGGGGGGC. | 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OsKUA1基因功能缺失的突变体,即为对低温敏感的水稻。分别命名为kua1-1突变体和kua1-2突变体。
实施例6、低温耐受和低温敏感表型鉴定
将野生型日本晴(WT)、OsKUA1的敲除突变体(kua1-1、kua1-2)和其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)的种子催芽5天后,水培液培养14天后,分别转移至正常温度(28℃)及低温(4℃)的水培液培养3天,观察各植株表型并拍照。结果发现在28℃条件下,野生型日本晴、OsKUA1的敲除突变体(kua1-1、kua1-2)和其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)的长势一致。而在4℃处理下,OsKUA1的敲除突变体(kua1-1、kua1-2)的植株叶片枯萎(如图4所示),植株存活率明显低于野生型日本晴和其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)(如图5所示)。而其过表达植株(KUA1-OE1、KUA1-OE2、KUA1-OE3)的叶片正常(如图4所示),植株存活率明显高于野生型日本晴和其敲除突变体(kua1-1、kua1-2)(如图5所示)。
本发明公开了水稻OsKUA1基因在提高植物对低温胁迫逆境耐受方面的应用。OsKUA1基因的敲除突变体导致水稻对低温敏感,植株存活率明显降低。OsKUA1基因过表达植物对低温耐受,植株存活率明显提高。本发明可为提高植物耐低温能力及培育耐低温水稻新品种提供保障。
Claims (8)
1.OsKUA1基因在培育耐低温水稻或低温敏感水稻中的应用,所述OsKUA1基因编码的氨基酸序列如SEQ ID NO:2所示。
2.如权利要求1所述的应用,其特征在于,所述OsKUA1基因的核苷酸序列如SEQ ID NO:1所示。
3.如权利要求1所述的应用,其特征在于,将OsKUA1基因导入水稻,得到对低温逆境胁迫耐受能力增强的转基因植株;或者利用CRISPER/Cas9技术定向敲除水稻OsKUA1基因,得到对低温逆境胁迫敏感的转基因植株。
4.如权利要求1至3任一项所述的应用,其特征在于,所述水稻是粳稻。
5.一种培育耐低温或低温敏感水稻的方法,其包括如下步骤:将OsKUA1基因导入水稻,得到对低温耐受能力增强的转基因植株;或者利用定向敲除水稻OsKUA1基因,得到对低温敏感的转基因植株。
6.如权利要求5所述的方法,其特征在于,培育耐低温水稻的方法是将OsKUA1基因导入水稻是通过导入含有OsKUA1基因的过表达重组载体,获得过表达阳性植株;任选地,进一步培育成耐低温水稻品种。
7.如权利要求5所述的方法,其特征在于,培育低温敏感水稻的方法通过CRISPER/Cas9敲除的OsKUA1基因,获得OsKUA1基因被敲除阳性植株;任选地,进一步培育成低温敏感的水稻品种。
8.如权利要求5-7任一项所述的方法,其特征在于,所述水稻是粳稻。
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