CN115125219A - OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用 - Google Patents
OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用 Download PDFInfo
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Abstract
本发明公开了一种水稻基因OsJMJ718及其编码蛋白在调控水稻粒型中的应用。本发明构建了OsJMJ718的CRISPR/Cas9敲除表达载体并转化受体植株愈伤组织,培养得到OsJMJ718基因突变的水稻植株;结果显示OsJMJ718基因突变的水稻植株(谷粒)比野生型种子粒长增加,粒宽减少,粒厚减少;表明OsJMJ718负调控水稻种子的粒宽,正调控种子粒长。本发明OsJMJ718基因可用于提高稻米长宽比,从而有助于优质水稻品种选育。
Description
技术领域
本发明涉及植物基因工程技术领域,更具体地,涉及OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用。
背景技术
水稻(Oryza sativa L.)是世界重要粮食作物之一,全世界半数以上的人口以其为主食,水稻生产是关系到国计民生的大事,与我国粮食安全保障密切相关。
水稻粒型包括粒长、粒宽、粒厚和长宽比,均为重要的农艺性状,共同影响稻米品质。水稻种子发育始于双受精作用,形成完整的胚和胚乳。在母体遗传信息、合子遗传信息和生长环境因素的影响下,种皮、胚和胚乳三个结构相互交流,协同控制种子的生长发育,最终决定种子的大小与形状。其遗传调控网络包括泛素-蛋白酶体途径、丝裂原活化蛋白激酶(MAPK)信号途径、G蛋白信号途径、转录因子及植物激素等多个调控通路。分离和克隆粒型相关基因是提高水稻产量的重要手段,目前已经定位到的与粒型相关数量性状位点(QTL)有600多个,并克隆了70多个基因,这些基因相互作用并与其他调控通路共同构成水稻粒型的调控网络。
包含Jumonji C(jmjC)结构域的蛋白质被认为具有组蛋白去甲基化酶的活性,基因组分析显示,水稻中有20个含有jmjC结构域的基因,例如中国专利CN106929498A公开了组蛋白脱乙酰化酶OsHDT701负调控种子发育过程,超量表达组蛋白脱乙酰化酶基因OsHDT701后减小种子的宽度和厚度。组蛋白去甲基化酶OsJMJ718就是JmjC组蛋白去甲基酶家族的成员之一。现有技术公开了OsJMJ718能特异去除组蛋白H3K9位点的甲基化修饰从而激活基因的表达。然而目前,OsJMJ718对水稻粒型方面的影响还未见相关报道。
发明内容
本发明的目的在于克服现有技术中存在的上述缺陷和不足,提供OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用。
本发明的上述目的是通过以下技术方案给予实现的:
本发明公开了OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用,所述水稻OsJMJ718基因核苷酸序列全长1140bp,编码379个氨基酸的组蛋白去甲基化酶OsJMJ718。
本发明构建了水稻OsJMJ718基因的CRISPR/Cas9敲除载体p YLCRISPR/Cas9-MH-OsJMJ718,并转化受体植株愈伤组织,培养筛选得到OsJMJ718基因突变的水稻植株;结果显示OsJMJ718基因突变的水稻植株(谷粒)比野生型种子粒长增加,粒宽减少,粒厚减少;表明OsJMJ718负调控水稻种子的粒宽,正调控种子粒长。从而可将水稻OsJMJ718基因应用于调控水稻粒型,有利于优质水稻品种选育,具体为:
水稻OsJMJ718基因在调控水稻粒长、粒宽、粒厚和/或长宽比中的应用。
具体地,为突变水稻中OsJMJ718基因,进而获得突变体植株。
优选地,为构建OsJMJ718基因的CRISPR/Cas9敲除载体并转化水稻植株。
进一步优选地,CRISPR/Cas9敲除载体为p YLCRISPR/Cas9-MH-OsJMJ718。
具体地,所述p YLCRISPR/Cas9-MH-OsJMJ718敲除载体的构建包括以下步骤:
S1.针对核苷酸序列如SEQ ID NO:1所示的OsJMJ718基因选择靶位点并设计PCR引物,进行PCR扩增,获得含所述靶位点序列的线性DNA片段;
S2.将上述线性DNA片段连入p YLCRISPR/Cas9-MH载体,构建得到p YLCRISPR/Cas9-MH-OsJMJ718敲除载体。
优选地,步骤S1所述靶位点的序列为:5'-TTGGGAGCCACCAGATATG-3'。
进一步优选地,步骤S1采用的接头引物为:
Os718-U6aF:5'-gccgTTGGGAGCCACCAGATATG-3';
Os718-U6aR:5'-aaacCATATCTGGTGGCTCCCAA-3'。
本发明还提供OsJMJ718基因的CRISPR/Cas9敲除载体或质粒在调控水稻粒型中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明提供了组蛋白去甲基化酶基因OsJMJ718及其编码蛋白在调控水稻粒型中的应用,本发明发现敲除OsJMJ718基因的水稻种子与野生型水稻种子相比粒长变长、粒宽和粒厚减少,表明OsJMJ718基因具有调控水稻种子的粒型的功能,OsJMJ718基因可用于提高稻米长宽比,为高产优质水稻新品种的培育提供新的目标基因资源。
附图说明
图1为OsJMJ718基因敲除表达载体的靶位点。
图2为T0代突变位点序列分析。
图3为T1代突变株系的突变位点序列分析。
图4为野生型水稻中花11(WT)和OsJMJ718敲除突变体水稻植株谷粒粒长粒宽表型图。A:野生型(WT)与OsJMJ718基因的敲除株系JMJ718-1、JMJ718-25和JMJ718-18种子的粒长;B:野生型(WT)与OsJMJ718基因的敲除株系JMJ718-1和JMJ718-25种子的粒宽。
图5为野生型水稻中花11(WT)和OsJMJ718敲除突变体水稻植株谷粒粒长、粒宽、粒厚和千粒重的统计图。A:与野生型(WT)相比,突变体JMJ718-1、JMJ718-25和JMJ718-18种子的粒长增加;B:与野生型(WT)相比,突变体JMJ718-1和JMJ718-25种子的粒宽减少;C:与野生型(WT)相比,突变体JMJ718-1、JMJ718-25和JMJ718-18种子的粒厚减少;D:与野生型(WT)相比,OsJMJ718基因的敲除株系JMJ718-1、JMJ718-25和JMJ718-18种子的千粒重减少。
具体实施方式
以下结合说明书附图和具体实施例来进一步说明本发明,但实施例并不对本发明做任何形式的限定。除非特别说明,本发明采用的试剂、方法和设备为本技术领域常规试剂、方法和设备。
除非特别说明,以下实施例所用试剂和材料均为市购。
实施例1组蛋白去甲基化酶基因OsJMJ718敲除转基因水稻的获得
1、CRISPR/Cas9水稻敲除载体的构建
利用CRISPR靶位点设计网站E-CRISPR(http://www.e-crisp.org/E-CRISP/)设计靶位点,用于构建单靶点敲除载体,如图1所示,对应引物为:
Os718-U6aF:5'-gccgTTGGGAGCCACCAGATATG-3';
Os718-U6aR:5'-aaacCATATCTGGTGGCTCCCAA-3'。
载体具体构建流程如下:
(1)制备双链接头:Os718-U6aF和Os718-U6aR各1μL,ddH2O 8μL,混合均匀,90℃30s后,移至室温冷却完成退火。
(2)连接gRNA载体,反应体系如下(10μL):
PCR反应程序37℃5min,20℃5min,5个循环。
(3)gRNA表达盒扩增:
取1μL连接产物作为模板,使用KOD-Plus高保真酶构建15μL体系进行扩增:10×KOD-Plus buffer1.5μL、dNTPs 0.6μL、MgSO4 0.6μL、KOD-Plus 0.3μL、9μL ddH2O,最后加入1μL模板,反应1引物或反应2引物2μL(上下游引物各1μL)其中每个表达盒2个PCR反应:
反应1引物序列:
U-F:5'-CTCCGTTTTACCTGTGGAATCG-3'
Os718-U6aR:5'-aaacCATATCTGGTGGCTCCCAA-3';
反应2引物序列:
Os718-U6aF:5'-gccgTTGGGAGCCACCAGATATG-3';
g RNA-R:5'-CGGAGGAAAATTCCATCCAC-3'
PCR反应程序为95℃2min;95℃10s,60℃15s,68℃20s,25个循环。
将反应1和反应2的PCR产物各稀释10倍,各取1μL混合为模板,进行第二轮PCR扩增,PCR体系为10×KOD-Plus buffer3μL、dNTPs1.2μL、MgSO41.2μL、KOD-Plus0.6μL、20μLddH2O,1μL模板,Cas-B2’和Cas-BL各1.5μL。
Cas-B2’:5'-TTCAGAggtctcTctgaCACTGGAATCGGCAGCAAAGG-3';
Cas-BL:5'-AGCGTGggtctcGaccgGGTCCATCCACTCCAAGCTC-3'
PCR反应程序为95℃2min;95℃10s,58℃15s,68℃20s,20个循环。PCR反应结束后对产物进行琼脂糖凝胶电泳,对符合大小的条带进行切胶回收。
(4)连接pYLCRISPR/Cas9-MH载体:
0.5μL上述步骤(3)的回收产物、1.5μL10×T4Ligase Buffer、1μL Bsa I、2μLpYLCRISPR/Cas9-MH质粒、10μLddH2O,37℃酶切10min后加入0.75μLATP和0.5μL T4DNAligase,混匀后放入PCR仪,37℃5min,10℃5min,20℃5min,共10个循环:
(5)转化大肠杆菌及质粒测序:
将上述连接产物转化到DH5α感受态中,涂布于含有卡那霉素的LB固体培养基上,37℃过夜培养。挑取平板上单菌落,用pYLCRISPR/Cas9-MH载体检测引物SP1和SP2序列在PCR中扩增,电泳出现目的条带后送往北京擎科生物科技有限公司进行测序验证,获得阳性菌株。提取质粒。
2、农杆菌介导的水稻CRISPR/Cas9敲除载体的转化
用农杆菌转化野生型中花11愈伤,经过预培养、侵染、共培养、筛选具有抗性的愈伤组织,分化、生根、炼苗移栽,得到转基因植株。
(1)挑选饱满的成熟种子去壳,用2.5%NaClO溶液摇45min,180rpm。无菌水漂洗3~5次,风干,铺在诱导培养基上,26℃暗培养4周,15天继代一次。
(2)将含有p YLCRISPR/Cas9-MH-OsJMJ718敲除载体的农杆菌在LB培养基上划线涂板,28℃暗培养3天。
(3)挑取单菌落接种到5ml含抗生素的LB液体培养基中,28℃震荡培养过夜。
(4)将新鲜的农杆菌菌液离心,收集(浓度适中)放入AAM液体培养基中,26℃避光培养2~5h。
(5)选择致密的愈伤组织颗粒(直径3~5mm)用于转化。将待转化的愈伤组织颗粒在准备好的AAM菌液中浸染5min,倒掉农杆菌悬浮液,利用无菌滤纸将愈伤组织上的多余菌液吸去,随后转移至铺有一层无菌滤纸的固体共培养基上,28℃暗培养3天。
(6)共培养后,用无菌水洗3次,用AAM培养液润洗一次,吹干愈伤组织,然后将愈伤组织转移到筛选培养基含抗生素上筛选1个月。
(7)筛选后的抗性愈伤组织转移到分化培养基含抗生素上在光照条件下26℃继续培养至分化出绿苗。把小苗转移到含抗生素的生根培养基中培养,将小苗从生根培养基中移除,洗净残留培养基,在清水中炼苗。当白色的新根长出时,将其移栽到温室或大田。
3、转基因阳性水稻株系检测及突变植株的筛选
利用农杆菌介导的水稻转基因法,转化中花11的愈伤组织,通过潮霉素选择,获得4株独立的转化株系。CTAB法提取水稻叶片的总DNA,使用2×Taq Master Mix和潮霉素检测引物hpt-t/F、hpt-t/R按如下体系进行PCR扩增,检测引物hpt-t/F、hpt-t/R的序列为:
hpt-t/F:5'-GATGTTGGCGACCTCGTATTGG-3';
hpt-t/R:5'-CGTGCTTTCAGCTTCGATGTAGGAG-3'。
PCR反应体系如下:
PCR程序设置:
程序中,第二步(变性)到第四步(延伸)进行30个循环。
PCR结束后对扩增产物进行1%琼脂糖凝胶电泳,观察每个植株对应的扩增结果是否产生一条600bp左右的条带,如果产生目的条带,则说明敲除载体已整合至该模版DNA对应植株的染色体上。用潮霉素特异引物检测T0转基因植株,获得转基因阳性单株3株。
鉴定突变体植株的类型,在靶位点序列上下游位置使用Primer Premier5设计了突变体检测引物718-F、718-R,其序列分别为:
718-F:5'-TGCCCTACATTGGATAATGG-3';
718-R:5'-ATAGCAGCACTGATCGCTC-3'。
提取阳性植株的DNA,使用2×金牌Mix(green)和引物718-F、718-R按下列体系进行PCR反应:
25μLPCR反应体系:0.5μLDNA模版,22.5μL 2×金牌Mix,1μL 718-F,1μL 718-R。PCR程序设置:98℃2min;98℃10s,56℃15s,72℃10s,72℃7min;16℃10min。第二步进行30-35个循环。
PCR扩增后进行1%琼脂糖凝胶电泳,使用试剂盒纯化回收目的条带后,连接T19simple载体,转化大肠杆菌DH5α,每个单株挑取10个单克隆,送往擎科生物技术(上海)有限公司测序;突变体和野生型的测序结果用Snapgene软件进行序列比对。
现有报道将CRISPR/Cas9技术获得的突变体分为三类:双等位基因纯合突变、双等位基因杂合突变和单条染色体突变。
结果发现,在转基因T0代植株获得1个双等位基因纯合突变株JMJ718-25,4个双等位基因杂合突变株,双等位突变株JMJ718-1缺失7,4bp;JMJ718-18缺失5,2bp;JMJ718-5株缺失1bp和替换1bp;JMJ718-25株缺失2bp,这些株系的突变类型包括碱基缺失和碱基突变,如图2所示,且所有的突变均出现在gRNA区间。
4、无转基因序列的突变株鉴定:
对突变株系的T1单株用引物Hpt-F/Hpt-R和Cas9-F/Cas9-R进行PCR扩增,2对引物都不能扩增出载体目的片段的植株为无转基因序列的突变株。同时用靶位点扩增引物检测T1植株的突变情况。共筛选到Cas9和Hpt基因检测阴性的单株8株,T1代突变株系的突变位点序列结果如图3所示,其中JMJ718-1的T1分别检测到2株纯合单株,均表现为缺失4bp;JMJ718-18检测到3种纯合单株,缺失5bp;JMJ718-25的T1分别检测到3株纯合单株,表现为缺失2bp。将8个筛选到的无转基因元件的纯合单株套袋收种。
实施例2敲除转基因水稻的谷粒表型观察
1、谷粒表型观察
水稻成熟后分别收取野生型和通过Cas9敲除OsJMJ718基因突变体T2代株系的种子,置于42℃烘干箱干燥至恒重后,测量粒长、粒宽、粒厚和千粒重。测定10株。
(1)粒长:每样品随机挑取10粒成熟饱满的谷粒,按照首尾相连、不重叠、不留间隙的方式,在坐标纸上紧靠排成一行,测量其长度,重复三次,计算每粒的平均长度即为粒长。结果如图4A、5A所示,与野生型相比,Cas9突变体植株JMJ718-1、JMJ718-25和JMJ718-18的谷粒长度均明显增加。
(2)谷粒粒宽:每样品随机挑取10粒成熟饱满的谷粒,将此10粒谷粒按照肩靠肩(即宽度方向)在坐标纸上紧靠排成一行,测量其宽度,重复三次,计算每粒的平均宽度即为粒宽。结果如图4B、5B所示,Cas9转基因植株JMJ718-18的谷粒宽度与野生型相比,变化不明显,但JMJ718-1和JMJ718-25的谷粒粒宽均小于野生型。
(3)谷粒粒厚:每样品随机挑取10粒成熟饱满的谷粒,测量其粒厚,重复三次,计算每粒的平均厚度即为粒厚。结果如图5C所示,跟野生型相比,JMJ718-1、JMJ718-25和JMJ718-18的谷粒粒厚减少。
(4)谷粒千粒重:每样品随机挑取100粒成熟饱满的谷粒,称其重量,重复三次,求其平均值,最后换算成千粒重。结果如图5D所示,跟野生型相比,JMJ718-1、JMJ718-25和JMJ718-18的谷粒千粒重减少。
序列表
<110> 广东省农业科学院农业生物基因研究中心
<120> OsJMJ718基因及其编码蛋白在调控水稻粒型中的应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1140
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 1
atgtcatgtt tgagttggga gccaccagat atgtggtcta aagtacatgg caccggcact 60
agtcctgaga tgaaaaacgt gaaggctatt gattgtttat cttgctgtga ggtcgagata 120
tgcactcaag acttcttcaa tgggtattat gaaggtcgga tgtatcaaaa tctatggcct 180
gaaatgctta aattgaagga ctggcctaca tcaaatcatt ttgaagagct tttgccttcc 240
catggagtta aatacatgaa ttctttacct tttcaaccat acacaaactt gaagtctggt 300
ttgctgaatg tctcaacctt gcttcctgat gacatcttaa agcttgacat gggcccaaaa 360
tcatatatag cttatggcta tgcacaggaa cttggtagag gagattctgt tacaaagctt 420
cactgcgatt tgtctgatgc agttaacgtt ttgatgcata ctgctgaagt tgacccttcc 480
gaggaacaaa tagatgcaat aaaaagtttg aaaagaagac atacagcgca aaatgaaaag 540
gagtgttctg ggaatgcaga cggaaattat acatctccta aaatctgtgg ggatgcaaat 600
gagttgtcct gtcctataaa cagtgagacc aacaagggag gtgctttatg ggatattttt 660
aggagggaag atgttccaaa actgaaattg tatctcgaca agcattctaa ggaatttcgc 720
catatatact gttctgcagt tcaaaaggta tgtaaccctg tacatgatga aacattttat 780
ctaacagaag aacacaagag aaaactcaag gaggagcatg gaattgagcc ttggacattt 840
gtacaaaaac ttggggaggc agtattcatt cctgctgggt gtcctcatca agtacggaat 900
cttaagtcct gcaccaagat tgccttggat tttgtatcac ccgagaatgt taaggagtgt 960
ctcagcttaa ccgaggactt ccgaagactt cctaagaacc acagggccaa agaagacaaa 1020
ttagagctag gtgtggttca aaatggaccc aagcccacat accctcgaga aactattcta 1080
aactctcaat gggacatctt gttgtttgca tatcatccaa atagttatga caaagtttaa 1140
<210> 2
<211> 379
<212> PRT
<213> 水稻(Oryza sativa L.)
<400> 2
Met Ser Cys Leu Ser Trp Glu Pro Pro Asp Met Trp Ser Lys Val His
1 5 10 15
Gly Thr Gly Thr Ser Pro Glu Met Lys Asn Val Lys Ala Ile Asp Cys
20 25 30
Leu Ser Cys Cys Glu Val Glu Ile Cys Thr Gln Asp Phe Phe Asn Gly
35 40 45
Tyr Tyr Glu Gly Arg Met Tyr Gln Asn Leu Trp Pro Glu Met Leu Lys
50 55 60
Leu Lys Asp Trp Pro Thr Ser Asn His Phe Glu Glu Leu Leu Pro Ser
65 70 75 80
His Gly Val Lys Tyr Met Asn Ser Leu Pro Phe Gln Pro Tyr Thr Asn
85 90 95
Leu Lys Ser Gly Leu Leu Asn Val Ser Thr Leu Leu Pro Asp Asp Ile
100 105 110
Leu Lys Leu Asp Met Gly Pro Lys Ser Tyr Ile Ala Tyr Gly Tyr Ala
115 120 125
Gln Glu Leu Gly Arg Gly Asp Ser Val Thr Lys Leu His Cys Asp Leu
130 135 140
Ser Asp Ala Val Asn Val Leu Met His Thr Ala Glu Val Asp Pro Ser
145 150 155 160
Glu Glu Gln Ile Asp Ala Ile Lys Ser Leu Lys Arg Arg His Thr Ala
165 170 175
Gln Asn Glu Lys Glu Cys Ser Gly Asn Ala Asp Gly Asn Tyr Thr Ser
180 185 190
Pro Lys Ile Cys Gly Asp Ala Asn Glu Leu Ser Cys Pro Ile Asn Ser
195 200 205
Glu Thr Asn Lys Gly Gly Ala Leu Trp Asp Ile Phe Arg Arg Glu Asp
210 215 220
Val Pro Lys Leu Lys Leu Tyr Leu Asp Lys His Ser Lys Glu Phe Arg
225 230 235 240
His Ile Tyr Cys Ser Ala Val Gln Lys Val Cys Asn Pro Val His Asp
245 250 255
Glu Thr Phe Tyr Leu Thr Glu Glu His Lys Arg Lys Leu Lys Glu Glu
260 265 270
His Gly Ile Glu Pro Trp Thr Phe Val Gln Lys Leu Gly Glu Ala Val
275 280 285
Phe Ile Pro Ala Gly Cys Pro His Gln Val Arg Asn Leu Lys Ser Cys
290 295 300
Thr Lys Ile Ala Leu Asp Phe Val Ser Pro Glu Asn Val Lys Glu Cys
305 310 315 320
Leu Ser Leu Thr Glu Asp Phe Arg Arg Leu Pro Lys Asn His Arg Ala
325 330 335
Lys Glu Asp Lys Leu Glu Leu Gly Val Val Gln Asn Gly Pro Lys Pro
340 345 350
Thr Tyr Pro Arg Glu Thr Ile Leu Asn Ser Gln Trp Asp Ile Leu Leu
355 360 365
Phe Ala Tyr His Pro Asn Ser Tyr Asp Lys Val
370 375
Claims (10)
1.OsJMJ718基因在调控水稻粒型中的应用,其特征在于,所述OsJMJ718基因的核苷酸序列如SEQ ID NO:1所示。
2.OsJMJ718蛋白在调控水稻粒型中的应用,其特征在于,所述OsJMJ718蛋白的氨基酸序列如SEQ ID NO:2所示。
3.根据权利要求1或2所述应用,其特征在于,所述调控水稻粒型为调控水稻种子粒长、粒宽、粒厚和/或长宽比。
4.根据权利要求1~3所述的应用,其特征在于,突变水稻中OsJMJ718基因,进而获得突变体植株。
5.根据权利要求4所述的应用,其特征在于,构建OsJMJ718基因的CRISPR/Cas9敲除载体并转化水稻植株。
6.根据权利要求5所述的应用,其特征在于,所述CRISPR/Cas9敲除载体为p YLCRISPR/Cas9-MH-OsJMJ718。
7.根据权利要求6所述的应用,其特征在于,所述p YLCRISPR/Cas9-MH-OsJMJ718敲除载体的构建包括以下步骤:
S1.针对核苷酸序列如SEQ ID NO:1所示的OsJMJ718基因选择靶位点并设计PCR引物,进行PCR扩增,获得含所述靶位点序列的线性DNA片段;
S2.将上述线性DNA片段连入p YLCRISPR/Cas9-MH载体,构建得到p YLCRISPR/Cas9-MH-OsJMJ718敲除载体。
8.根据权利要求7所述的应用,其特征在于,步骤S1所述靶位点的序列为:5'-TTGGGAGCCACCAGATATG-3'。
9.根据权利要求8所述的应用,其特征在于,步骤S1采用的接头引物为:
Os718-U6aF:5'-gccgTTGGGAGCCACCAGATATG-3';
Os718-U6aR:5'-aaacCATATCTGGTGGCTCCCAA-3'。
10.OsJMJ718基因的CRISPR/Cas9敲除载体或质粒在调控水稻粒型中的应用。
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