CN115011609B - BnaFANCM基因、应用及获得油菜雄性不育突变体的方法 - Google Patents
BnaFANCM基因、应用及获得油菜雄性不育突变体的方法 Download PDFInfo
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Classifications
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
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- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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Abstract
本发明提供BnaFANCM基因、应用及获得油菜雄性不育突变体的方法，涉及植物基因编辑和植物育种技术领域；本发明首先获得2个FANCM的同源序列BnaFANCM‑C05和BnaFANCM‑A05；根据BnaFANCM‑C05基因的序列设计CRISSPR/Cas9的靶点Target1和Target2；然后构建Target1和Target2分别与sgRNA融合的重组载体pKSE401‑BnaFANCM‑CRISPR；含有pKSE401‑BnaFANCM‑CRISPR载体的GV3101侵染油菜，获得转基因植株。本发明利用CRISPR/Cas9系统对甘蓝型油菜BnaFANCM基因进行编辑，进而创制新的雄性不育的油菜种质资源，其不仅在油菜杂种优势利用中具有广阔的应用前景，而且在新种质创制方面也有广阔的应用前景。
Description
技术领域
本发明涉及BnaFANCM基因、应用及获得油菜雄性不育突变体的方法，属于植物基因编辑和植物育种技术领域。
背景技术
甘蓝型油菜(Brassica napus L.)属于十字花科、芸薹属，是中国最重要的油料作物之一。甘蓝型油菜原产于欧洲地中海地区，我国约20世纪20年代从日本引进，后来又从欧洲引入原产品种由于我国油菜的栽种历史只有近百年，缺乏优异种质资源，而油菜种质资源是科学研究、品种改良和产业发展不可或缺的物质基础，开展优异种质资源的研究是保障作物新品种不断育成和农业生产可持续发展的重要领域，也是一个国家在作物育种领域核心竞争力的体现。
目前，我国油菜杂种优势利用的雄性不育系统，主要围绕细胞质雄性不育和细胞核雄性不育系统，但是这2种系统在实际生产应用上均存在一些缺陷；近年来，我国在油菜雄性不育研究领域不断取得新的进展，一些分子标记的开发、育种相关基因的克隆和研究，不断推动油菜雄性不育培育的研究。
FANCM基因参与减数分裂细胞正常的同源染色体交叉、有丝分裂重组和生育，在拟南芥中，FANCM(哺乳动物Fanconi贫血互补组M的同源物)抑制重组。
近几年，CRISPR/Cas9(Clustered regulularly interspaced shortpalindromic repeats and CRISPR associated)系统是一种能够高效且精准编辑基因组的新兴基因工程技术，目前利用CRISPR/Cas9系统通过对油菜FANCM基因改造尚未有报道。
发明内容
针对现有技术的一些缺点与不足，本发明提供一种新的雄性不育油菜种质资源的创制方法；本发明还利用CRISPR/Cas9系统对甘蓝型油菜BnaFANCM基因进行定点突变，获得了一种雄性不育的转基因植株。
为达到上述技术目的，本发明采用的技术方案如下：
本发明首先提供油菜雄性不育相关基因BnaFANCM，所述基因BnaFANCM包括基因BnaFANCM-C05和BnaFANCM-A05，所述BnaFANCM-C05的核苷酸序列如SEQ.ID.NO.8所示，所述BnaFANCM-A05的核苷酸序列如SEQ.ID.NO.9所示。
本发明还提供BnaFANCM基因在油菜雄性不育育种中的应用。
本发明还提供一种基因编辑载体pKSE401-BnaFANCM-CRISPR，所述基因编辑载体包含用于甘蓝型油菜BnaFANCM基因编辑的CRISPR/Cas9系统序列元件组，该系统序列包括所述序列元件组包括U6-26p-Target1-gRNA、U6-26p-Target2-gRNA和根据密码子优化后的Cas9基因以及抗性基因Kana。
本发明中还提供了用于甘蓝型油菜BnaFANCM基因编辑的基因工程菌，所述基因工程菌采用上述基因编辑载体pKSE401-BnaFANCM-CRISPR转化宿主细菌GV3101得到。
本发明首先提供一种获得油菜雄性不育突变体的方法，所述方法利用CRISPR/Cas9系统对甘蓝型油菜BnaFANCM基因进行编辑，进一步的，所述方法包括：
(1)针对甘蓝型油菜中的BnaFANCM基因序列设计并筛选sgRNA靶点Target1和Target2，所述Target1的核苷酸序列如SEQ.ID.NO.1所示，所述Target2的核苷酸序列如SEQ.ID.NO.2所示，并合成构建载体所需的引物。
(2)构建双靶点CRISPR/Cas9基因编辑载体：通过PCR扩增以及酶切将2个靶点Target1和Target2分别与sgRNA序列连接，构建出双靶点基因编辑载体pKSE401-BnaFANCM-CRISPR。
(3)将pKSE401-BnaFANCM-CRISPR载体转化农杆菌，获得含有基因编辑表达载体pKSE401-BnaFANCM-CRISPR的农杆菌；
(4)利用农杆菌转化的方式将pKSE401-BnaFANCM-CRISPR载体转至受体材料中，经过油菜下胚轴侵染、诱导愈伤组织、再分化、生根培养等步骤获得转基因植株。
进一步的，为验证所得到的植株为目标植株，在转基因植株中筛选阳性苗植株，利用U626F/R以及Cas9F/R进行扩增鉴定，并通过PCR扩增阳性苗FANCM基因靶点序列并连接T载体送生工生物工程有限公司进行测序，将测序结果进行比对，同时确定编辑形式。
其中，所述甘蓝型油菜BnaFANCM基因包括BnaFANCM-C05和BnaFANCM-A05，所述sgRNA的核苷酸序列如SEQ.ID.NO.3所示，所述Target1、Target2为基因BnaFANCM-C05和BnaFANCM-A05的共同靶点；
所述BnaFANCM-C05的核苷酸序列如SEQ.ID.NO.8所示，氨基酸序列如SEQ.ID.NO.10所示；
所述BnaFANCM-A05的核苷酸序列如SEQ.ID.NO.9所示，氨基酸序列如SEQ.ID.NO.11所示。
步骤(1)中所述引物序列如下：
DT1-BsF(SEQ.ID.NO.4)：
ATATATGGTCTCGATTGAGTCCACACGAATTACTTAGTT
DT2-BsR(SEQ.ID.NO.5)：
ATTATTGGTCTCGAAACGCTGAACTTTTTGCGACTGCAA
DT1-F0(SEQ.ID.NO.6)：
TGAGTCCACACGAATTACTTAGTTTTAGAGCTAGAAATAGC
DT2-R0(SEQ.ID.NO.7)：
AACGCTGAACTTTTTGCGACTGCAATCTCTTAGTCGACTCTAC
步骤(3)中所述农杆菌为农杆菌GV3101，所述受体材料为Y127。
本发明还提供FANCM基因或上述方法在获得油菜雄性不育突变体中的应用，进一步的，所述应用为采用CRISPR/Cas9系统编辑FANCM基因，进一步获得油菜雄性不育突变体；进一步的，所述油菜FANCM基因包括BnaFANCM-C05和BnaFANCM-A05。
本发明还提供了通过CRISPR/Cas9编辑BnaFANCM，获得BnaFANCM编辑的突变体fancm-1、fancm-2、fancm-3，进一步观察基因编辑BnaFANCM突变体的表型，发现突变体相对于野生型表现出雄性不育的表型；利用CRISPR/Cas9系统定向突变BnaFANCM获得新的雄性不育植株的方法为甘蓝型油菜的杂种选育提供了新的种质资源，在油菜分子设计育种中具有广阔的应用前景。
本发明中对BnaFANCM基因的编辑，主要的编辑类型是T碱基的插入导致基因移码突变。
本发明具有以下优点与有益效果：
目前有对自然突变的材料进行筛选鉴定不育系，还有人工进行物理射线诱变或者化学诱变，然后筛选不育系；自然突变以及物理化学诱变的突变率很低，需要大量的筛选材料，人力物力会有很大的损耗。所以，本发明聚焦于基因编辑技术，对育性基因进行编辑产生不育系材料。
(1)本发明中通过编辑BnaFANCM基因解除了限制基因重组或限制新基因型的产生的限制因素之一，从而产生更多的基因型；基因型决定表型，所以最终能够产生很多不同表型的植株，便于筛选有利表型进行繁育。
(2)采用本发明的方法在甘蓝型油菜中特异性敲除BnaFANCM基因，获得BnaFANCM不同突变类型的单株，其可育性相比受体材料Y127显著降低；
(3)本发明利用CRISPR/Cas9系统定向突变BnaFANCM获得新的不育植株的方法为甘蓝型油菜的杂种选育提供了新的种质资源；
(4)本发明的方法为杂交种子生产中的杂种优势利用提供有利材料，具有重要的理论和实践意义；
(5)本发明中的BnaFANCM突变植株后代可以不断产生新的基因型，对比自然突变，加快了新的表型的产生；
(6)本发明构建的基因编辑载体pKSE401-BnaFANCM-CRISPR转化油菜后获得的转化株，为研究基因BnaFANCM的功能及作用机制提供了实验材料。本发明的方法既可以在杂种优势、三系杂交中应用，所获得的突变体植株本身还是不断变异，即植株本身不育，但是在不育纯合的时候通过增加基因重组，产生更多基因型。
附图说明
图1为pKSE401-BnaFANCM-CRISPR载体示意图；图中LB：左边界；RB：右边界；Kan：卡那霉素抗性基因；35S：CaMV35启动子；U6-26p-Target1-gRNA：gRNA表达元件组，其包括启动子U6-26p、靶点1(Target1)和gRNA骨架结构；U6-26p-Target2-gRNA：gRNA表达元件组，包括启动子U6-26p、靶点2(Target2)和gRNA骨架结构；Cas9：根据密码子优化后的Cas9基因。
图2为经过转化得到阳性株中提取叶片基因组的PCR鉴定胶图；图中，WT：野生型；fancm-1、fancm-2、fancm-3：突变体转基因植株；+：正对照，pKSE401-BnaFANCM-CRISPR质粒；-：负对照，ddH2O；Marker：Takara DL2000 DNA Marker。
图3为与野生型相比fancm突变体中BnaFANCM-C05靶点具体位置(a)、BnaFANCM-C05基因(b)和BnaFANCM-A05基因(c)的测序结果汇总分析示意图。
图4为fancm突变体靶点1处T插入导致的移码突变简析示意图；图中(a)为与野生型相比突变体中BnaFANCM-C05基因发生的改变；(b)为与野生型相比突变体中BnaFANCM-A05基因发生的改变。
图5为突变体材料T0代部分株系自交角果表型观察与正反交表型观察，其中(a)为T0代部分株系自交角果表型观察；(b)为fancm突变体与野生型(WT)的正反交表型观察。
具体实施方式
为了使本领域技术人员更好的理解本发明的技术方案，下面对本发明的较佳实施例进行详细的阐述，但是如下实施例并不限制本发明的保护范围。
本发明的实施例中，没有多作说明的都是采用常规实验方法完成，实施例中所涉及过程都是本领域技术人员根据产品说明书或本领域基础知识可以理解并且容易实现的，没有特殊说明的材料或试剂，均为常规市售材料，因此不再详细描述。
本发明中所涉及的培养基及其配方如下所示：
LB液体培养基：称取胰蛋白胨10g、酵母提取物5g、氯化钠10g溶于800mL ddH2O中，定容至1L，然后分装至10个锥形瓶用封口膜封口，121℃高温高压灭菌15min，冷却后放至4℃保存。
LB固体培养基：称取胰蛋白胨10g、酵母提取物5g、氯化钠10g、琼脂粉15g溶于800mL ddH2O中，然后定容至1L，然后分装至10个锥形瓶用封口膜封口，121℃高温高压灭菌15min，冷却后放至4℃保存。使用时放入微波炉中加热至融化，待液体冷却到50℃左右加入抗生素，摇匀后立刻倒至无菌平皿中，每皿约10mL。
M0培养基(1L)：MS粉4.4g，蔗糖30g，800ml ddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，最后加凝固剂Agar 10g，灭菌后分装到无菌平皿中,放于4℃冰箱待用。
DM培养基(1L)：MS粉4.4g，蔗糖30g，800ml ddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，灭菌,等培养基冷却后加入AS，1L中加入1mL AS(母液100μmol/mL),放于4℃冰箱待用。
M1培养基(1L)：MS粉4.4g，蔗糖30g，甘露醇18g，2,4-D 1mg，KT 0.3mg，800mlddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，最后加凝固剂Agar 10g，灭菌后等培养基快冷却后加入AS，1L中加入1mL AS(母液100μmol/mL),然后分装到无菌平皿中,放于4℃冰箱待用。
M2培养基(1L)：MS粉4.4g，蔗糖30g，甘露醇18g，2,4-D 1mg，KT 0.3mg，800mlddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，再加凝固剂Agar 10g，灭菌后等培养基快冷却后加入：特美汀300mg，STS 150μmol，卡那霉素25mg，然后分装到无菌平皿中,放于4℃冰箱待用。
M3培养基(1L)：MS粉4.4g，葡萄糖10g，木糖0.25g，MES 0.6g，800ml ddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，再加凝固剂Agar 10g，灭菌后等培养基快冷却后加入：ZT 2mg，IAA 0.1mg，特美汀300mg，AgNO3 150μmol，卡那霉素25mg，然后分装到无菌平皿中,放于4℃冰箱待用。
M4培养基(1L)：MS粉4.4g，蔗糖10g，800ml ddH2O充分溶解，调节pH值为5.84-5.88，定容至1L，凝固剂Agar 10g，灭菌后等培养基快冷却后加入：特美汀300mg，然后分装,放于4℃冰箱待用。
STS：[Ag(SO3)2]3-现用现配，时间过长有沉淀；母液：硫代硫酸钠，0.1M(2.48g溶于100ml ddH2O)；AgNO3，0.1M(1.7g溶于100ml ddH2O)VNa2SO3:VAgNO3＝4：1，将AgNO3溶于硫代硫酸钠中。
2，4-D(1mg/ml)：取1ml 10mg/ml的母液于12ml无菌管中，加入9ml灭菌后的双蒸水，混匀，保存于-20℃。
KT(1mg/ml)：0.02g KT先溶于1M HCL中，加水定容至20mL，保存于-20℃。
AS(100umol/ml)：0.392gAS先溶于少量甲醇中，再加二甲基亚砜，定容至20m，抽滤分装，保存于-20℃。
ZT(2mg/ml)：称0.02gZT粉末溶于少量75％酒精中，加水定容至10ml，抽滤分装，保存于-20℃。
IAA(1mg/ml):0.1gIAA溶于少量1mol/LNaOH中，再加ddH2O定容至100ml，抽滤分装，保存于-20℃。
TMT(300mg/ml):3.2g特美汀粉末溶于10.66ml灭菌的ddH2O中，震荡混匀，抽滤分装，保存于-20℃。
HYG(50mg/ml)：1g潮霉素B溶于20ml灭菌的ddH2O中，震荡混匀，抽滤分装，保存于-20℃。
Kana(100mg/ml)：10g卡那粉末溶于100ml灭菌的ddH2O中，震荡混匀，抽滤分装，保存于-20℃。
实施例1：基于CRISPR/Cas9系统定向突变甘蓝型油菜BnaFANCM载体的构建
根据甘蓝型油菜参考基因组网站(http://cbi.hzau.edu.cn/bnapus/)中参考基因组ZS11的序列信息，获得两个FANCM同源序列，分别为BnaFANCM-C05和BnaFANCM-A05，其核苷酸序列如SEQ ID NO.8、SEQ ID NO.9所示，其氨基酸序列如SEQ ID NO.10、SEQ IDNO.11所示。
SEQ ID NO.8、SEQ ID NO.9具体序列信息请见序列表。
SEQ ID NO.10：
MGVDGVSYHRVSWGRYKDWAAVRDACKSNSSSSSTHTNDNGTKAKRSTDRDRSTDVVSDSCGTNDNTSVGDTAKTWYVNVRDYATKTASNTVATGGKTAAVVMYNYRWGKVAASRVMACHNVGWTDTGTCSKRASWKTKRVVTVKDSGTCVTNCVCVDAHRAGNYSYCVVVRMAVVRATATGSKTAGDNSTYRNSDHDVCYVHDRKVVGKDADVSKRDVRYAVRKNGVSRDYTSHMARDKRAVGHSHGDVSCAATYHRKSSHGRAYMKGARMSKNADRMTKMRSNGASKSKMVDHYKKDRTSRVSNRGSVRDMDASNGDVVKATGSSGKTKGSKVAVKRSGGNVVATSGGDMVDVCDANVSRMRMGRTGRKNNGRVACGSKNSYMRKKANGAKKHMRNGGMNSNHSRMHVYKVHVKSRGKKDAATAKKKTMDMAKYKNKWRVSAHTSKVHKVMHSRTSDAMHTTTSKSKYGAARDDAGRVGDDKDSDDDVNTSRKAKVVSTSTTTKASSTHCYNSCASVNTGKVVSSSNVGSDYVGRKSSNKSHDVVMDSSSKHRDSCKKGDCANTSSKWHSTDVGKGDNCSGMSSDDDCDSGSTSSRVAHSMTKHTKTKKTHRV
SEQ ID NO.11：
MGVDGDWAAVRDACKSNSSSSSTHTNGNGTKAKRSTDRARADHKYVVSDSCGTNDNTSVGDTAKTWYMNVRDYATKTASNTVATGGKTAAVVMYNYRWGKVAASRVMACHNVGWTDTGTCSKRASWKTKRVVTVKDSGTCVTNCVCVDAHRAGNYSYCVVVRMAVVRATATGSKTAGDNSTYRNSDHDVCYVHDRKVVGKDADVSKRDVRYAVRKNGVSRDYTSHMARDKRAVGHSHGDVSCAATYHRKSSHGRAYMKGARMSKNDRMTKMRSNGASKSKMVDHYKKDRTSRVSNRGSVRDMDASNDVVKATGSSGKTKGSKVAVKRSGGNVVATSGGDMVDVCDANVSRMRMGRTGRKNNGRVVVACGSKNSYMRKKANGAKKHMRNGGMNSNHSRMHVYKVHVKSRGKKDATTAKKKTMDMAKYKNKWRVSAHTSKVHKVMHSRTSDAMHTTTSKSKYGAARDDAGRVGDDKDSSDDDVNTSRKAKSTSTTTKDASSTHCYSSCASVDTGKVVVSSSNVGSDCVGRKSSNKSHTDVVDSSSKHRDNSCKKGDCANTSSKRHSTDVGKGDNCAGMSSDDDCDSRTNKSGVVDSVYDGVAYANRDDTSTNASTKKVHASTANRTKDANTSAVSMWRTANTNASSSASKDWRVSSGKSTRRKKRRRGDCSSAVKNNGAKTDHRSRSRSVKNRGKKKRADNNARAAVSSSMSVDNVDTSDSDSDDGTMTANTACAKVDMMAVYRRSSSARRDVAASSSYSSGKTNSRSDSDKSSSRTTTNNSNKDAVATGDVASTDSRKRKSCNSANVVNNKAHAATKSHGRSNAGASYKDDDDDAYATDDAMAHATSKRSTKTKDASVKHGNRNDGKDGSDGW
将BnaFANCM-C05基因序列提交至CRISPR-P(http://cbi.hzau.edu.cn/cgi-bin/CRISPR)网站，筛选确定脱靶率低且能同时编辑BnaFANCM-C05与BnaFANCM-A05的基因编辑靶点序列Target1(核苷酸序列如SEQ ID NO.1所示，TGG为PAM序列)、Target2(核苷酸序列如SEQ ID NO.2所示，AGG为PAM序列)和sgRNA(核苷酸序列如SEQ ID NO.3所示，TGG为PAM序列)。
Target1(核苷酸序列如SEQ ID NO.1所示，TGG为PAM序列)：
GTTGCTTTACCAACAGGCCTTGG
Target2(核苷酸序列如SEQ ID NO.2所示，AGG为PAM序列)：
GGTTTGCTTGGTGATCGACGAGG
sgRNA(核苷酸序列如SEQ ID NO.3所示，TGG为PAM序列)：
GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT
根据筛选的靶点设计CRISPR/Cas9载体靶点引物，引物序列如表1所示，确保设计的2个靶点可以同时敲除BnaFANCM-C05和BnaFANCM-A05。
表1.CRISPR/Cas9载体靶点引物
| 引物 | 序列5’-3’ |
| DT1-BsF(SEQ.ID.NO.4) | ATATATGGTCTCGATTGAGTCCACACGAATTACTTAGTT |
| DT2-BsR(SEQ.ID.NO.5) | ATTATTGGTCTCGAAACGCTGAACTTTTTGCGACTGCAA |
| DT1-F0(SEQ.ID.NO.6) | TGAGTCCACACGAATTACTTAGTTTTAGAGCTAGAAATAGC |
| DT2-R0(SEQ.ID.NO.7) | AACGCTGAACTTTTTGCGACTGCAATCTCTTAGTCGACTCTAC |
随后以入门载体pCBC-DT1T2(来自华中农业大学郭亮教授)为模板，用表1中2对引物在高保真酶2*Phanta Max Master Mix(购自南京诺唯赞生物科技股份有限公司)的作用下进行PCR扩增反应，PCR反应体系如表2。
表2.高保真酶PCR扩增反应体系
| PCR成分 | 体积 |
| 2*Phanta Max Master Mix | 25μL |
| DT1-BsF(10μM) | 1μL |
| DT2-BsR(10μM) | 1μL |
| DT1-F0(0.5μM) | 1μL |
| DT2-R0(0.5μM) | 1μL |
| pCBC-DT1T2(100-200ng) | 1μL |
| ddH2O | 20μL |
PCR反应程序为：
95℃预变性3min；95℃变性30s、61℃退火30s、72℃延伸30s，共进行34个循环；72℃终延伸5min。
PCR反应结束后，PCR产物在1％琼脂糖凝胶(质量体积分数)中120V 400mA下进行凝胶电泳27min，然后在紫外凝胶成像仪下照相，记录结果。
结果显示，有626bp大小片段，是构建的载体靶位点和gRNA的片段，切胶，利用FastPure Gel DNA Extraction Mini Kit试剂盒纯化回收(购自南京诺唯赞生物科技股份有限公司)该PCR产物。然后建立酶切连接体系，具体连接体系见表3。
表3.酶切-连接体系
反应结束后，取5μL连接产物转化感受态大肠杆菌DH5α(购自南京诺唯赞生物科技股份有限公司)，将转化后的DH5α涂布于含有50mg/mL Kan的固体LB平板培养基上进行筛选，37℃过夜培养后，挑取阳性克隆于400μL含有50mg/mL Kan的液体LB培养基中并在摇床中进行震荡培养4-6h，然后取2μL菌液作为模板PCR扩增进行鉴定，PCR扩增体系见表4，PCR反应程序为：94℃预变性3min；94℃变性30s、57℃退火30s、72℃延伸40s，共进行28个循环；72℃终延伸10min。引物使用通用引物U626-IDF和U629R，具体引物序列如下所示
U626-IDF:TGTCCCAGGATTAGAATGATTAGGC(SEQ.ID.NO.12)
U629-IDR：AGCCCTCTTCTTTCGATCCATCAAC(SEQ.ID.NO.13)；
表4.菌液PCR反应体系
| 反应成分 | 体积 |
| 菌液 | 2μL |
| U626-IDF | 1μL |
| U629R | 1μL |
| rTaq酶 | 10μL |
| ddH2O | 6μL |
PCR反应程序为：94℃预变性3min；94℃变性30s、57℃退火30s、72℃延伸40s，共进行28个循环；72℃终延伸10min。
经过PCR跑胶鉴定后得到的片段大小为726bp，将含有靶序列以及sgRNA的序列连接到终载体pKSE401(来自华中农业大学郭亮教授，可市场购买)上，因终载体pKSE401上还有一段sgRNA，所以片段长度增加，吸取片段大小正确的阳性克隆菌液150μL送至生工生物工程(上海)股份有限公司进行测序，测序引物使用U626-IDF和U629-IDF，引物序列如下：
U626-IDF：TGTCCCAGGATTAGAATGATTAGGC(SEQ.ID.NO.12)
U629-IDF：TTAATCCAAACTACTGCAGCCTGAC(SEQ.ID.NO.14)；
测序正确的克隆，靶点与sgRNA连接并存在于终载体中，提取质粒，转化农杆菌感受态GV3101(购买于上海唯地生物技术有限公司)，挑取阳性单克隆，用于农杆菌介导的遗传转化，载体pKSE401-BnaFANCM-CRISPR示意图如图1所示。
实施例2：pKSE401-BnaFANCM-CRISPR基因编辑重组载体转化至甘蓝型油菜下胚轴
(1)种子消毒与播种
挑选籽粒饱满的快生型油菜Y127(来自华中农业大学洪登峰老师)置于12mL无菌管中，在超净台中用75％酒精消毒45s，随后用灭菌ddH2O清洗种子2-3遍，再用15％Bleach溶液(配制为8.115mL无菌水+1.875mL次氯酸钠+10μL曲拉通)消毒7min30s，再用灭菌ddH2O清洗种子至水中无明显泡沫为止，最后将消毒完毕的种子均匀的播种至M0培养基上，暗培养。
(2)菌液准备
播种后的第7天，将实施例1中得到的含pKSE401-BnaFANCM-CRISPR重组载体的GV301农杆菌进行扩大培养：50μL重组菌液加入5mL的抗性LB(加入50mg/L Kan+50mg/L Gen+50mg/L Rif)于28℃、220rpm摇床中培养约14-16h。
(3)侵染与共培养
采用分光光度计测步骤(2)中所述的抗性LB培养基中菌的OD值，OD值为0.4左右时的菌液较好，一般摇菌14-16小时即可。吸取2mL培养好的菌液到2ml无菌离心管中，5000rpm10min离心，弃上清；然后加入2mL DM(AS+)液悬浮，5000rpm 10min离心，弃上清；再加入2mL的DM(AS+)液悬浮，放4℃冰箱备用。
取出暗培养7天的幼苗，用经高温杀菌的镊子和剪刀切下下胚轴置于装有18ml DM(AS+)一次性平皿中，切取时尽量快速斜向下切下，保证切口平整，长度在0.8-1cm为好。切好后，加入事先准备好的菌液，浸染12min30 s，隔段时间摇晃1次，4～5次即可；用枪头吸净DM(AS+)与菌液，将下胚轴外植体铺于事先灭菌的滤纸上，吸净表面过多的菌液。用镊子将外植体均匀摆到M1培养基上，摆放时要让外植体两端充分接触到培养基，24℃暗培养36-48h。
(4)选择培养及愈伤诱导
将共培养(转化后的重组农杆菌与下胚轴共培养)后的M1中外植体转到选择及诱导愈伤的M2培养基上，M2培养基中外植体倒置光下正常培养，培养条件为24℃下白天16h、晚上8h的方式交替培养，2-3周诱导愈伤。
(5)再分化
将选择出来的外植体转到分化M3培养基上，每2-3周继代一次，期间淘汰褐化死亡的外植体，其中两端膨大并有绿点的外植体为佳，直至出现绿芽。
(6)生根培养
将分化出来的芽苗，用高温灭菌的手术刀对芽苗基部切出一部分创口，然后转入M4培养基中，进行生根培养。待生根后，可将其放置培养间内进行炼苗，待苗状态稳定后，从培养基中取出，取苗过程中尽量不要破坏植物的根系，然后根部用1％多菌灵浸泡一遍，最后将苗移到土壤中培养，培养时需要用保鲜膜保湿1周左右，即可获得等待鉴定的转基因油菜。
实施例3：转基因植株编辑情况检测
(1)转基因植株DNA的提取
采取CTAB法提取转基因油菜叶片中的DNA，具体步骤如下：
取拇指盖大小叶片放入1.5mL离心管中，于液氮中制冷用研磨枪进行研磨，研磨成干粉后，加入600μL CTAB，充分摇晃然后将样品放入65℃水浴锅中孵育60min；孵育完成后，在管中加入600μL氯仿/异戊醇(体积比为24:1)溶液，剧烈震荡，充分除去蛋白质，然后放入离心机中12000g离心10min；将离心好的离心管按顺序摆放在操作板上，吸取400μL上清液到新的1.5mL离心管中，注意不要将位于上清液下面的残留组织吸到新离心管中；然后向上清中加入400μL异丙醇，轻轻颠倒混匀，接着将样品放入-20℃冰箱中冷却至少10min，以使DNA沉淀完全；将离心管放入离心机中，室温下12000g离心10min；弃上清，加入700μL 70％冰乙醇洗涤沉淀(弹起管底沉淀)，12000g瞬旋；弃去上清并用移液枪吸净剩余液体，置于超净台中风干沉淀10min；向离心管中加入50μL ddH2O溶解沉淀，放入37℃水浴锅中60min，得到基因组DNA样品；最后取1μL基因组样品测定浓度，检测合格后(OD260/OD280在1.6-1.8之间)，将基因组样品放入-20℃冰箱中备用。
(2)阳性苗的鉴定
以步骤(1)中提取的DNA为模板，利用引物U626-IDF、U626-IDR以及Cas9-F、Cas9-R，分别进行PCR扩增，水和野生型Y127的DNA为阴性对照，质粒pKSE401-BnaFANCM-CRISPR为阳性对照，退火温度分别为57℃和62℃，其他PCR扩增反应程序以及条件与表4菌液PCR反应相同，引物序列如下：
U626-IDF:TGTCCCAGGATTAGAATGATTAGGC(SEQ.ID.NO.12)
U629-IDR:AGCCCTCTTCTTTCGATCCATCAAC(SEQ.ID.NO.13)；
Cas9-F：TGCAGGAGATTTTCTCCAACGA(SEQ.ID.NO.15)
Cas9-R：AGCCTTCGTAATCTCGGTGTTCA(SEQ.ID.NO.16)
PCR完成后，扩增产物在1％的琼脂糖凝胶中进行电泳，使用紫外凝胶成像仪拍照，记录结果。图2为经过转化筛选从14株中得到3株阳性株中提取叶片基因组的PCR鉴定胶图；图中，WT：野生型；fancm-1、fancm-2、fancm-3：突变体转基因植株；+：阳性对照，pKSE401-BnaFANCM-CRISPR质粒；-：阴性对照，ddH2O；Marker：Takara DL2000 DNA Marker；从图中可见，fancm-1、fancm-2、fancm-3为转基因阳性植株。
(3)TA克隆检测阳性苗编辑位点
使用高保真酶在引物C05OEF/C4C5R的作用下，对上述鉴定成功的阳性株的基因组进行PCR扩增、跑胶、胶回收以及连接pMD19-T载体，转化大肠杆菌，挑菌鉴定，送单克隆菌液至生工生物工程(上海)股份有限公司进行测序，具体实验操作及方法如下：
以油菜品种Y127(来源于华中农业大学)、fancm-1、fancm-2、fancm-3的叶片DNA分别为模板，使用高保真酶2*Phanta Max Master Mix(购自南京诺唯赞生物科技股份有限公司)扩增BnaFANCM基因组的Target1和Target2附近1000bp片段，PCR反应如表5所示。
表5.高保真酶扩增BnaFANCM基因组反应体系
| PCR成分 | 体积 |
| 2*Phanta Max Master Mix | 25μL |
| C05OEF(10μM) | 2μL |
| C4C5R(10μM) | 2μL |
| DNA(100-200ng) | 1μL |
| ddH2O | 20μL |
PCR反应程序为：95℃预变性3min；95℃变性30s、57℃退火30s、72℃延伸1min30s，共进行34个循环；72℃终延伸5min。
PCR反应结束后，PCR产物在1％琼脂糖凝胶(质量体积分数)中120V 400mA下进行凝胶电泳27min，然后在紫外凝胶成像仪下照相，记录结果。结果显示，有1322bp大小片段，切胶，利用FastPure Gel DNA Extraction Mini Kit试剂盒纯化回收(购自南京诺唯赞生物科技股份有限公司)该PCR产物。然后将回收的PCR扩增产物进行末端加A，加A体系为：回收产物7.5μL、dNTPs 1μL、PCR Buffer(Mg+free)1μL、rTaq 0.5μL，反应条件72℃ 30min。接着将加A产物连接pMD19-T载体上，连接体系为：加A产物4.5μL、pMD-19T载体0.5μL、SolutionⅠ5μL，在16℃下连接过夜，得到连接产物(TA克隆使用试剂均购自宝生物工程(大连)有限公司)。
向30μL大肠杆菌感受态细胞(购自南京诺唯赞生物科技股份有限公司)中加入5μL的连接产物，通过热激法将连接产物转入大肠杆菌中，然后利用含终浓度为30mg/mL的Amp的LB培养基筛选阳性菌落，并挑取12个单菌落震荡培养12-16h，取2μL菌液作为模板PCR扩增进行鉴定，引物用C05OEF/C4C5R，其他PCR反应条件与程序参照表4，PCR反应的引物序列如下：
C05OEF：GGTACCATGGGACCCGAGTTTCCGAT(SEQ.ID.NO.17)
C4C5R：CTGAAAAATGGCATCCAACAAG(SEQ.ID.NO.18)
送阳性单克隆菌液至生工生物工程(上海)股份有限公司进行测序，对得到的测序结果进行分析，测序结果汇总如图3所示，可以发现较多的单克隆在靶点1处表现出T碱基的插入，故此对其进行进一步分析，分析结果见图4。
图4为fancm突变体靶点1处插入T导致的移码突变简析示意图；图中(a)为与野生型相比突变体中BanFANCM-C05基因发生的改变；(b)为与野生型相比突变体中BanFANCM-A05基因发生的改变。从图(a)(b)中可以看出，fancm突变体的BanFANCM-C05和BanFANCM-A05基因中均会发生移码突变，导致FANCM基因翻译过程提前终止，FANCM蛋白不能正常合成，这证实了基因编辑载体成功在目的靶点行使功能，BanFANCM-C05和BanFANCM-A05基因在甘蓝型油菜中被成功敲除。
实施例4：基因编辑植株表型观察
对实施例3中获得的3株基因编辑油菜进行每天进行自交授粉，一个星期后发现fancm突变体表现出不育的表型，如图5(a)，为进一步确定fancm突变体是雄性不育还是雌性不育的，对突变体材料进行正反交实验。如图5(b)所示，突变体材料花粉授到受体材料Y127柱头上是不结角果的，Y127花粉授到突变体上是结角果的，证明Bnafancm突变体是雄性不育的。正反交角果数如表6所示。
表6.fancm突变体与野生型(WT)的正反交结角果数统计
| ♀WT(结角果数/去雄数) | ♂WT(结角果数/去雄数) | |
| fancm-1 | 0/5 | 4/6 |
| fancm-2 | 0/5 | 4/5 |
| fancm-3 | 0/5 | 4/5 |
应当理解的是，上述具体实施方法仅用于示例性说明或解释本发明的基本原理，本发明不受上述实施例的限制，在不脱离本发明精神和范围的前提下，本发明还会有各种变化和改进，这些变化和改进都落入要求保护的本发明范围内，本发明要求保护范围由所附的权利要求书及其等效物界定。
序列表
<110> 江苏大学
<120> BnaFANCM基因、应用及获得油菜雄性不育突变体的方法
<160> 18
<170> SIPOSequenceListing 1.0
<210> 1
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
gttgctttac caacaggcct tgg 23
<210> 2
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 2
ggtttgcttg gtgatcgacg agg 23
<210> 3
<211> 83
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gttttagagc tagaaatagc aagttaaaat aaggctagtc cgttatcaac ttgaaaaagt 60
ggcaccgagt cggtgctttt ttt 83
<210> 4
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atatatggtc tcgattgagt ccacacgaat tacttagtt 39
<210> 5
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
attattggtc tcgaaacgct gaactttttg cgactgcaa 39
<210> 6
<211> 41
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
tgagtccaca cgaattactt agttttagag ctagaaatag c 41
<210> 7
<211> 43
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
aacgctgaac tttttgcgac tgcaatctct tagtcgactc tac 43
<210> 8
<211> 2922
<212> DNA
<213> 甘蓝型油菜(Brassica napus)
<400> 8
atgggacccg agtttccgat cgaactcgtt gaagaagaag atggagtgag ttaccaccga 60
gtctctttct ggctgggtca attcagattt ccttatttaa aattattttt gtttttgcag 120
ttcgattggg aagcagcagt cagagaaatc gacttggctt gcctcaaatc cttaaaccct 180
tcttcttctt cttcgaccca tttcaccaac gacaatggca ctaaacctgc taaaagacaa 240
tctactctcg atcgattcat cgatcgatct actccggatc ctcctgttgt ttccgacccg 300
agtttcgagt gtggtactaa cgacaacact cccagcgtcg ggattgatcc tgagacagct 360
aaaacttgga tttatccagt aaacgttcct ctaagagatt atcagtttgc tataaccaag 420
actgctttgt tttcaaacac attagttgct ttaccaacag gccttggtaa aacgctcata 480
gctgcagttg taatgtataa ttacttcaga tggtttccac aaggtaaaat tgtctttgcc 540
gcaccttcta ggcctcttgt gatgcagcag attgaggcct gccataatat cgtggggata 600
ccacaagaat ggacgattga cttgacgggt cagacttgcc cttccaaaag agcttccttg 660
tggaaaacca aaagggtttt cttcgtcact ccacaagttc ttgagaagga tatacagtca 720
ggaacgtgtg ttaccaactg cttggtttgc ttggtgatcg acgaggcaca tcgagcttta 780
gggaattatt cttattgtgt tgtagttcgt gagttgatgg cagtaccagt gcagttgaga 840
atattggctc ttactgcaac tcctggatca aagacacagg ccatacaggg tatccttgat 900
aatttgcaga tatcaacact tgaatatcga aacgagagtg accatgatgt ctgcccttat 960
gtccacgaca gaaaagtaga actaatcgag gttcccttgg gtaaagatgc agatgaggta 1020
tctaaacgcc tattagatgt tatacgtcca tatgcagtca ggcttaaaaa tttcggggtc 1080
attctaagca gggattatca aactttgagt ccacacgaat tacttatggc aagggataag 1140
tttcgtgaag cacctgtacc aggcattccc catataagtc acggagatgt agaatcttgc 1200
tttgcagctc ttatcacgct ttatcacatt cgcaagcttc tttctagtca tggaataagg 1260
ccagcgtatg agatgcttga agaaaaactc caggaagggc catttgctag gttgatgagt 1320
aagaatgcag atattaggat gacgaagctt ttgatgcagc aaaggttgtc gaacggagca 1380
ccaagcccga aattgtccaa gatgttggag attctagttg atcactacaa aataaaagat 1440
ccgaggacat cacgggtcat tattttctcg aatttcagag gaagcgtaag agacataatg 1500
gacgcattaa gtaatattgg agatgttgtc aaagcaactg agtttattgg tcaaagttca 1560
ggtaagacac tgaagggaca gtcgcaaaaa gttcagcagg ctgttctgga gaaatttaga 1620
tctggtgggt ttaatgttat tgttgcaaca tctatcggcg aagaaggctt ggatatcatg 1680
gaagtcgact tagttatatg ttttgatgct aatgtatccc ctctgaggat gatccaacgc 1740
atgggaagaa ctggaaggaa aaataatggc cgacctttac tagttcttgc ttgtgaagga 1800
tctgaaaaga atagctatat gcgaaagaaa gcaaatggcc aagccattaa aaaacacatg 1860
cggaatggag gaatgaatag ttttaatttt catcctagtc caaggatgat tccccatgtt 1920
tataagccag aagttcagca tgttaagttt tcgatcgagc aattcattcc acgtggaaag 1980
aagctacaag atgagcctgc cgctgagact ccagctttca agaaaaagct tacaccggaa 2040
gagatggata tgctcgccaa gtatttcaaa cccaacgagg aaaagtggag agtttccttg 2100
attgctttcc ctcacttcca aacattgcca tccaaagtgc acaaagtaat gcattcacgc 2160
caaacaagca tattaattga tgctatgcag catctgcaag agacaacttt gacagagcaa 2220
agtaaaagct tcttcattaa gtatggagct cctttggctg aaagagatga gcttgacgca 2280
ggtctgaggg ttggtgatga tccgaaagat ttaccctctt tcgatgattt ggatgtcaac 2340
acatcacaga gaaaggcaaa acaagttgta gaatctccca caagcacatt agagactaca 2400
gagaaggaat tcgaagcatc ttcaccaaca cactgttatc ttttcaattc ggaatgtgcg 2460
tccgttaata ctctggggaa ggtctttata ttgccggttc ctctttcatt atcttctaat 2520
gtaccagggt cagactatgt gggaagagaa aaagaacttt cttccccgaa taaatcccac 2580
attgacgttg ttccgatgga tagttcctca aaacatcggc aagatagtat tccatgcaag 2640
ttaaagcaag gattcttgcc agattgtgcc aacgagactt tggagtccca aagccttttg 2700
aaatggcact ccaccgatgt aggtaaagga gatatagaga attgttctgg agaaattatg 2760
atatcatcgg atgaagaaga cgactgtgag gatttggagc ttagtcaagg ctcactaact 2820
tcatcaagag tggcgttgtt ccagattcac ctgtctatga ccaaggagtt gcatacgaag 2880
caaacagaga agaagacctt gatcttccac ccacgagttt aa 2922
<210> 9
<211> 3993
<212> DNA
<213> 甘蓝型油菜(Brassica napus)
<400> 9
atgggacccg agtttccgat cgaactcgtt gaagaagaag atggattcga ttgggaagca 60
gcagtcagag aaatcgactt ggcttgcctc aaatccttaa acccttcttc ttcttcttcg 120
acccatttca ccaacggcaa tggcactaaa cctgctaaaa gacaatctac tcttgatcga 180
ttcatcgcaa gagccgacca caagcctcct cctccgtatc ctcctgttgt ttccgacccg 240
agtttcgagt gtggtactaa cgacaacact cccagcgtcg ggattgatcc tgagacagct 300
aaaacttgga tttatccaat gaacgttcct ctaagagatt atcagtttgc tataacgaag 360
actgctttgt tttcaaacac attagttgct ttaccaacag gccttggtaa aacgctcata 420
gctgcagttg taatgtataa ttacttcaga tggtttccac aaggtaaaat tgtctttgcc 480
gcaccttcta ggcctcttgt gatgcagcag attgaggcct gccataatat cgtggggata 540
ccacaagaat ggacgattga cttgacgggt cagacttgcc cttccaaaag agcttccttg 600
tggaaaacca aaagggtttt cttcgtcact ccacaagttc ttgagaagga tatacagtca 660
ggaacgtgtg ttaccaactg cttggtttgc ttggtgatcg acgaggcaca tcgagcttta 720
gggaattatt cttattgtgt tgtagttcgt gagttgatgg cagtaccagt gcagttgaga 780
atattggctc ttactgcaac tcctggatca aagacacagg ccatacaggg tatccttgat 840
aatttgcaga tatcaacact tgaatatcga aacgagagtg accatgatgt ctgcccttat 900
gtccacgaca gaaaagtaga actaatcgag gttcccttgg gtaaagatgc agatgaggta 960
tctaaacgcc tattagatgt tatacgtcca tatgctgtca ggcttaaaaa tttcggggtc 1020
attctaagca gggattatca aactttgagt ccacacgaat tacttatggc aagggataag 1080
tttcgtgaag cacctgtacc aggcattccc catataagtc acggagatgt agaatcttgc 1140
tttgcagctc ttatcacgct ttatcacatt cgcaagcttc tttctagtca tggaataagg 1200
ccagcgtatg agatgcttga agaaaaactt caggaagggc catttgctag gttgatgagt 1260
aagaatgaag atattaggat gacgaagctt ttgatgcagc aaaggttgtc gaacggagca 1320
ccaagcccga aattgtccaa gatgttggag attctagttg atcactacaa aataaaagat 1380
ccgaggacat cacgggtcat tattttctcg aatttcagag gaagcgtaag agacataatg 1440
gacgcattaa gtaatattga agatgttgtc aaagcaactg agtttattgg tcaaagttca 1500
ggtaagacac tgaagggaca gtcgcaaaaa gttcagcagg ctgttctgga gaaatttaga 1560
tctggtgggt ttaatgttat tgttgcaaca tctatcggcg aagaaggctt ggatatcatg 1620
gaagtcgact tagttatatg ttttgatgct aatgtatccc ctctgaggat gatccaacgc 1680
atgggaagaa ctggaaggaa aaataatggc cgagttgtag ttcttgcttg tgaaggatct 1740
gaaaagaata gctatatgcg aaagaaagca aatggccaag ccattaaaaa acacatgcgg 1800
aatggaggaa tgaatagttt taattttcat cctagtccaa ggatgattcc ccatgtttat 1860
aagccagaag ttcagcatgt taagttttcg atcgagcaat tcattccacg tggaaagaag 1920
ctacaagatg agcctgccac tgagactcca gctttcaaga aaaagcttac accggaagag 1980
atggatatgc tcgccaagta tttcaaaccc aacgaggaaa agtggagagt ttccttgatt 2040
gctttccctc acttccaaac attgccatcc aaagtgcaca aagtaatgca ttcacgccaa 2100
acaagcatat taattgatgc tatgcagcat ctgcaagaga caactttgac agagcaaagt 2160
aaaagtttct tcattaagta tggagctcct ttggctgaaa gagatgagct tgacgcaggt 2220
ctgagggttg gtgatgatcc gaaagattta ccctcttccg atgatttgga tgtcaacaca 2280
tcacagagaa aggcaaaaca aattttagaa tctcccacaa gcacattaga gactacagag 2340
aaggatttcg aagcatcttc acccacacac tgttatcttt tcagttcaga atgtgcgtcc 2400
gttgatactc tggggaaggt ctttgtattg ccggttcctc tctcattctc ttctaatgta 2460
ccagggtcag actgcgtggg aagagaaaaa gaactttctt ccccgaataa gtcccacact 2520
gacgttgttc cgatagatag ttcctcaaaa catcggcaag ataatatttc atgcaagtta 2580
aagcaaggat tcttgccaga ttgtgccaac gagactttgg agtcccaaag ccttttgaaa 2640
aggcactcca ccgatgtagg taaaggagat atagagaatt gtgctggaga aattatgata 2700
tcatcggatg aagaagacga ctgtgaggat ttggagctta gtccaaggct cactaacttc 2760
atcaagagtg gcgttgttcc agattcacct gtctatgacc aaggagttgc atacgaagca 2820
aacagagaag aagaccttga tcttccaccc acgagtttaa ctaatgaatt ggcagaagag 2880
ccatcgacac ctgagaaaaa ggttcacatt gcttctacgg ccaatgaatt cagaactcct 2940
cagaaggaag aagatttagc caacgaaaca gaaagcttcg ctgtttctcc aatgcctgag 3000
gagtggagaa ctcccttggc gaatatcacc aacgcaagca gcagcgctag caaagattgg 3060
cgcgtgagtt cgggagaaaa gtcagaaact cttcgacagc ctcgcaagtt gaagagactt 3120
cgtagacttg gagattgctc gagtgctgtg aaggagaata atcctggtat tgcaaagaca 3180
gaccatatca gatctcgttc tcgcagtgta aagaacataa gaggcaagaa gaagatacgc 3240
gcggataata atgctagaat cttcattgaa gcggaagctg aggtgtcttc ggaatcagaa 3300
atgtcggttg atgagaacgt agatttgacc agcgattcat ttgaagatag cttcatagat 3360
gacggtacaa tgcctacagc aaatactcaa gccgagtgtg ctaaagttga catgatggcc 3420
gtttacagac gttctctact cagccaatca ccattaccgg caagatttcg tgatgtagct 3480
gcatcaagtc cgagtcctta ttcttctggt ctcttgaaga caataaatga gagcagaagc 3540
gactcagata aatcattgtc ttctcttaga accccacaaa caacgaacaa cgagtcaaac 3600
aaggatgcag tggccacagg agactttttg gtagcacaaa tctcaacaga cagccggaaa 3660
aggaaattca gcttatgcaa ctcagcgaat gtcccagtga ttaacttgga aaacaagttt 3720
gaagctcatg cacaagccac ggagaaggaa agccatgaag gtccgagaag caatgcaggt 3780
gcatcacagt acaaggatga ggatgaagat gatgatgcat tctacgcgac actggacttt 3840
gatgccatgg aagcgcatgc gacattgcta ttgtcgaaac aaaggtcaga aacgaaaaca 3900
aaagaagatg catcggtgaa acctcatttg ggcaatcaga ggaatgatgg tttgccgaag 3960
gatgggccat cttttgatct tggtttgtgg tga 3993
<210> 10
<211> 615
<212> PRT
<213> 甘蓝型油菜(Brassica napus)
<400> 10
Met Gly Val Asp Gly Val Ser Tyr His Arg Val Ser Trp Gly Arg Tyr
1 5 10 15
Lys Asp Trp Ala Ala Val Arg Asp Ala Cys Lys Ser Asn Ser Ser Ser
20 25 30
Ser Ser Thr His Thr Asn Asp Asn Gly Thr Lys Ala Lys Arg Ser Thr
35 40 45
Asp Arg Asp Arg Ser Thr Asp Val Val Ser Asp Ser Cys Gly Thr Asn
50 55 60
Asp Asn Thr Ser Val Gly Asp Thr Ala Lys Thr Trp Tyr Val Asn Val
65 70 75 80
Arg Asp Tyr Ala Thr Lys Thr Ala Ser Asn Thr Val Ala Thr Gly Gly
85 90 95
Lys Thr Ala Ala Val Val Met Tyr Asn Tyr Arg Trp Gly Lys Val Ala
100 105 110
Ala Ser Arg Val Met Ala Cys His Asn Val Gly Trp Thr Asp Thr Gly
115 120 125
Thr Cys Ser Lys Arg Ala Ser Trp Lys Thr Lys Arg Val Val Thr Val
130 135 140
Lys Asp Ser Gly Thr Cys Val Thr Asn Cys Val Cys Val Asp Ala His
145 150 155 160
Arg Ala Gly Asn Tyr Ser Tyr Cys Val Val Val Arg Met Ala Val Val
165 170 175
Arg Ala Thr Ala Thr Gly Ser Lys Thr Ala Gly Asp Asn Ser Thr Tyr
180 185 190
Arg Asn Ser Asp His Asp Val Cys Tyr Val His Asp Arg Lys Val Val
195 200 205
Gly Lys Asp Ala Asp Val Ser Lys Arg Asp Val Arg Tyr Ala Val Arg
210 215 220
Lys Asn Gly Val Ser Arg Asp Tyr Thr Ser His Met Ala Arg Asp Lys
225 230 235 240
Arg Ala Val Gly His Ser His Gly Asp Val Ser Cys Ala Ala Thr Tyr
245 250 255
His Arg Lys Ser Ser His Gly Arg Ala Tyr Met Lys Gly Ala Arg Met
260 265 270
Ser Lys Asn Ala Asp Arg Met Thr Lys Met Arg Ser Asn Gly Ala Ser
275 280 285
Lys Ser Lys Met Val Asp His Tyr Lys Lys Asp Arg Thr Ser Arg Val
290 295 300
Ser Asn Arg Gly Ser Val Arg Asp Met Asp Ala Ser Asn Gly Asp Val
305 310 315 320
Val Lys Ala Thr Gly Ser Ser Gly Lys Thr Lys Gly Ser Lys Val Ala
325 330 335
Val Lys Arg Ser Gly Gly Asn Val Val Ala Thr Ser Gly Gly Asp Met
340 345 350
Val Asp Val Cys Asp Ala Asn Val Ser Arg Met Arg Met Gly Arg Thr
355 360 365
Gly Arg Lys Asn Asn Gly Arg Val Ala Cys Gly Ser Lys Asn Ser Tyr
370 375 380
Met Arg Lys Lys Ala Asn Gly Ala Lys Lys His Met Arg Asn Gly Gly
385 390 395 400
Met Asn Ser Asn His Ser Arg Met His Val Tyr Lys Val His Val Lys
405 410 415
Ser Arg Gly Lys Lys Asp Ala Ala Thr Ala Lys Lys Lys Thr Met Asp
420 425 430
Met Ala Lys Tyr Lys Asn Lys Trp Arg Val Ser Ala His Thr Ser Lys
435 440 445
Val His Lys Val Met His Ser Arg Thr Ser Asp Ala Met His Thr Thr
450 455 460
Thr Ser Lys Ser Lys Tyr Gly Ala Ala Arg Asp Asp Ala Gly Arg Val
465 470 475 480
Gly Asp Asp Lys Asp Ser Asp Asp Asp Val Asn Thr Ser Arg Lys Ala
485 490 495
Lys Val Val Ser Thr Ser Thr Thr Thr Lys Ala Ser Ser Thr His Cys
500 505 510
Tyr Asn Ser Cys Ala Ser Val Asn Thr Gly Lys Val Val Ser Ser Ser
515 520 525
Asn Val Gly Ser Asp Tyr Val Gly Arg Lys Ser Ser Asn Lys Ser His
530 535 540
Asp Val Val Met Asp Ser Ser Ser Lys His Arg Asp Ser Cys Lys Lys
545 550 555 560
Gly Asp Cys Ala Asn Thr Ser Ser Lys Trp His Ser Thr Asp Val Gly
565 570 575
Lys Gly Asp Asn Cys Ser Gly Met Ser Ser Asp Asp Asp Cys Asp Ser
580 585 590
Gly Ser Thr Ser Ser Arg Val Ala His Ser Met Thr Lys His Thr Lys
595 600 605
Thr Lys Lys Thr His Arg Val
610 615
<210> 11
<211> 872
<212> PRT
<213> 甘蓝型油菜(Brassica napus)
<400> 11
Met Gly Val Asp Gly Asp Trp Ala Ala Val Arg Asp Ala Cys Lys Ser
1 5 10 15
Asn Ser Ser Ser Ser Ser Thr His Thr Asn Gly Asn Gly Thr Lys Ala
20 25 30
Lys Arg Ser Thr Asp Arg Ala Arg Ala Asp His Lys Tyr Val Val Ser
35 40 45
Asp Ser Cys Gly Thr Asn Asp Asn Thr Ser Val Gly Asp Thr Ala Lys
50 55 60
Thr Trp Tyr Met Asn Val Arg Asp Tyr Ala Thr Lys Thr Ala Ser Asn
65 70 75 80
Thr Val Ala Thr Gly Gly Lys Thr Ala Ala Val Val Met Tyr Asn Tyr
85 90 95
Arg Trp Gly Lys Val Ala Ala Ser Arg Val Met Ala Cys His Asn Val
100 105 110
Gly Trp Thr Asp Thr Gly Thr Cys Ser Lys Arg Ala Ser Trp Lys Thr
115 120 125
Lys Arg Val Val Thr Val Lys Asp Ser Gly Thr Cys Val Thr Asn Cys
130 135 140
Val Cys Val Asp Ala His Arg Ala Gly Asn Tyr Ser Tyr Cys Val Val
145 150 155 160
Val Arg Met Ala Val Val Arg Ala Thr Ala Thr Gly Ser Lys Thr Ala
165 170 175
Gly Asp Asn Ser Thr Tyr Arg Asn Ser Asp His Asp Val Cys Tyr Val
180 185 190
His Asp Arg Lys Val Val Gly Lys Asp Ala Asp Val Ser Lys Arg Asp
195 200 205
Val Arg Tyr Ala Val Arg Lys Asn Gly Val Ser Arg Asp Tyr Thr Ser
210 215 220
His Met Ala Arg Asp Lys Arg Ala Val Gly His Ser His Gly Asp Val
225 230 235 240
Ser Cys Ala Ala Thr Tyr His Arg Lys Ser Ser His Gly Arg Ala Tyr
245 250 255
Met Lys Gly Ala Arg Met Ser Lys Asn Asp Arg Met Thr Lys Met Arg
260 265 270
Ser Asn Gly Ala Ser Lys Ser Lys Met Val Asp His Tyr Lys Lys Asp
275 280 285
Arg Thr Ser Arg Val Ser Asn Arg Gly Ser Val Arg Asp Met Asp Ala
290 295 300
Ser Asn Asp Val Val Lys Ala Thr Gly Ser Ser Gly Lys Thr Lys Gly
305 310 315 320
Ser Lys Val Ala Val Lys Arg Ser Gly Gly Asn Val Val Ala Thr Ser
325 330 335
Gly Gly Asp Met Val Asp Val Cys Asp Ala Asn Val Ser Arg Met Arg
340 345 350
Met Gly Arg Thr Gly Arg Lys Asn Asn Gly Arg Val Val Val Ala Cys
355 360 365
Gly Ser Lys Asn Ser Tyr Met Arg Lys Lys Ala Asn Gly Ala Lys Lys
370 375 380
His Met Arg Asn Gly Gly Met Asn Ser Asn His Ser Arg Met His Val
385 390 395 400
Tyr Lys Val His Val Lys Ser Arg Gly Lys Lys Asp Ala Thr Thr Ala
405 410 415
Lys Lys Lys Thr Met Asp Met Ala Lys Tyr Lys Asn Lys Trp Arg Val
420 425 430
Ser Ala His Thr Ser Lys Val His Lys Val Met His Ser Arg Thr Ser
435 440 445
Asp Ala Met His Thr Thr Thr Ser Lys Ser Lys Tyr Gly Ala Ala Arg
450 455 460
Asp Asp Ala Gly Arg Val Gly Asp Asp Lys Asp Ser Ser Asp Asp Asp
465 470 475 480
Val Asn Thr Ser Arg Lys Ala Lys Ser Thr Ser Thr Thr Thr Lys Asp
485 490 495
Ala Ser Ser Thr His Cys Tyr Ser Ser Cys Ala Ser Val Asp Thr Gly
500 505 510
Lys Val Val Val Ser Ser Ser Asn Val Gly Ser Asp Cys Val Gly Arg
515 520 525
Lys Ser Ser Asn Lys Ser His Thr Asp Val Val Asp Ser Ser Ser Lys
530 535 540
His Arg Asp Asn Ser Cys Lys Lys Gly Asp Cys Ala Asn Thr Ser Ser
545 550 555 560
Lys Arg His Ser Thr Asp Val Gly Lys Gly Asp Asn Cys Ala Gly Met
565 570 575
Ser Ser Asp Asp Asp Cys Asp Ser Arg Thr Asn Lys Ser Gly Val Val
580 585 590
Asp Ser Val Tyr Asp Gly Val Ala Tyr Ala Asn Arg Asp Asp Thr Ser
595 600 605
Thr Asn Ala Ser Thr Lys Lys Val His Ala Ser Thr Ala Asn Arg Thr
610 615 620
Lys Asp Ala Asn Thr Ser Ala Val Ser Met Trp Arg Thr Ala Asn Thr
625 630 635 640
Asn Ala Ser Ser Ser Ala Ser Lys Asp Trp Arg Val Ser Ser Gly Lys
645 650 655
Ser Thr Arg Arg Lys Lys Arg Arg Arg Gly Asp Cys Ser Ser Ala Val
660 665 670
Lys Asn Asn Gly Ala Lys Thr Asp His Arg Ser Arg Ser Arg Ser Val
675 680 685
Lys Asn Arg Gly Lys Lys Lys Arg Ala Asp Asn Asn Ala Arg Ala Ala
690 695 700
Val Ser Ser Ser Met Ser Val Asp Asn Val Asp Thr Ser Asp Ser Asp
705 710 715 720
Ser Asp Asp Gly Thr Met Thr Ala Asn Thr Ala Cys Ala Lys Val Asp
725 730 735
Met Met Ala Val Tyr Arg Arg Ser Ser Ser Ala Arg Arg Asp Val Ala
740 745 750
Ala Ser Ser Ser Tyr Ser Ser Gly Lys Thr Asn Ser Arg Ser Asp Ser
755 760 765
Asp Lys Ser Ser Ser Arg Thr Thr Thr Asn Asn Ser Asn Lys Asp Ala
770 775 780
Val Ala Thr Gly Asp Val Ala Ser Thr Asp Ser Arg Lys Arg Lys Ser
785 790 795 800
Cys Asn Ser Ala Asn Val Val Asn Asn Lys Ala His Ala Ala Thr Lys
805 810 815
Ser His Gly Arg Ser Asn Ala Gly Ala Ser Tyr Lys Asp Asp Asp Asp
820 825 830
Asp Ala Tyr Ala Thr Asp Asp Ala Met Ala His Ala Thr Ser Lys Arg
835 840 845
Ser Thr Lys Thr Lys Asp Ala Ser Val Lys His Gly Asn Arg Asn Asp
850 855 860
Gly Lys Asp Gly Ser Asp Gly Trp
865 870
<210> 12
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
tgtcccagga ttagaatgat taggc 25
<210> 13
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agccctcttc tttcgatcca tcaac 25
<210> 14
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
ttaatccaaa ctactgcagc ctgac 25
<210> 15
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
tgcaggagat tttctccaac ga 22
<210> 16
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agccttcgta atctcggtgt tca 23
<210> 17
<211> 26
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
ggtaccatgg gacccgagtt tccgat 26
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
ctgaaaaatg gcatccaaca ag 22
Claims (9)
1.基因BnaFANCM在油菜雄性不育育种中的应用，所述应用为对BnaFANCM基因编辑；所述编辑类型是T碱基的插入导致基因移码突变。
2.一种基因编辑载体pKSE401-BnaFANCM-CRISPR，所述基因编辑载体包括BnaFANCM基因序列和BnaFANCM基因编辑的CRISPR/Cas9系统序列元件组，所述序列元件组包括U6-26p-Target1-gRNA、U6-26p-Target2-gRNA和根据密码子优化后的Cas9基因以及抗性基因Kana；
其中，Target1的核苷酸序列如SEQ.ID.NO.1所示；Target2的核苷酸序列如SEQ.ID.NO.2所示；sgRNA的核苷酸序列如SEQ.ID.NO.3所示。
3.一种基因工程菌，用于BnaFANCM基因定点突变，所述基因工程菌包括权利要求2所述的基因编辑载体pKSE401-BnaFANCM-CRISPR，所述基因工程菌的宿主菌为农杆菌GV3101。
4.一种获得油菜雄性不育突变体的方法，其特征在于，所述方法利用CRISPR/Cas9系统对甘蓝型油菜BnaFANCM基因进行编辑；所述编辑类型是T碱基的插入导致基因移码突变。
5.根据权利要求4所述的方法，其特征在于，所述方法包括：
（1）针对BnaFANCM基因序列设计并筛选sgRNA靶点Target1和Target2，所述Target1的核苷酸序列如SEQ.ID.NO.1所示，所述Target2的核苷酸序列如SEQ.ID.NO.2所示，并合成构建载体所需的引物；
（2）构建双靶点CRISPR/Cas9基因编辑载体pKSE401-BnaFANCM-CRISPR；
（3）抽提质粒，转化宿主菌，获得含有基因编辑表达载体pKSE401-BnaFANCM-CRISPR的基因工程菌；
（4）利用农杆菌介导转化的方式将pKSE401-BnaFANCM-CRISPR载体转至受体材料中，获得转基因植株。
6.根据权利要求5所述的方法，其特征在于，步骤（1）中所述引物序列如下：
DT1-BsF(SEQ.ID.NO.4)：
ATATATGGTCTCGATTGAGTCCACACGAATTACTTAGTT
DT2-BsR(SEQ.ID.NO.5)：
ATTATTGGTCTCGAAACGCTGAACTTTTTGCGACTGCAA
DT1-F0(SEQ.ID.NO.6)：
TGAGTCCACACGAATTACTTAGTTTTAGAGCTAGAAATAGC
DT2-R0(SEQ.ID.NO.7)：
AACGCTGAACTTTTTGCGACTGCAATCTCTTAGTCGACTCTAC。
7.根据权利要求5所述的方法，其特征在于，所述BnaFANCM基因包括BnaFANCM-C05和BnaFANCM-A05，所述sgRNA的核苷酸序列如SEQ.ID.NO.3所示，所述Target1、Target2为基因BnaFANCM-C05和BnaFANCM-A05的共同靶点。
8.根据权利要求5所述的方法，其特征在于，步骤（3）中所述宿主菌为农杆菌GV3101。
9.根据权利要求5所述的方法，其特征在于，步骤（4）中所述受体材料为Y127。
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| FR2980212A1 (fr) * | 2011-09-16 | 2013-03-22 | Agronomique Inst Nat Rech | Augmentation de la recombinaison meiotique chez les plantes par inhibition de la proteine fancm |
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