CN112481292A - 创制OsNRR基因突变体的方法及其应用 - Google Patents
创制OsNRR基因突变体的方法及其应用 Download PDFInfo
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- CN112481292A CN112481292A CN201910851049.0A CN201910851049A CN112481292A CN 112481292 A CN112481292 A CN 112481292A CN 201910851049 A CN201910851049 A CN 201910851049A CN 112481292 A CN112481292 A CN 112481292A
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Abstract
本申请提供了创制OsNRR基因突变体的方法及其应用。本申请通过CRISPR/Cas9***对OsNRR基因中的靶序列进行基因编辑,使所述OsNRR基因不能表达正常功能的OsNRR蛋白,由此改良水稻根系生长。
Description
技术领域
本发明涉及基因编辑技术领域,具体地,涉及创制OsNRR基因突变体的方法及其应用,更具体地,涉及以CRISPR/Cas9基因编辑技术创制OsNRR基因突变体来改良水稻根系生长。
背景技术
作物根系从土壤中吸收营养物质和水分,确保作物能够在各种复杂环境下存活并结实,是影响作物产量的关键因素之一。当前,由于全球气候变化的不确定性,灌溉水资源的减少,化肥的过度使用,环境污染、水土流失等因素,使得农业生产的外界环境在不断恶化,严重威胁农业生产的可持续性发展。在这种情景下,培育具有发达、健壮根系的作物品种,以适应各种复杂的土壤、生态环境,就显得尤其重要。但是,由于作物的根系生长在地下,它常常被育种家和生物学家所忽视,因而非常缺乏对作物根系的了解。近年来,由于分子生物学、功能基因组学等领域的快速发展,在模式作物拟南芥中有关根生长发育的研究取得了丰富的成果,逐步克隆和鉴定了一批关键的控制根系生长发育的基因,这些成果为作物根系的研究和改良提供了很好的基础。
随着水稻功能基因组的快速发展,对水稻根系结构和功能的了解也在不断地加深。已鉴定和克隆了几个关键的基因或QTL,包括控制根深度的DRO1基因,控制磷吸收的PSTOL1基因,营养应答和根生长基因OsNRR,以及OsARD4等。这些基因的克隆和鉴定,为通过育种或生物技术手段改良水稻根系奠定了基础。
Zhang等通过分析大量营养元素氮、磷、钾充足或缺乏情况下的基因表达差异,发现一个新的基因,他们将之命名为NRR(nutrition response and root growth)(LOC_Os05g51690)。研究发现,NRR基因在水稻基因组中只有一个拷贝,在水稻根、茎、叶和穗中表达,在N,P,K营养不足的情况下,苗期根系表达水平升高。进一步地研究表明,NRR的转录物通过选择性剪接产生NRRa和NRRb两个成熟mRNA,NRRa定位在细胞核,而NRRb定位在细胞膜上,表明二者在功能上可能有不同的分工。超表达NRRa导致水稻根系生长迟缓,而RNAi下调NRRa或者NRRb都促进水稻在各种不同营养条件下的根系生长,表明二者负调节水稻根生长。
CRISPR/Cas9是在ZFN,TALEN之后发现的一种新型流行的基因编辑技术。该技术由于操作简单,突变效率高,成本低等优势,目前被应用在人类细胞、动物、植物等多方面,为作物育种家提供了精准、高效、快速改良作物性状的技术手段。
发明内容
一方面,本申请基于CRISPR/Cas9基因编辑技术,提供了创制OsNRR基因突变体的方法。
在一实施方案中,本申请提供的创制OsNRR基因突变体的方法,其通过CRISPR/Cas9***对OsNRR基因中的靶序列进行基因编辑,使所述OsNRR基因不能表达正常功能的OsNRR蛋白。
另一方面,本申请基于CRISPR/Cas9基因编辑技术,提供了改良水稻根系生长的方法。
在一实施方案中,本申请提供的改良水稻根系生长的方法,包括以下步骤:通过CRISPR/Cas9***对水稻OsNRR基因中的靶序列进行基因编辑,使所述OsNRR基因不能表达正常功能的OsNRR蛋白;以及筛选具有OsNRR基因突变体的水稻进行性状分析。又一方面,本申请提供了CRISPR/Cas9***在创制OsNRR基因突变体或改良水稻根系生长中的应用。
在具体实施方案中,本申请提供的上述方法或应用中所述的OsNRR基因包含SEQIDNO.1所示核苷酸序列或其片段或其变体或其互补序列。
在具体实施方案中,本申请提供的上述方法或应用中所述的靶序列包含SEQIDNO.4所示的核苷酸序列或其片段或其变体或其互补序列。在具体实施方案中,本申请提供的上述方法或应用中所述的CRISPR/Cas9***包含Cas9表达盒和gRNA表达盒。在具体实施方案中,所述Cas9表达盒包含甘蔗Ubi4启动子,所述gRNA表达盒包含U3启动子。
在具体实施方案中,本申请以水稻品种空育131为例,利用基因编辑技术,对水稻品种空育131的NRR基因进行敲除突变,获得非转基因、编辑位点纯合的敲除突变体,并且在各种营养条件下测试突变体植株根系生长,表明敲除NRR基因是快速改良水稻品种根系生长的有效途径。
可见,本申请提出了一种基因编辑技术的巧妙运用,利用CRISPR/Cas9基因编辑技术获得OsNRR基因突变体。通过在各种营养条件下(1/2MS、无氮、无磷、无钾、无氮磷、无氮磷钾)对突变体进行性状分析,发现突变体的主根长度、冠根数量和根生物量显著高于野生型植株,表明NRR基因的敲除能够显著改良水稻的根系生长。
此外,与TALEN和ZFN技术相比,本申请提供的方法操作过程简单方便,节约成本,一般实验室都可操作,是一种快速获得根系生长旺盛水稻品种或种质资源的高效生物技术育种方法。
附图说明
图1示出了OsNRR基因结构和编辑植株OsNRR基因的DNA序列。其中,WT,野生型空育131;CR-32、CR-50,编辑植株;下划线表示PAM序列;虚线表示缺失序列;方框表示外显子;垂直箭头指示靶位置;倾斜箭头指示序列在第1外显子中。
图2示出了野生型和编辑植株NRR基因翻译的氨基酸序列,WT,野生型;CR-32、CR-50,编辑植株;虚线表示缺失的氨基酸。
图3示出了在不同营养条件下NRR基因敲除突变体植株与野生型植株的生长比较。其中,A、B、C、D、E、F分别为1/2MS培养基、无氮培养基、无磷培养基、无钾培养基、无氮磷培养基和无氮磷钾培养基;1、2为野生型,3、4为CR-32,5、6为CR-50。
具体实施方式
提供以下定义和方法用以更好地界定本申请以及在本申请实践中指导本领域普通技术人员。除非另作说明,术语按照相关领域普通技术人员的常规用法理解。本文所引用的所有专利文献、学术论文、行业标准及其他公开出版物等,其中的全部内容整体并入本文作为参考。
本申请提出了一种以CRISPR/Cas9基因编辑技术创制OsNRR基因突变体来改良水稻根系生长的方法。通过改良水稻根系生长,可以获得高水肥利用率的优质水稻品种,促进农业的绿色发展。
具体地,本申请通过CRISPR/Cas9基因编辑技术创制OsNRR基因敲除突变体来改良水稻根系生长,适用于所有含有功能型OsNRR基因的水稻品种或者亲本,例如籼稻亲本华占、93-11、黄华占、隆科638S、天丰A等恢复系和不育系,籼稻品种美香占2号、玉珍香、粤农丝苗等,粳稻品种龙粳31、中粳616、吉大319等。
在一实施方案中,本申请所提供的方法包括以下步骤:1)对拟改良水稻品种OsNRR基因的序列进行分析,确定基因突变类型,选择该基因移码突变位置或者提前终止位置进行基因编辑靶点设计;2)构建打靶载体,将拟改良水稻品种的愈伤组织作为遗传转化的受体材料,用农杆菌介导法将打靶载体导入愈伤组织细胞,并再生成水稻植株;3)对OsNRR基因编辑体的基因型进行检测和分析,选取具有移码突变基因型的编辑体进行性状分析,考察根系生长情况。
在具体实施方案中,本申请所提供的方法包括以下步骤:1)水稻中OsNRR基因的检测;2)水稻中OsNRR基因cas9敲除表达载体的构建;3)将粳稻空育131的愈伤组织作为遗传转化的受体材料,鉴定转基因植株OsNRR基因编辑情况,获得不同突变类型的OsNRR等位基因,并筛选功能失活突变基因型,最终获得根系生长改良品系。
在一具体实施方案中,所述OsNRR基因为编码OsNRR蛋白的基因。在另一具体实施方案中,所述OsNRR基因编码的OsNRR蛋白为:由SEQIDNO.3所示的氨基酸序列组成的蛋白质。
在又一具体实施方案中,所述OsNRR基因具有:1)如SEQIDNO.1所示的DNA序列;或2)如SEQIDNO.2所示的编码区的DNA序列;或3)与1)或2)限定的DNA序列杂交且编码OsNRR蛋白的DNA分子;或4)与1)或2)限定的DNA序列至少具有70%以上同源性,例如70%,75%,80%,85%,90%,91%,92%,93%,94%,95%,96%,97%,98%,99%,99.5%或99.9%,且编码所述OsNRR功能蛋白的DNA分子。
在本申请中,所述水稻中OsNRR基因的表达是通过对水稻中OsNRR基因进行基因编辑实现的。所述基因编辑是借助CRISPR/Cas9***实现的。所述CRISPR/Cas9***中,cas9蛋白的表达由甘蔗Ubiquitin4启动子启动,gRNA由U3启动子启动。gRNA的靶序列如下:ATGTACGCCGCCATGTACC(SEQIDNO.4)。所述gRNA的编码序列如SEQIDNO.1中第208-226位核苷酸所示,该序列靶定OsNRR等位基因的第1个外显子。
本申请利用基因组编辑技术对OsNRR基因功能的定点敲除,成功地获得了根系生长显著改良的粳稻品种。通过本申请所获得的非转基因纯合NRR突变体将是非常有价值的种质资源,可以利用它们改良水稻品种的根系结构和功能。此外,本申请也为利用CRISPR/Cas9基因编辑技术快速改良水稻品种的根系生长提供一种有效的策略。
以下实施例用于说明本发明,但不用来限制本发明的范围。在不背离本发明精神和实质的情况下,对本发明方法、步骤或条件所作的修改或替换,均属于本申请的范围。
若无特别指明,实施例按照常规实验条件,如Sambrook等人的《分子克隆实验手册》(Sambrook J&Russell DW,Molecular cloning:a laboratory manual,2001),或按照制造厂商说明书建议的条件。
若未特别指明,实施例中所用的化学试剂均为常规市售试剂,实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
以下实施例所涉及的水稻品种材料由中国种子集团有限公司提供。
实施例1水稻OsNRR基因的序列分析
水稻OsNRR基因的基因组序列如SEQIDNO.1所示序列分析显示,该基因含有4个外显子,分别为SEQIDNO.1中第1-339位(第1外显子)、第1844-2377位(第2外显子)、第2860-3024位(第3外显子)和第3213-3770(第4外显子)。
实施例2基因编辑靶点设计和打靶载体构建及水稻遗传转化
本实施例采用基因编辑技术为第三代基因编辑技术CRISPR/cas9。根据Liu等人(CRISPR-P2.0:An improved CRISPR/Cas9 tool for genome editing in plants[J].Molecular plant,2017,10(3):530-532)的方法,利用CRISPR-P2.0(http://crispr.hzau.edu.cn/CRISPR2/)在线工具设计、优化和筛选出水稻品种空育131的OsNRR基因的第1外显子208-226处的碱基(即SEQIDNO.4)为靶序列,如图1所示。
植物表达载体包含甘蔗Ubi4启动子驱动的Cas9表达盒,以及水稻U3启动子驱动的gRNA表达盒。利用Asc1和Pme1将含有OsNRR基因靶序列的gRNA表达盒组装入表达载体,挑选出正确的阳性克隆,并通过测序确认。
将所构建的编辑载体通过电击法转入农杆菌菌株中。
将灭菌好的水稻品种空育131种子放在诱导培养基上于28℃暗培养30天诱导愈伤组织。愈伤组织每2周继代1次。农杆菌侵染水稻愈伤组织及筛选、分化流程参照Nishimura等的文献报道(A protocol for Agrobacterium-mediated transformation inrice.Nature protocols,2006,6:2 796-2 802)。
待分化出2~3cm的小苗后,选取单株小苗转入生根盒进行生根培养。
实施例3水稻植株编辑位点区域碱基长度检测
以T0代水稻幼苗基因组DNA为模板,用NRR基因靶位点特异性检测引物(正向引物为5’端含有FAM荧光修饰的csp5931:5’-TGTCTCCGTTCATTGTCACG-3’;反向引物csp5932:5’-GGATTAGCTAGTGTGCAAGG-3’)和Q5超保真DNA聚合酶进行PCR扩增。待反应结束后,取5μLPCR产物电泳检测扩增成功与否。
之后将剩余的PCR产物稀释800倍后加入HiDi试剂,利用ABI3730XL测序仪进行毛细管电泳,之后用软件分析扩增片段的碱基长度。
对40株T0代转基因植株的NRR基因编辑靶区域分别进行PCR扩增,检查编辑靶片段的DNA序列长度变化,结果显示有32株植株的OsNRR基因在靶区域发生长度变化,突变率为80%,变化主要以单个碱基的***或缺失为主,最长的缺失为18bp。另外,所有发生编辑的植株中,OsNRR基因的两个拷贝都被成功编辑。这些数据表明,编辑载体能够高效地编辑OsNRR基因。
实施例4非转基因纯合NRR基因敲除株系的获得
通过对32株T0代编辑植株在温室种植过程中的各种农艺性状进行观察,选取其中2个编辑株系026和035,其生长、发育、结实率等性状表现良好,与野生型相比无显著差异。使其自交T1代种子(约100粒)进行发芽、育苗,经转基因成分检测和靶序列长度变化检测,去掉含有转基因成分、没有编辑或者编辑位点杂合的植株,保留不携带转基因成分,并且突变位点纯合的植株,将其分别命名为CR-32和CR-50。
为确认CR-32和CR-50突变体株系中NRR基因编辑情况,对其靶位点区域进行PCR扩增和测序。
结果显示,CR-32株系缺失了包含起始密码子ATG和PAM序列在内的18bp核苷酸(图1),从而导致突变后的基因翻译提前终止(图2)。另外一个株系CR-50则在PAM序列前3位和4位碱基间***了1个碱基T(图1),导致从第6个氨基酸开始读框移码,翻译出与野生型NRR基因完全不同的氨基序列,而且在下游的第41个氨基酸后遇到了终止密码子,提前终止了翻译(图2)。
实施例5突变体根系生长的表型鉴定
将CR-32和CR-50T2代纯合突变体株系及对照种子灭菌后放入37℃的培养箱中催芽,待种子露白后每个株系取30粒种子置于i)含有氮磷钾的1/2MS液体培养基、ii)无氮液体培养基、iii)无磷液体培养基、iv)无钾液体培养基、v)无氮磷液体培养基、或者vi)无氮磷钾液体培养基中,于27℃、70%湿度、12h光/12h暗的条件下培养。
培养15天后,取样进行根系表型的观察,用直尺测定最长根的长度,数出单株根的数量,取10个单株用吸水纸吸干表面水分后立即称重得出10株的根鲜重,重复3次,用SPSS软件分析差异显著性。
结果显示,在所有6种营养条件下,敲除突变体水稻幼苗根的数量、长度和鲜重方面均高于野生型对照,差异达到极显著水平(表1),且其根的平均生物量是野生型植株的2~3倍,但地上部分的生长没有明显差异(图3)。这说明水稻NRR基因敲除突变体在各种营养条件下,特别是在大量营养元素缺失的情况下,根系生长更旺盛。
表1在不同营养条件下NRR基因敲除突变体植株与野生型植株的根系生长比较
其中,同一营养条件下同列数据后带相同字母表示在0.01水平上差异不显著。
以上所述,仅为本发明较佳的具体实施方式,但本发明的保护范围并不局限于此,任何熟悉本技术领域的技术人员在本发明揭露的技术范围内,根据本发明的技术方案及其发明构思加以等同替换或改变,都应涵盖在本发明的保护范围之内。
序列表
<110> 中国种子集团有限公司
<120> 创制OsNRR基因突变体的方法及其应用
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<170> SIPOSequenceListing 1.0
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taagccctct ccctgcttcc gccttgttca ataatcaata tactcttctc ctcgatctct 180
tctcttgtcc atctccaata attctccatg tacgccgcca tgtacccgga gaccttcggc 240
ttctctgctt acccccagca gcagcagccg ccgcccgacg ccgcctcctg catctacacc 300
accgccttgc cgctcatcgc cgacccgcct gacatcctcg tgagctgcct cctcctcctc 360
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accacaacat gtatgtacct cagacatttc atgagtgaga tgactaatcg aatcgatcca 720
tccatcatcc ttaattgctg ctgttgcttt cctaatttct cttcaaacgc aaacgcttag 780
ctagcttcct aaacagagca actaaaaatt cactcactca cctttgagat ttcttttctt 840
cttccaccac tactctacta catacatggc gtggcgaatg aatgaatgaa ttaattaaaa 900
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tggtgcaagc aactggtcat gttggctgca acttttcctt tcctttttgt ctttttcatt 1020
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tggcagggag gggggatcta attaggttga gtcttaatag agcttttttg gccaggattt 1200
ctgatgcccc tcctgtccat cagtatatgc tctcggagca tgacagggac ctgtaagttg 1260
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caatggacca gccaggccaa acttcctgga cttccaagga ttggactttg agacagcgtt 2340
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cctagggaaa cccaagtaca actaccggcc atggagaagg agaagccatc agttcctgaa 720
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ggaccagcca ggccaaactt cctggacttc caaggattgg actttgagac agcgtttggg 840
ttgaggaggg catacagcga aggagacatt cagaatcttg gagctagcac ccctcgaccc 900
gggaactcag gaaacgctca attagcatct tgcgagaggc ttgtaaccat cagtgacctg 960
aaatctgaag aaaggaagca gaagctatct aggtacagaa agaagaaggt gaagagaaac 1020
tttggcagaa agatcaagta tgcttgcagg aaggctcttg cagacagcca gccaagagtg 1080
cgagggaggt ttgccaagat cgaagaaggc gacttgctca agccaaggaa gtagtaagag 1140
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ggcttctaga agcttctagc tggtggagga ggaggaggag gaggaggagg agaggaaagg 1260
atccaaggtg caaggcccag cctttgtgtg tagggggagg gcgaggccaa ggtggaagag 1320
aggcgccgta gtcgcggctg cacttgtcgg aattaatttt agctgctttg gatcgtctcc 1380
ttctactact agactagtac ttgtaattct attttccttc caatgcatgt gtaataataa 1440
ttagtaataa tgctagtagt cggtattact tgctgcgagc tgaaggccgc ccatccatgt 1500
acaaatggct cgatgaagaa acttatcact agtagtaata aacttcagct cccttcaggc 1560
ttcgcactgg cactggaagg catccattat ctctct 1596
<210> 3
<211> 308
<212> PRT
<213> 水稻(Oryza.sativa L)
<400> 3
Met Tyr Ala Ala Met Tyr Pro Glu Thr Phe Gly Phe Ser Ala Tyr Pro
1 5 10 15
Gln Gln Gln Gln Pro Pro Pro Asp Ala Ala Ser Cys Ile Tyr Thr Thr
20 25 30
Ala Leu Pro Leu Ile Ala Asp Pro Pro Asp Ile Leu Gly Asn Met Ala
35 40 45
Gln Pro Ser Phe Leu Ser Glu Tyr Asp Leu Gly Gly Glu Gly Asp Leu
50 55 60
Phe Lys Ala Pro Glu Pro Ile Ile Glu Glu Pro Val Leu Ser Leu Asp
65 70 75 80
Pro Val Ala Ala Ala Ile Ser Met Met Ser Gly Ser Glu Asn Val Met
85 90 95
Asp Glu Thr Ile Glu Val Ala Asp Ile Ser Asp Ile Gln Asn Asp Ser
100 105 110
Leu Leu Ser Glu Val Leu Tyr Glu Cys Glu Lys Glu Leu Met Glu Lys
115 120 125
Ser Ala Ile Glu Glu Thr Ile Ser Glu Leu Leu Asp Val Lys Ile Pro
130 135 140
Met Leu Gln Val Glu Glu Phe Pro Arg Glu Thr Gln Val Gln Leu Pro
145 150 155 160
Ala Met Glu Lys Glu Lys Pro Ser Val Pro Glu Cys Cys Ser Leu Gln
165 170 175
Lys Ser Val Ser Ser Gly Cys Leu Asn Ser Ala Asp Trp Ile Asn Gly
180 185 190
Pro Ala Arg Pro Asn Phe Leu Asp Phe Gln Gly Leu Asp Phe Glu Thr
195 200 205
Ala Phe Gly Leu Arg Arg Ala Tyr Ser Glu Gly Asp Ile Gln Asn Leu
210 215 220
Gly Ala Ser Thr Pro Arg Pro Gly Asn Ser Gly Asn Ala Gln Leu Ala
225 230 235 240
Ser Cys Glu Arg Leu Val Thr Ile Ser Asp Leu Lys Ser Glu Glu Arg
245 250 255
Lys Gln Lys Leu Ser Arg Tyr Arg Lys Lys Lys Val Lys Arg Asn Phe
260 265 270
Gly Arg Lys Ile Lys Tyr Ala Cys Arg Lys Ala Leu Ala Asp Ser Gln
275 280 285
Pro Arg Val Arg Gly Arg Phe Ala Lys Ile Glu Glu Gly Asp Leu Leu
290 295 300
Lys Pro Arg Lys
305
<210> 4
<211> 19
<212> DNA
<213> 水稻(Oryza.sativa L)
<400> 4
atgtacgccg ccatgtacc 19
Claims (10)
1.创制OsNRR基因突变体的方法,其通过CRISPR/Cas9***对OsNRR基因中的靶序列进行基因编辑,使所述OsNRR基因不能表达正常功能的OsNRR蛋白。
2.如权利要求1所述的方法,其中所述OsNRR基因包含SEQIDNO.1所示核苷酸序列或其片段或其变体或其互补序列。
3.如权利要求1或2所述的方法,其中所述靶序列包含SEQIDNO.4所示的核苷酸序列或其片段或其变体或其互补序列。
4.如权利要求1至3中任一项所述的方法,其中所述CRISPR/Cas9***包含Cas9表达盒和gRNA表达盒;优选地,其中所述Cas9表达盒包含甘蔗Ubi4启动子;所述gRNA表达盒包含U3启动子。
5.改良水稻根系生长的方法,其包括以下步骤:
-通过CRISPR/Cas9***对水稻OsNRR基因中的靶序列进行基因编辑,使所述OsNRR基因不能表达正常功能的OsNRR蛋白;以及
-筛选具有OsNRR基因突变体的水稻进行性状分析。
6.如权利要求5所述的方法,其中所述OsNRR基因包含SEQIDNO.1所示核苷酸序列或其片段或其变体或其互补序列。
7.如权利要求5或6所述的方法,其中所述靶序列包含SEQIDNO.4所示核苷酸序列或其片段或其变体或其互补序列。
8.如权利要求5至7中任一项所述的方法,其中所述CRISPR/Cas9***包含Cas9表达盒和gRNA表达盒;优选地,其中所述Cas9表达盒包含甘蔗Ubi4启动子;所述gRNA表达盒包含U3启动子。
9.CRISPR/Cas9***在创制OsNRR基因突变体或改良水稻根系生长中的应用;其中所述CRISPR/Cas9***的靶序列包含SEQIDNO.4所示的核苷酸序列或其片段或其变体或其互补序列。
10.如权利要求9所述的应用,其中所述CRISPR/Cas9***包含Cas9表达盒和gRNA表达盒;优选地,其中所述Cas9表达盒包含甘蔗Ubi4启动子;所述gRNA表达盒包含U3启动子。
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Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6156954A (en) * | 1998-07-21 | 2000-12-05 | The Salk Institute For Biological Studies | Receptor-like protein kinase, RKN, and method of use for increasing growth and yield in plants |
CN108486146A (zh) * | 2018-03-16 | 2018-09-04 | 中国农业科学院作物科学研究所 | LbCpf1-RR突变体用于CRISPR/Cpf1***在植物基因编辑中的应用 |
CN108823236A (zh) * | 2018-05-25 | 2018-11-16 | 厦门大学 | 一种基因编辑技术打靶OsELF3 基因延长水稻抽穗的方法 |
-
2019
- 2019-09-10 CN CN201910851049.0A patent/CN112481292A/zh active Pending
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6156954A (en) * | 1998-07-21 | 2000-12-05 | The Salk Institute For Biological Studies | Receptor-like protein kinase, RKN, and method of use for increasing growth and yield in plants |
CN108486146A (zh) * | 2018-03-16 | 2018-09-04 | 中国农业科学院作物科学研究所 | LbCpf1-RR突变体用于CRISPR/Cpf1***在植物基因编辑中的应用 |
CN108823236A (zh) * | 2018-05-25 | 2018-11-16 | 厦门大学 | 一种基因编辑技术打靶OsELF3 基因延长水稻抽穗的方法 |
Non-Patent Citations (1)
Title |
---|
王海明等: "利用CRISPR/Cas9 基因编辑技术敲除水稻NRR 基因促进根系生长的研究", 杂交水稻, vol. 34, no. 5, pages 39 - 45 * |
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