CN111560371B - 一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料 - Google Patents
一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料 Download PDFInfo
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Abstract
本发明公开了一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料,利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝(BnTFL1‑1,BnTFL1‑2,BnTFL1‑3,BnTFL1‑4,BnTFL1‑5,BnTFL1‑6)其中的一个或一个以上,即获得开花时间提前,主枝和所有侧枝均产生终端花、花序结构由无限花序变为有限花序的甘蓝型油菜基因编辑材料;不同拷贝被编辑的基因编辑材料均表现出相同的开花时间、花器官和花序结构表型,表明甘蓝型油菜BnTFL1基因的这6个拷贝均具有调控开花时间、花器官发育以及花序结构的功能,且无加性效应。这些基因编辑材料可作为适合机械化收割的油菜材料或品种,为甘蓝型油菜育种提供了宝贵的基因资源和种质资源。
Description
技术领域
本发明属于植物基因工程和生物技术领域,涉及甘蓝型油菜BnTFL1基因定点突变的方法和应用,特别涉及一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料。
背景技术
油菜产油效率高、抗逆性强、适应范围广,是当今世界最重要油料作物之一。油菜作为国产食用植物油的第一大来源,占国产油料作物产油量的55%以上,是维护国家食用油供给安全的核心,其种植面积和产量均居世界第一。油菜的主要产品是菜籽油和菜籽粕。菜籽油在我国食用植物油供给中占植物油总量的50%以上,富含人体所需的不饱和脂肪酸、维生素等营养物质。菜籽粕富含大量的优质蛋白质、矿物质和氨基酸等营养物质,可作为优质家畜饲料蛋白的主要来源(殷艳,王汉中.我国油菜产业发展成就,问题与科技对策[J].中国农业科技导报,2012,14(04):1-7;傅廷栋.油菜的品种改良[J].作物研究,2007(03):159-162)。
油菜作为我国南方的主要冬季作物,可利用冬季闲田发展种植,在轮作复种中占有重要的地位。目前,我国大多数的油菜品种生育期较长,导致油菜与换茬作物在种植过程中存在换茬矛盾的问题,使南方地区冬季农田多有闲置,油菜种植面积减少;且熟期较长的油菜品种在生育后期常常遭遇高温逼熟和干热风等自然灾害,使作物产量受损(李新,肖麓,杜德志.油菜开花期的遗传调控及QTL研究进展[J].中国油料作物学报,2019,41(02):283-291)。此外,无限生长习性的油菜花序随着花序轴的生长不断地产生花芽,花期时间长且熟期不一致,植株底部过熟的荚果在收割时容易开裂落粒,上部未完全成熟的荚果脱粒不干净,导致收获时产量损失严重,这严重制约油菜的机械化收割。因此,培育早熟且花期较为一致的油菜品种,不仅有利于机械化收割,而且能够及时的为后茬作物腾地,保证后茬作物的高产稳产。
花作为被子植物特有的繁殖器官,是形成雌雄配子进行有性生殖的场所。开花是植物从营养生长向生殖生长的转变,受光周期途径、春化途径、赤霉素途径、自主途径和年龄途径等多条遗传途径共同调控。多条成花诱导途径的信号整合在一起,转录调控开花基因FLOWERING LOCUS T(FT),APETALA1(AP1),LEAFY(LFY),FLOWERING LOCUS C(FLC),TERMINAL FLOWER1(TFL1),从而精确控制开花时间。拟南芥中,FT蛋白在叶片中被诱导表达后通过韧皮部运输到顶端分生组织。在顶端分生组织中,FT与bZIP转录因子FLOWERINGLOCUS D(FD)相互作用,形成转录复合物激活开花基因AP1的表达,从而诱导植物成花。在拟南芥中过量表达LFY或AP1基因导致茎尖分生组织转变为花序分生组织,花序顶端形成终端花,开花时间提前;AP1基因不仅调控开花时间,也会影响花瓣和萼片的发育,拟南芥ap1突变体花分生组织转变为花序分生组织,花萼转变为花苞,并形成不正常的花瓣。FLC是MADS-box家族的转录因子,通过抑制开花促进因子FT,SOC1的表达抑制开花(Hanano S,GotoK.Arabidopsis TERMINAL FLOWER1 is involved in the regulation of floweringtime and inflorescence development through transcriptional repression[J].Plant Cell,2011,23(9):3172-3184;Wolabu T W,Zhang F,Niu L F,et al.ThreeFLOWERING LOCUS T-like genes function as potential florigens and mediatephotoperiod response in sorghum[J].New Phytologist,2016,210:946-959)。
TERMINAL FLOWER1(TFL1)与FT蛋白同属于磷脂酰乙醇胺结合蛋白(PEBP)家族,其氨基酸序列与FT保持高度的同源性,但是在开花调控过程中功能完全相反。FT促进植物开花,而TFL1抑制植物开花。TFL1与FT之间的生物学功能差异是由于一些关键的氨基酸残基存在的差异所决定的,TFL1蛋白位于88位的组氨酸改变为酪氨酸或123位的苯丙氨酸改变为缬氨酸都能使TFL1转变为开花促进因子(Hanzawa Y.A single amino acid converts arepressor to an activator of flowering[J].P NatlAcadSci USA,2005,102)。TFL1蛋白位于细胞核和细胞膜,在细胞核中,TFL1作为协助转录因子发挥作用。TFL1与FT竞争性结合FD,抑制开化基因LFY和AP1的表达,进而抑制开花;同时,LFY和AP1也会抑制TFL1的表达。因此,TFL1和LFY、AP1的相互平衡决定了植物的开花时间(Hanano S,Goto K.ArabidopsisTERMINAL FLOWER1 is involved in the regulation of flowering time andinflorescence development through transcriptional repression[J].Plant Cell,2011,23(9):3172-3184)。
TFL1在参与开花时间调控的同时,也参与花序结构的形成和花器官发育的调控。野生型拟南芥营养生长阶段TFL1表达水平较低,但在营养生长向生殖生长转变的过程中,顶端分生组织将转变为花序分生组织,TFL1在花序分生组织的中心区域表达,表达量快速升高。此时,TFL1蛋白从其表达部位移动到花分生组织表皮细胞,分布到整个分生组织,将开花基因LFY和AP1的表达限制在花序分生组织的侧翼,从而在侧翼启动花分生组织的发育,维持植物无限生长的特性,延迟开花。(Conti L,Bradley D.TERMINAL FLOWER1is amobile signal controlling Arabidopsis architecture[J].Plant Cell,2007,19(3):767-778)。与野生型相比,拟南芥tfl1突变体顶端分生组织中没有TFL1基因的表达,在成花转变过程中,开花基因LFY和AP1在顶端分生组织表达,花序的正常发育受到抑制,花序分生组织转变为花分生组织,花序结构由无限花序转变为有限花序,在花序顶端形成终端花,结束花序的正常生长,且开花时间提前,叶片和开花数量减少(Ho W W H,WeigelD.Structural features determining flower-promoting activity of ArabidopsisFLOWERING LOCUS T[J].Plant Cell,2014,26(2):552-564)。
甘蓝型油菜作为异源四倍体作物,其基因组比较复杂,同源拷贝较多,不同的同源拷贝之间往往存在基因冗余现象和基因加性效应。利用常规育种技术培育生育期较短、具有有限花序且花期一致的甘蓝型油菜品种往往需要较长时间,且耗时耗力;诱变育种存在较大盲目性,诱变的方向和性质不能控制,有利变异少,改良的数量性状较差。因此,利用可以同时修饰目标性状基因多个拷贝的新技术,是实现目标性状改良,培育新材料的有效途径。
CRISPR/Cas9作为最新出现的基因编辑技术,是由RNA引导Cas9蛋白核酸酶在特定的位点对靶向基因组进行切割和修饰,从而实现基因敲除、插入、替换和染色体重组。其作为反向遗传学中研究基因功能的一种重要工具,具有载体构建简单、靶向特异性高等特点。且利用CRISPR/Cas9基因编辑技术创造的突变体可以通过自交繁殖去除载体,这类去除载体的突变体有期望用于植物生产,目前已有许多物种利用CRISPR/Cas9技术创造出了一系列的突变体。
发明内容
本发明的目的在于,提供一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料,为甘蓝型油菜育种提供宝贵的基因资源和种质资源。
为了实现上述任务,本发明采取如下的技术解决方案:
一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料,其特征在于,利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝即:BnTFL1-1,BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6其中的一个或一个以上,即获得开花时间提前,主枝和所有侧枝产生终端花、花序结构由无限花序变为有限花序的甘蓝型油菜基因编辑材料;
所述甘蓝型油菜BnTFL1基因6个拷贝的全长编码区核酸序列如下所示:
BnTFL1-1
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGCTTTTCCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 160
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTGTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGTGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAG 537
BnTFL1-2
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGTTTTTGCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATACAT 180
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTTTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGCGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAA 537
BnTFL1-3
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-4
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGCTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-5
ATGGAGAATATGGGAAGTAGAGTGATAGAGCCATTGATAGTGGGAAGAGTGGTAGGAGAG 60
GTTCTTGATTTTTTCACTCAAACAATTGAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TGCAATGGCCATGAGCTTTTCCCTTCCTTTGTCTCCTCAAAGCCTAGGGTTGAGATCCAT 180
GGCGGTGATCTCAGATCTTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGCGACCCCTTTTTAAAAGAACACCTGCATTGGATTGTGACAAACATCCCCGGTACAACA 300
GATGCAACATTTGGAAAAGAGGTGGTGAGCTATGAGTTCCCAAGGCCAAATATAGGGATA 360
CACAGGTTCGTGTTTGTTCTCTTCAAGCAGAAGCAAAGACATGTTATCGATATCTCCCCA 420
AACATTCCTTCGAGAGATAAGTTCAATACTCGCAAATTTGCGATCGAGCATGATCTTGGT 480
CTCCCTGTCGCGGCTGTCTTCTTCAACGCACAGAGAGAAACCGCAGCTCGCAGACGTTAA 540
BnTFL1-6
ATGGGAGAAAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGATGTTCTCGAT 60
TTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACATGAAGCAAGTCTCCAACAGC 120
CATGAGCTTTTTCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCATGGTGGTGAT 180
CTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCTAGTGACCCC 240
TTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACCGATGCTACA 300
TTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATACACAGGTTC 360
GTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATTCCTTCGAGA 420
GATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCTGTCGCTGCT 480
GTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 525
所述利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝即:BnTFL1-1,BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6其中的一个或一个以上的具体步骤如下:
步骤一,甘蓝型油菜BnTFL1基因CRISPR/Cas9表达载体的构建:
1)sgRNA靶位点的选择:
针对甘蓝型油菜自交系K407中BnTFL1基因6个不同拷贝的结构和同源关系,基于CRISPRdirect网站(http://crispr.dbcls.jp/)设计了两个sgRNA序列,第一个sgRNA序列位于BnTFL1基因第一个外显子,所选序列为5’-CCAAGCCTAGGGTTGAGATC-3’,命名为sgR-BnTFL1-Target1;第二个sgRNA序列位于BnTFL1基因第四个外显子,所选序列为5’-GAGCTGCCAAAGCCAAACAT-3’,命名为sgR-BnTFL1-Target2;
2)sgRNA上下游引物设计:
sgR-BnTFL1-Target1的上游引物BnTFL1-Target1-F:5’-ATTGGATCTCAACCCTAGGCTTGG-3’;
sgR-BnTFL1-Target1的下游引物BnTFL1-Target1-R:5’-AAACCCAAGCCTAGGGTTGAGATC-3’;
sgR-BnTFL1-Target2的上游引物BnTFL1-Target2-F:5’-ATTGGAGCTGCCAAAGCCAAACAT-3’;
sgR-BnTFL1-Target2的下游引物BnTFL1-Target2-R:5’-AAACATGTTTGGCTTTGGCAGCTC-3’;
3)CRISPR/Cas9表达载体的构建与农杆菌转化:
取上述步骤2)所述的sgR-BnTFL1-Target1和sgR-BnTFL1-Target2的上下游引物混合,经PCR仪退火得到双链DNA;利用T4连接酶将双链DNA与经过AarI酶切的CRISPR/Cas9载体进行T4连接,将连接产物转化大肠杆菌并做菌落PCR鉴定以及测序验证,得到表达载体;将验证含有sgRNA的载体转化农杆菌,进行菌落PCR验证得到含有表达载体的农杆菌菌株。
步骤二,甘蓝型油菜BnTFL1基因CRISPR/Cas9基因编辑突变体的获得与鉴定:
1)基于农杆菌介导的方法遗传转化甘蓝型油菜自交系K407,通过潮霉素抗性筛选获得基因编辑突变体植株;
2)利用PCR检测基因编辑突变体植株中的CRISPR/Cas9表达载体,并克隆基因编辑突变体植株中BnTFL1基因6个不同拷贝的基因组序列,经过测序后,检测每个CRISPR/Cas9基因编辑突变体植株中各BnTFL1拷贝的编辑状况。
步骤三,验证甘蓝型油菜BnTFL1基因对开花时间、花器官发育和花序结构的影响:
1)比较甘蓝型油菜自交系K407和不同CRISPR/Cas9基因编辑突变体在开花时间、花器官和花序结构的差异;
2)分析BnTFL1基因6个不同拷贝的功能。
本发明的基于CRISPR/Cas9的甘蓝型油菜基因编辑材料,利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝(BnTFL1-1,BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6)其中的一个或一个以上,获得具有有限花序,开花时间提前且花期相对一致的甘蓝型油菜CRISPR/Cas9基因编辑编辑材料。不同拷贝被编辑的基因编辑材料均表现出相同的开花时间、花器官和花序结构表型,表明甘蓝型油菜BnTFL1基因的这6个拷贝均具有调控开花时间、花器官发育以及花序结构的功能,且无加性效应。这些基因编辑材料可作为适合机械化收割的油菜材料或品种,为甘蓝型油菜育种提供了宝贵的基因资源和种质资源。
附图说明
图1A为由CRISPR/Cas9诱导的BnTFL1基因的两个靶标位点示意图;
图1B为两个靶标位点敲除BnTFL1基因各拷贝示意图;
图1C为CRISPR/Cas9编辑BnTFL1-2拷贝纯合突变体靶标位点区域测序结果;
图2A为BnTFL1-2拷贝2个纯合编辑突变体花器官图片;
图2B为BnTFL1-2拷贝2个纯合编辑突变体株型图片;
图3为BnTFL1多个CRISPR/Cas9纯合突变体中不同拷贝编辑对开花时间和花器官发育影响的图片;
以下结合附图和实施例对本发明作进一步地详细说明。
具体实施方式
首先需要说明的是,在以下的实施例中,所使用的试剂如未作特殊说明,均来源于商业渠道,所涉及试验方法、检测方法等,若无特别说明,均为现有技术中已有的常规试验方法、检测方法。
为了解析甘蓝型油菜BnTFL1基因对开花时间、花器官发育以及花序结构的影响,本实施例给出一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料,利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝(BnTFL1-1,BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6)其中的一个或一个以上,即获得开花时间提前,主枝和所有侧枝产生终端花、花序结构由无限花序变为有限花序的甘蓝型油菜基因编辑材料。
具体实施过程如下:
1、甘蓝型油菜BnTFL1基因CRISPR/Cas9表达载体的构建
1.1甘蓝型油菜自交系K407中BnTFL1基因全长编码区核酸序列的克隆:
以甘蓝型油菜自交系K407的cDNA为模板,利用下表2所示的BnTFL1基因克隆引物BnTFL1-X F1和BnTFL1-X R1(X代表1,2,3,4,5,6),克隆自交系K407中BnTFL1基因。
将克隆得到的PCR产物进行1%的琼脂糖凝胶电泳,并对与目的片段长度一致的电泳条带按照OMEGA胶回收试剂盒进行纯化回收,纯化回收的PCR产物送至上海生工生物工程股份有限公司进行测序,得到甘蓝型油菜自交系K407中BnTFL1基因6个拷贝的全长编码区核酸序列如下所示:
BnTFL1-1
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGCTTTTCCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 160
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTGTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGTGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAG 537
BnTFL1-2
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGTTTTTGCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATACAT 180
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTTTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGCGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAA 537
BnTFL1-3
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-4
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGCTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-5
ATGGAGAATATGGGAAGTAGAGTGATAGAGCCATTGATAGTGGGAAGAGTGGTAGGAGAG 60
GTTCTTGATTTTTTCACTCAAACAATTGAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TGCAATGGCCATGAGCTTTTCCCTTCCTTTGTCTCCTCAAAGCCTAGGGTTGAGATCCAT 180
GGCGGTGATCTCAGATCTTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGCGACCCCTTTTTAAAAGAACACCTGCATTGGATTGTGACAAACATCCCCGGTACAACA 300
GATGCAACATTTGGAAAAGAGGTGGTGAGCTATGAGTTCCCAAGGCCAAATATAGGGATA 360
CACAGGTTCGTGTTTGTTCTCTTCAAGCAGAAGCAAAGACATGTTATCGATATCTCCCCA 420
AACATTCCTTCGAGAGATAAGTTCAATACTCGCAAATTTGCGATCGAGCATGATCTTGGT 480
CTCCCTGTCGCGGCTGTCTTCTTCAACGCACAGAGAGAAACCGCAGCTCGCAGACGTTAA 540
BnTFL1-6
ATGGGAGAAAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGATGTTCTCGAT 60
TTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACATGAAGCAAGTCTCCAACAGC 120
CATGAGCTTTTTCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCATGGTGGTGAT 180
CTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCTAGTGACCCC 240
TTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACCGATGCTACA 300
TTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATACACAGGTTC 360
GTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATTCCTTCGAGA 420
GATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCTGTCGCTGCT 480
GTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 525
1.2sgRNA靶位点的选择与上下游引物的合成:
针对甘蓝型油菜自交系K407中BnTFL1基因6个不同拷贝的结构和同源关系,利用CRISPRdirect(http://crispr.dbcls.jp/)设计两个sgRNA序列如图1A、图1B所示。
第一个sgRNA序列位于BnTFL1基因第一个外显子,所选序列为5’-CCAAGCCTAGGGTTGAGATC-3’,命名为sgR-BnTFL1-Target1;
第二个sgRNA序列位于BnTFL1基因第四个外显子,所选序列为5’-GAGCTGCCAAAGCCAAACAT-3’,命名为sgR-BnTFL1-Target2。
这两个sgRNA序列分别合成两对接头引物,用于构建两个不同靶标位点的CRISPR/Cas9表达载体。
根据上述两个sgRNA序列,设计如表2所示的接头引物(分别命名为BnTFL1-Target1-F、BnTFL1-Target1-R、BnTFL1-Target2-F、BnTFL1-Target2-R),接头引物前四位碱基为AarI限制性内切酶的黏性末端。
表2:引物序列表
1.3双链gDNA的获得:
将上述两对接头引物送至上海生工生物工程股份有限公司进行引物合成,合成好的接头引物稀释到10μM,每对引物分别取10μL混合加入PCR管中95℃保温5min,合成双链gDNA用于后续载体构建。
1.4双链gDNA与CRISPR/Cas9载体的连接:
用AarI限制性内切酶将CRISPR/Cas9质粒在37℃恒温培养箱中酶切10小时,酶切反应体系如表3所示。
表3:酶切反应体系
酶切反应结束后,酶切产物按照OMEGA胶回收试剂盒进行纯化后,使用NanoDropND-1000检测产物浓度和质量。将酶切纯化的CRISPR/Cas9线性化质粒与上述双链gDNA加入PCR管16℃孵育5-6小时进行T4连接,连接反应体系如表4所示。
表4:连接反应体系
1.5大肠杆菌的热激转化与表达载体的鉴定
将上述T4连接产物加入大肠杆菌感受态细胞DH5α,轻轻吸打混匀放置于冰上20min,42℃水浴90s,迅速置于冰上2min,加入700μL的LB培养液,放置于37℃摇床振荡培养30分钟后将菌液涂布于含100ug/ml壮观霉素的LB固体培养基平板上,37℃恒温培养箱倒置过夜。
挑取单克隆菌落PCR,使用表2所示的引物Cas9-F和BnTFL1-Target1-R或BnTFL1-Target2-R分别进行菌落PCR验证,单克隆PCR反应体系如表5所示,PCR反应条件如表6所示。
表5:单克隆PCR反应体系
表6:PCR反应条件
PCR反应结束后将产物利用1%的琼脂糖凝胶电泳进行验证。将PCR检测条带大小正确的菌落选取3个分别加到含有100ug/ml壮观霉素的LB液体培养基中,吸打混匀之后放置于37℃/220rpm摇床过夜培养,挑选单克隆菌液送至上海生工生物工程股份有限公司进行测序,将测序结果正确的单克隆菌液按照OMEGA质粒提取试剂盒进行质粒提取,成功构建sgR-BnTFL1-Target1和sgR-BnTFL1-Target2两个靶标位点不同的CRISPR/Cas9基因编辑载体。
1.6农杆菌转化
将上述成功构建的两个CRISPR/Cas9基因编辑载体sgR-BnTFL1-Target1和sgR-BnTFL1-Target2分别吸取5μL加入农杆菌感受态细胞GV3101,轻轻吸打混匀放置于冰上30min,液氮速冻5min后立即放入37℃水浴锅中孵育5min,孵育结束后放置于冰上3min,加入700μL LB培养液(含25ug/ml利福平),放置于28℃摇床振荡培养2小时后将菌液涂布于含100ug/ml壮观霉素和25ug/ml利福平的LB固体培养基平板上,28℃恒温培养箱倒置过夜。挑取单克隆菌落PCR,使用表2所示的引物Cas9-F和BnTFL1-Target1-R或BnTFL1-Target2-R分别进行菌落PCR验证,PCR反应体系如表5所示,PCR反应条件如表6所示。PCR反应结束后将产物利用1%的琼脂糖凝胶电泳进行验证。将PCR检测的阳性菌落挑取3个分别加到含有100ug/ml壮观霉素和25ug/ml利福平的LB液体培养基中,吸打混匀之后放置于28℃/220rpm摇床振荡培养。最后将阳性菌株的菌液保存在33%的甘油中放置于-80℃进行长期保存。
2、甘蓝型油菜BnTFL1基因CRISPR/Cas9基因编辑突变体的获得与鉴定
对上述两个CRISPR/Cas9基因编辑载体基于植物组织培养结合农杆菌介导的遗传转化方法进行油菜遗传转化,均获得多个潮霉素抗性的CRISPR/Cas9基因编辑突变体油菜株系。在此基础上,提取每个突变体油菜株系的基因组DNA,然后以基因组DNA为模板,以表2所示的BnTFL1-X-Cas9-F和BnTFL1-X-Cas9-R(X代表1,2,3,4,5)为扩增引物,对BnTFL1基因的6个拷贝进行PCR扩增,将扩增产物切胶纯化,检测其质量和浓度,送至上海生工生物工程股份有限公司进行测序,以检测每个CRISPR/Cas9基因编辑突变体中各BnTFL1拷贝的编辑情况。
3、验证甘蓝型油菜BnTFL1-2对开花时间和花器官发育的影响
综合分析上述突变体中BnTFL1基因6个不同拷贝的测序结果,如图1C部分所示,目前已获得2株不同编辑位点且只编辑了BnTFL1-2这个拷贝的油菜纯合编辑突变体,分别命名为#Target1-20-1和#Target2-13-3,这两个纯合编辑突变体在靶标位点均发生了碱基A的插入。其中,#Target1-20-1的靶标位点位于第一个外显子,#Target2-13-3的靶标位点位于第四个外显子,这两个纯合突变体在不同靶标位点敲除甘蓝型油菜BnTFL1-2,如图2A、图2B所示,其纯合突变体较野生型K407相比,开花时间提前,主枝和所有侧枝表现出花序顶部花瓣萼片卷曲、柱头开裂,主枝和所有侧枝均产生终端花、花序结构由无限花序变为有限花序,表明甘蓝型油菜中BnTFL1-2突变后影响甘蓝型油菜开花时间、花器官发育以及花序结构发育。
4、验证甘蓝型油菜多个突变体中不同拷贝编辑对开花时间和花器官发育的影响
通过综合分析T2代不同突变体中BnTFL1基因6个拷贝的测序结果,发现除上述2个只敲除BnTFL1-2拷贝的纯合突变体之外(如下表1所示),申请人还获得了一些同时敲除甘蓝型油菜BnTFL1基因不同拷贝的纯合突变体,如图3所示。同时敲除甘蓝型油菜BnTFL1基因不同拷贝的纯合突变体与只敲除BnTFL1-2拷贝的纯合突变体相比,都表现为开花时间提前,主枝和所有侧枝表现出花序顶部花瓣萼片卷曲、柱头开裂,主枝和所有侧枝均产生终端花、花序结构由无限花序变为有限花序;不同拷贝被编辑的纯合突变体均表现出相同的开花时间、花器官和花序结构表型,表明甘蓝型油菜BnTFL1基因的6个拷贝(BnTFL1-1,BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6)均具有调控开花时间、花器官发育以及花序结构的功能,且无加性效应。
表1:BnTFL1多个CRISPR/Cas9纯合突变体中不同拷贝编辑情况
综上所述,通过CRISPR/Cas9基因编辑技术获得的具有有限花序,开花时间提前且花期相对一致的甘蓝型油菜CRISPR/Cas9基因突变体,经过载体分离后可作为适合机械化收割的油菜材料或品种。
核苷酸序列表
<110>西北农林科技大学
<120>一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料
<160>
<210> 1
<211>537
<212> BnTFL1-1
<213> 全长编码区核酸序列
<220>
<400>
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGCTTTTCCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 160
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTGTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGTGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAG 537
<210> 2
<211>537
<212> BnTFL1-2
<213> 全长编码区核酸序列
<220>
<400>
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TCCAATGGCCATGAGTTTTTGCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATACAT 180
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT 240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA 360
CACAGGTACGTTTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT 420
ATTATTTCGAGGGATCAGTTCAACACTCGCGAATTTGCGATCGAGAATGATCTTGGTCTC 480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAA 537
<210> 3
<211>534
<212> BnTFL1-3
<213> 全长编码区核酸序列
<220>
<400>
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
<210> 4
<211>534
<212> BnTFL1-4
<213> 全长编码区核酸序列
<220>
<400>
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC 120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT 180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC 300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA 360
CACAGGTTCGTGTTTGCTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT 420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT 480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
<210> 5
<211>540
<212> BnTFL1-5
<213> 全长编码区核酸序列
<220>
<400>
ATGGAGAATATGGGAAGTAGAGTGATAGAGCCATTGATAGTGGGAAGAGTGGTAGGAGAG 60
GTTCTTGATTTTTTCACTCAAACAATTGAAATGAACGTGAGTTACAACAAGAAGCAAGTC 120
TGCAATGGCCATGAGCTTTTCCCTTCCTTTGTCTCCTCAAAGCCTAGGGTTGAGATCCAT 180
GGCGGTGATCTCAGATCTTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT 240
AGCGACCCCTTTTTAAAAGAACACCTGCATTGGATTGTGACAAACATCCCCGGTACAACA 300
GATGCAACATTTGGAAAAGAGGTGGTGAGCTATGAGTTCCCAAGGCCAAATATAGGGATA 360
CACAGGTTCGTGTTTGTTCTCTTCAAGCAGAAGCAAAGACATGTTATCGATATCTCCCCA 420
AACATTCCTTCGAGAGATAAGTTCAATACTCGCAAATTTGCGATCGAGCATGATCTTGGT 480
CTCCCTGTCGCGGCTGTCTTCTTCAACGCACAGAGAGAAACCGCAGCTCGCAGACGTTAA 540
<210> 6
<211>525
<212> BnTFL1-6
<213> 全长编码区核酸序列
<220>
<400>
ATGGGAGAAAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGATGTTCTCGAT 60
TTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACATGAAGCAAGTCTCCAACAGC 120
CATGAGCTTTTTCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCATGGTGGTGAT 180
CTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCTAGTGACCCC 240
TTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACCGATGCTACA 300
TTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATACACAGGTTC 360
GTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATTCCTTCGAGA 420
GATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCTGTCGCTGCT 480
GTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 525
<210> 7
<211>20
<212> BnTFL1-1 F1
<213>引物序列
<220>
<400>
ATGGAGAATATGGGAACTAG
<210> 8
<211>19
<212> BnTFL1-1 R1
<213>引物序列
<220>
<400>
ATGCATTTGATGGACACAG
<210> 9
<211>19
<212> BnTFL1-2 F1
<213>引物序列
<220>
<400>
AAGAAACCTTTACAAAATA
<210> 10
<211>18
<212> BnTFL1-2 R1
<213>引物序列
<220>
<400>
TACGAAAACAATATTTTA
<210> 11
<211>20
<212> BnTFL1-3 F1
<213>引物序列
<220>
<400>
CCACATTTAGTTAGAAGCAG
<210> 12
<211> 18
<212> BnTFL1-3 R1
<213>引物序列
<220>
<400>
TTTGTCAGTCATTATTCA
<210> 13
<211> 20
<212> BnTFL1-4 F1
<213>引物序列
<220>
<400>
CCACATTTAGTTAGAAGCAG
<210> 14
<211> 18
<212> BnTFL1-4 R1
<213>引物序列
<220>
<400>
TTTGTCAGTCATTATTCA
<210> 15
<211> 18
<212> BnTFL1-5 F1
<213>引物序列
<220>
<400>
TGGTATCCCACACCACTA
<210> 16
<211> 19
<212> BnTFL1-5 R1
<213>引物序列
<220>
<400>
ATCCTTGTCACCCAGTCTT
<210> 17
<211> 18
<212> BnTFL1-6 F1
<213>引物序列
<220>
<400>
CTCTCCCGTAGCTCACTT
<210> 18
<211> 18
<212> BnTFL1-6 R1
<213>引物序列
<220>
<400>
ACTCATTTTTCTCAGTCT
<210> 19
<211> 21
<212> BnTFL1-1-Cas9-F
<213>引物序列
<220>
<400>
TCTTGGTTGCCTTTGGTATCC
<210> 20
<211> 21
<212> BnTFL1-1-Cas9-R
<213>引物序列
<220>
<400>
AAGGTCTTGTCACTTTGACCG
<210> 21
<211> 22
<212> BnTFL1-2-Cas9-F
<213>引物序列
<220>
<400>
CATCGATCTCTCTCCAGCTTAC
<210> 22
<211> 22
<212> BnTFL1-2-Cas9-R
<213>引物序列
<220>
<400>
CGATTTGACCTATCGAGACATA
<210> 23
<211> 21
<212> BnTFL1-3-Cas9-F
<213>引物序列
<220>
<400>
CTTAATCTACGTTCATAGCTG
<210> 24
<211> 23
<212> BnTFL1-3-Cas9-R
<213>引物序列
<220>
<400>
GACCTATTAAACCATAAATAAGG
<210> 25
<211> 21
<212> BnTFL1-4-Cas9-F
<213>引物序列
<220>
<400>
CTTAATCTACGTTCATAGCTG
<210> 26
<211> 23
<212> BnTFL1-4-Cas9-R
<213>引物序列
<220>
<400>
GACCTATTAAACCATAAATAAGG
<210> 27
<211> 24
<212> BnTFL1-5-Cas9-F
<213>引物序列
<220>
<400>
ATTCATTCTTTTAGATTTGCCCGA
<210> 28
<211> 23
<212> BnTFL1-5-Cas9-R
<213>引物序列
<220>
<400>
AGGGTTTAGTTAGTATGGTAGCC
<210> 29
<211> 24
<212> BnTFL1-6-Cas9-F
<213>引物序列
<220>
<400>
GTAAATTAATGGACTCAGTTATTG
<210> 30
<211> 24
<212> BnTFL1-6-Cas9-R
<213>引物序列
<220>
<400>
ATCATATATCATATAAGAACAAGC
<210> 31
<211> 24
<212> BnTFL1-Target1-F
<213>引物序列
<220>
<400>
ATTGGATCTCAACCCTAGGCTTGG
<210> 32
<211> 24
<212> BnTFL1-Target1-R
<213>引物序列
<220>
<400>
AAACCCAAGCCTAGGGTTGAGATC
<210> 33
<211> 24
<212> BnTFL1-Target2-F
<213>引物序列
<220>
<400>
ATTGGAGCTGCCAAAGCCAAACAT
<210> 34
<211> 24
<212> BnTFL1-Target2-R
<213>引物序列
<220>
<400>
AAACATGTTTGGCTTTGGCAGCTC
<210> 35
<211> 24
<212> Cas9-F
<213>引物序列
<220>
<400>
CTGTGGTTGGCATGCACATAC
Claims (2)
1.一种基于CRISPR/Cas9的甘蓝型油菜基因编辑材料的构建方法,其特征在于,利用CRISPR/Cas9基因编辑技术敲除甘蓝型油菜BnTFL1基因6个拷贝即:BnTFL1-1, BnTFL1-2,BnTFL1-3,BnTFL1-4,BnTFL1-5,BnTFL1-6中的一个或一个以上,获得开花时间提前,主枝和所有侧枝产生终端花、花序结构由无限花序变为有限花序的甘蓝型油菜基因编辑材料;
所述甘蓝型油菜BnTFL1基因6个拷贝的全长编码区核酸序列如下所示:
BnTFL1-1
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC120
TCCAATGGCCATGAGCTTTTCCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT160
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA360
CACAGGTACGTGTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT420
ATTATTTCGAGGGATCAGTTCAACACTCGTGAATTTGCGATCGAGAATGATCTTGGTCTC480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAG 537
BnTFL1-2
ATGGAGAATATGGGAACTAGAGTGATAGAGCCATTAATAGTGGGAAGAGTGGTCGGAGAT 60
GTTCTTGATAATTTCACTCCAACAATTAAAATGAACGTGAGTTACAACAAGAAGCAAGTC120
TCCAATGGCCATGAGTTTTTGCCTTTAGCTGTCTCCTCCAAGCCTAGGGTTGAGATACAT180
GATGGTGATCTCAGATCTTTCTTCACCCTGGTGATGACAGACCCTGATGTTCCAAATCCT240
AGTGACCCCTTTCTAAAAGAACGCCTGCATTGGCTCGTCATGAACATCCCCGGCACAACA300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGCTGCCAAAGCCAAACATAGGGATA360
CACAGGTACGTTTTTGTTCTGTTCAGGCAGAAGCAAAGACGTGTTAAGTTCCCAAGCAAT420
ATTATTTCGAGGGATCAGTTCAACACTCGCGAATTTGCGATCGAGAATGATCTTGGTCTC480
CCTGTCGCGGCTGTCTTCTTCAACGCTCAGAGAGAAACCGCATCTCGCAGACGTTAA 537
BnTFL1-3
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA360
CACAGGTTCGTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-4
ATGGAGAATATGGGAGTTAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGAT 60
GTTCTTGATTTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACAAGAATCAAGTC120
TCCAACGGCCATGAGCTTTTGCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCAT180
GGTGGTGATCTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT240
AGTGACCCCTTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACC300
GATGCTACATTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATA360
CACAGGTTCGTGTTTGCTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATT420
CCTTCGAGAGATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCT480
GTCGCTGCTGTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 534
BnTFL1-5
ATGGAGAATATGGGAAGTAGAGTGATAGAGCCATTGATAGTGGGAAGAGTGGTAGGAGAG 60
GTTCTTGATTTTTTCACTCAAACAATTGAAATGAACGTGAGTTACAACAAGAAGCAAGTC120
TGCAATGGCCATGAGCTTTTCCCTTCCTTTGTCTCCTCAAAGCCTAGGGTTGAGATCCAT180
GGCGGTGATCTCAGATCTTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCT240
AGCGACCCCTTTTTAAAAGAACACCTGCATTGGATTGTGACAAACATCCCCGGTACAACA300
GATGCAACATTTGGAAAAGAGGTGGTGAGCTATGAGTTCCCAAGGCCAAATATAGGGATA360
CACAGGTTCGTGTTTGTTCTCTTCAAGCAGAAGCAAAGACATGTTATCGATATCTCCCCA420
AACATTCCTTCGAGAGATAAGTTCAATACTCGCAAATTTGCGATCGAGCATGATCTTGGT480
CTCCCTGTCGCGGCTGTCTTCTTCAACGCACAGAGAGAAACCGCAGCTCGCAGACGTTAA540
BnTFL1-6
ATGGGAGAAAGAGTGATAGAGCCATTGATAATGGGAAGAGTGGTAGGAGATGTTCTCGAT 60
TTCTTCACTCCAACAATTAAAATGAATGTGAGCTACAACATGAAGCAAGTCTCCAACAGC120
CATGAGCTTTTTCCTTCCTCTGTCTCCTCCAAGCCTAGGGTTGAGATCCATGGTGGTGAT180
CTCAGATCCTTCTTCACCTTGGTGATGATAGACCCTGATGTTCCAGGTCCTAGTGACCCC240
TTTCTAAAAGAGCACCTGCATTGGATAGTAACAAACATCCCCGGTACAACCGATGCTACA300
TTTGGAAAAGAGGTGGTGAGCTATGAGTTGCCAAGGCCTAGCATAGGGATACACAGGTTC360
GTGTTTGTTCTGTTCAAGCAGAAGCAAAGACGTGTTATCTTCCCAAACATTCCTTCGAGA420
GATAACTTCAACACTCGAAAATTTGCGATCGAGTATGATCTTGGTCTTCCTGTCGCTGCT480
GTCTTCTTTAACGCCCAGAGAGAAACTGCAGCTCGAAGACGTTAA 525
具体步骤如下:
步骤一,甘蓝型油菜BnTFL1基因CRISPR/Cas9表达载体的构建:
1)sgRNA靶位点的选择:
针对甘蓝型油菜自交系K407中BnTFL1基因6个不同拷贝的结构和同源关系,基于CRISPRdirect 网站设计两个sgRNA序列,第一个sgRNA序列位于BnTFL1基因第一个外显子,所选序列为5’-CCAAGCCTAGGGTTGAGATC-3’,命名为sgR-BnTFL1-Target1;第二个sgRNA序列位于BnTFL1基因第四个外显子,所选序列为5’-GAGCTGCCAAAGCCAAACAT-3’,命名为sgR-BnTFL1-Target2;
2)sgRNA上下游引物设计:
sgR-BnTFL1-Target1的上游引物BnTFL1-Target1-F:5’-ATTGGATCTCAACCCTAGGCTTGG-3’;
sgR-BnTFL1-Target1的下游引物BnTFL1-Target1-R:5’-AAACCCAAGCCTAGGGTTGAGATC-3’;
sgR-BnTFL1-Target2的上游引物BnTFL1-Target2-F:5’-ATTGGAGCTGCCAAAGCCAAACAT-3’;
sgR-BnTFL1-Target2的下游引物BnTFL1-Target2-R:5’-AAACATGTTTGGCTTTGGCAGCTC-3’;
3)CRISPR/Cas9表达载体的构建与农杆菌转化:
取上述步骤2)所述的sgR-BnTFL1-Target1和sgR-BnTFL1-Target2的上下游引物混合,经PCR仪退火得到双链DNA;利用T4连接酶将双链DNA与经过AarI酶切的CRISPR/Cas9质粒进行T4连接酶连接,将连接产物转化大肠杆菌并做菌落PCR鉴定以及测序验证,得到表达载体;将验证含有sgRNA的载体转化农杆菌,进行菌落PCR验证得到含有表达载体的农杆菌菌株;
步骤二,甘蓝型油菜BnTFL1基因CRISPR/Cas9基因编辑突变体的获得与鉴定:
1)基于农杆菌介导的方法遗传转化甘蓝型油菜自交系K407,通过潮霉素抗性筛选获得基因编辑突变体植株;
2)利用PCR检测基因编辑突变体植株中的CRISPR/Cas9表达载体,并克隆基因编辑突变体植株中BnTFL1基因6个不同拷贝的基因组序列,经测序后,检测每个CRISPR/Cas9基因编辑突变体植株中各BnTFL1拷贝的编辑状况;
步骤三,验证甘蓝型油菜BnTFL1基因对开花时间、花器官发育和花序结构的影响:
1)比较甘蓝型油菜自交系K407和不同CRISPR/Cas9基因编辑突变体在开花时间、花器官和花序结构的差异;
2)分析BnTFL1基因6个不同拷贝的功能。
2.权利要求1所述方法获得的甘蓝型油菜基因编辑材料在甘蓝型油菜育种中的应用。
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Publication number | Priority date | Publication date | Assignee | Title |
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CN110777164A (zh) * | 2019-11-20 | 2020-02-11 | 扬州大学 | 一种基于CRISPR/Cas9技术获得甘蓝型油菜黄籽种质的方法 |
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Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
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Non-Patent Citations (3)
Title |
---|
Sukarkarn Sriboon et al.."Knock-out of TERMINAL FLOWER 1 genes altered flowering time and plant architecture in Brassica napus".《BMC Genetics》.2020,第21卷第1-13页. * |
Yuan Guo et al.."Mutations in single FT - and TFL1 -paralogs of rapeseed ( Brassica napus L.) and their impact on flowering time and yield components".《frontiers in PLANT SCIENCE》.2014,第5卷第1-12页. * |
刘凯歌 等."甘蓝型油菜BnTTG1-1基因的功能分析".《植物学报》.2017,第52卷(第6期),第713-722页. * |
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