CN108913714A - 一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法 - Google Patents
一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法 Download PDFInfo
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Abstract
本发明公开了一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，属于水稻育种技术领域，涉及应用水稻甜菜醛脱氢酶基因BADH2创制香稻的方法。在香稻中，BADH2基因功能缺失，造成化学物质2‑乙酰‑1‑吡咯啉(2‑AP)积累而产生香味，所述CRISPR/Cas9系统采用的sgRNA引导序列为SEQ ID No.2或SEQ ID No.3所示的任一序列。使用本发明方法，能够成功敲除BADH2基因，快速创制香稻。方法操作简单，省时省力，缩短香稻育种年限。
Description
技术领域
本发明涉及水稻育种技术领域，具体是一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法。
背景技术
香味是水稻的重要品质指标之一，主要受第8染色体上的一对隐性基因(BADH2)控制，在香稻中，BADH2基因功能缺失，造成化学物质2-乙酰-1-吡咯啉(2-AP)积累而产生香味。目前，水稻香味改良主要通过杂交育种和基因工程育种。在杂交选育过程中，需要进行大量的反复多代回交或者自交，才能选育出综合性状优良且纯和的香稻材料，育种周期长，工作量大。基因工程育种开辟了水稻育种的新途径，突破物种间的界限，缩短育种进程。RNAi技术和基因组编辑技术是重要的基因工程育种方法。RNAi技术利用体外合成的短双链RNA抑制细胞内BADH2基因的表达，快速获得香味水稻[Niu X L,Tang W,Huang W Z,etal.RANi-directed downregulation of OsBADH2 results in aroma(2-acetyl-1-pyrroline)production in rice(Oryza sativa L.).BMC Plant Biol,2008,8:10；Chen Ml,Wei X J,Shao G N,Tang S Q,et al.Fragrance of the rice grain achieved viaartificial microRNA-induced down-regulation of OsBADH2.Plant Breeding,2012,131:584-590.]，然而RNAi主要靶标成熟的RNA，是一种基因下调方法，无法完全去除基因功能。基因编辑技术主要包括锌脂核酸酶(ZFN)、类转录激活因子效应物核酸酶(TALEN)及CRISPR系统[Gaj,T.,et al.ZFN,TALEN,and CRISPR/Cas-based methods for genomeengineering.Trends Biotechnol,2013,31(7):397-405]。CRISPR/Cas9是目前使用最为广泛且最为高效的基因组编辑系统，与ZFN和TALEN技术相比，操作更简单，成本更低。CRISPR/Cas9系统由两部分组成，即Cas9蛋白(一种核酸酶)和sgRNA(引导Cas9蛋白至靶位点的向导序列)，欲敲除某个基因时，只需设计sgRNA上针对靶基因处特异的20bp碱基序列即可，该系统已成功应用于水稻基因组编辑。
发明内容
本发明目的在于克服现有技术的不足，适应现实发展，提供一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法。
本发明是通过如下技术方案实现的：
一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，利用CRISPR/Cas9系统对普通水稻中的BADH2基因进行定点敲除，所述BADH2基因DNA序列如SEQ ID No.1所示，或为与SEQ ID No.1所示序列存在90％以上序列相似性的序列。
进一步，所述CRISPR/Cas9系统采用的sgRNA引导序列为SEQ ID No.2或SEQ IDNo.3所示的任一序列。
进一步，所述BADH2基因转化子如SEQ ID No.2或SEQ ID No.3所示序列位点的突变，采用如下引物检测：
U3/U6-F：5’-AGCCTTCCAGTCTAAATGACAGTAA-3’
U3/U6-R：5’-GCTGCTAGGTACAATTTGTGAGACA-3’
上述的利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，包括如下步骤：
(1)设计基于BADH2基因的sgRNA序列；
(2)设计并合成sgRNA引物；
(3)引物退火成双链；
(4)连接；
(5)转化大肠杆菌；
(6)提取质粒；
(7)凝胶电泳及测序；
(8)转化农杆菌；
(9)转化愈伤组织；
(10)对转化子中BADH2基因的敲除效果进行检测；
(11)突变判定；
(12)香味物质2-AP相对定量分析；
(13)观察突变体农艺性状。
进一步，所述步骤(10)对转化子中BADH2基因的敲除效果进行检测：提取T0代叶片DNA，利用引物U3/U6-F(AGCCTTCCAGTCTAAATGACAGTAA)和U3/U6-R(GCTGCTAGGTACAATTTGTGAGACA)对转化子BADH2基因DNA中SEQ ID No.2和SEQ ID No.3所在位点序列进行PCR扩增，将获得的PCR产物目标片段克隆至pMD-18-T载体，转化大肠杆菌，挑取单克隆测序，测10个单克隆以上，保证获得5个以上阳性克隆。
更进一步，PCR扩增程序为：95℃预变性3min；94℃变性30s、60℃退火30s、72℃延伸1min，共32个循环；最后72℃延伸2min。
本发明的优点和积极效果：
1、使用本发明，能够成功敲除BADH2基因，快速创制香稻。
2、方法操作简单，省时省力，缩短香稻育种年限。
附图说明
图1：通过CRISPR/Cas9定点敲除获得的BADH2基因突变植株的靶序列突变情况；其中，WT-U6表示SEQ ID No.2对应的野生型BADH2基因的位点附近的序列(展示了正义链)，WT-U3表示SEQ ID No.3对应的野生型BADH2基因的位点附近的序列(展示了正义链)，E1-1，E2-1，E2-2，E3-1，E3-2，E4-3和E7-1为测序时不同样品的编号并以此代表不同的突变类型。
图2：为图1中展示序列所对应的测序峰图，为对突变体中靶序列测序所获得，(+)表示测序时测到的是靶序列的正义链，(-)表示测序时测到的是靶序列的负义链，中括号内数字范围表示测序峰图中此数字范围内的碱基对应于图1中的对应序列。
图3：野生型和突变体中的香味物质2-AP含量，采集时间5.5min处为2-AP峰。
具体实施方式
下面对本发明的具体实施例做详细说明。
一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其包括如下步骤：
(1)设计基于BADH2基因的sgRNA序列
利用在线公开软件CRISPR-P(http://cbi.hzau.edu.cn/crispr/)设计sgRNA引导序列，选择分值较高且位于BADH2基因序列合适位置的引导序列，本实施例所选的sgRNA引导序列为SEQ ID No.2和SEQ ID No.3所示的序列。
(2)设计并合成sgRNA引物
根据在线软件设计的sgRNA原始序列及载体构建要求，对于如SEQ ID No.2所示的sgRNA序列，设计的正反引物为sgU6-F
(cttgACTAGAGACGCTTGATTGT)和sgU6-R
(aaacACAATCAAGCGTCTCTAGT)；对于如SEQ ID No.3所示的sgRNA序列，设计的正反引物为sgU3-F(ggcaAGAGCCTATCGGTGTAGTT)和sgU3-R(aaacAACTACACCGATAGGCTCT)。将设计好的引物交由金斯瑞生物科技有限公司合成。
(3)引物退火成双链
将合成的正反引物分别用1倍TE缓冲液溶解成100pmol/μl，各取10μL混合，加入0.4μL 5M NaCl，充分混匀，于PCR仪中95℃200s，取消低温保存设置，30min后取出并稀释10倍成5pmol/μl接头。
(4)连接
CRISPR/Cas9载体利用发明人所在实验室构建的以U6和U3为启动子的载体即pCUbi1390Cas9-U6和pCUbi1390Cas9-U3(参见中国专利201710531581.5)。CRISPR/Cas9载体(经Aar I酶切)，1μL对应接头，1μL T4连接酶，1μL T4连接酶缓冲液，5μL ddH2O，16℃连接2h。
(5)转化大肠杆菌
①将10μL连接产物接入100μL大肠杆菌感受态，混匀，冰浴30min，42℃水浴50s，冰浴3min。
②接种于LB液体培养基，混匀，37℃振荡培养1h。
③转接于LB固体培养基，37℃倒置培养12～16h。
④挑取菌落至LB液体培养基(含卡那霉素)中，37℃振荡培养12～16h。转入无菌离心管中，3500rpm离心5min，去上清液，收集菌体以用于质粒提取。
(6)提取质粒
按照生产商(成都福际生物技术有限公司)提供的质粒小量提取试剂盒(货号DE-01021)中的说明方法(Plasmid Mini Kit Instruction Manual)提取。
(7)凝胶电泳及测序
质粒样品在通过琼脂糖凝胶电泳鉴定浓度后，送测序服务公司进行测序，测序引物如下：
OsU6-F(TTGAGCGATTACAGGCGAAAGTG)(检测克隆正确性，启动子为OsU6时用)。
OsU3-F(GGCATGCATGGATCTTGGAGGAAT)(检测克隆正确性，启动子为OsU3时用)。
35S-F(TGACGCACAATCCCACTATCCTTC)(35S正向引物，检测载体完整性)。
Cas9-R-1(TCGAGCCTGCGGGACTTAGAG)(cas9 5′端引物，检测载体完整性)。
C126(TCGTGAAGAAGACCGAGGTT)(cas9 3′端引物，检测载体完整性)。
(8)转化农杆菌
在质粒序列鉴定正确后，将质粒转化农杆菌，具体步骤如下：
①取-80℃保存的农杆菌感受态EHA105于冰上融化。
②将10μL质粒加入100μL感受态中，混匀，依次于冰上静置10min、液氮5min、水浴37℃5min、冰浴5min。
③加入900μLYEP液体培养基，28℃、150rpm震荡避光培养2.5h。
④将上述1ml液体涂布于YEP固体培养基(含卡那霉素、利福平、潮霉素)，28℃倒置培养至形成单菌落。
(9)转化愈伤组织
通过农杆菌转化法进行转化，具体步骤为：
①将无菌的去壳的水稻种子放置在N6D固体培养基上30℃光照培养5d。
②农杆菌悬浮AAM液体培养基，菌液浓度大约为0.1OD600。
③将经过预培养的种子浸入农杆菌悬浮液，摇晃1.5min，用无菌滤纸吸除过量菌液，转移到垫有用AAM培养基浸湿的无菌滤纸的2N6-AS培养基上，25℃避光培养3d。
④种子用无菌水冲洗5次，接种于N6D培养基(含潮霉素和特美汀)于30℃培养。
⑤愈伤组织长出后转移至RE-III培养基(含潮霉素和特美汀)于30℃培养。
⑥幼苗长出后转移到HF培养基(含潮霉素和特美汀)上长根。
(10)对转化子中BADH2基因的敲除效果进行检测
提取T0代叶片DNA，利用引物U3/U6-F
(AGCCTTCCAGTCTAAATGACAGTAA)和U3/U6-R
(GCTGCTAGGTACAATTTGTGAGACA)对转化子BADH2基因DNA中SEQ ID No.2和SEQ IDNo.3所在位点序列进行PCR扩增；
PCR反应体系
PCR扩增程序为：95℃预变性3min；94℃变性30s、60℃退火30s、72℃延伸1min，共32个循环；最后72℃延伸2min。
将获得的PCR产物目标片段克隆至pMD-18-T载体(厂商Takara，货号6011)，转化大肠杆菌，挑取单克隆测序，测10个单克隆以上，保证获得5个以上阳性克隆。
(11)突变判定
将测序结果与野生型序列进行比对、分析，结果存在四种可能，无突变(野生型)，纯合突变、杂合突变和双等位基因突变。
本实施例中，获得的转化子T0代发生突变的株系其基因组DNA序列突变情况如表1及附图1和附图2所示，进而导致蛋白质序列发生变化如表2所示。
表1 BADH2基因靶位点突变情况
表2转化子中基因突变对蛋白质序列及功能的影响
| 核酸突变类型 | 氨基酸序列的改变 | 说明 | 可能后果 |
| E1-1 | T97_C99delinsLD | 保守序列的缺失/插入 | 功能失活 |
| E2-1 | V155fs | 移码 | 功能失活 |
| E2-2 | R79_W161delinsAIAAK | 保守序列的缺失/插入 | 功能失活 |
| E3-1 | C100fs | 移码 | 功能失活 |
| E3-2 | L95fs | 移码 | 功能失活 |
| E4-3 | C100fs | 移码 | 功能失活 |
| E7-1 | G153_V155delinsV | 保守序列的缺失/插入 | 功能失活 |
(12)突变体香味物质2-AP相对定量分析
固相微萃取(SPME)技术收集挥发物。称取2g糙米粉装入4ml样品瓶中，置于SPME专用采样台上90℃预热30min，然后用含有65μm PDMS/DVB萃取头(SPME；Supelco)的SPME针头刺穿瓶垫，暴露出SPME纤维头吸附顶部空间的挥发物，90℃持续收集30min。收回纤维头后收回针头，立即在色谱进样口250℃、5min解吸附，不分流模式进样，进样口内置内径0.75mm玻璃衬管(SGE Ltd.)。色谱柱为HP-5MS毛细管柱(30m×0.25mm,0.25mm厚涂层,Agilent)，载气为99.999％氮气，流速1.2ml/min。色谱升温程序为：40℃保留3min，以20℃/min升温至230℃，230℃保留2min。质谱条件为：连接线温度230℃，离子源温度230℃，能量70eV，检测器150℃。
质谱定性分析采用全扫描模式(fullscan)收集信号，利用NIST谱库查询及其与标样比较的方法确认HPL挥发产物成分。定量分析采用选择例子检测(SIM)模式。选用质荷比(m/z)为69，83和111的离子碎片对2-乙酰-1-吡咯啉进行定量，如附图3。
(13)观察突变体农艺性状
本实施例中，突变体的农艺性状与野生型相比，除E2株系的结实率降低外，其它性状无显著差异。
上述实施例仅是本发明的较优实施方式，凡是依据本发明的技术实质对以上实施例所做的任何简单修饰、修改及替代变化，均属于本发明技术方案的范围内。
序列表
<110> 江西省超级水稻研究发展中心
<120> 一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法
<130> 1110000000
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5860
<212> DNA
<213> （水稻）Oryza sativa
<400> 1
atggccacgg cgatcccgca gcggcagctc ttcgtcgccg gcgagtggcg cgcccccgcg 60
ctcggccgcc gcctccccgt cgtcaacccc gccaccgagt cccccatcgg taccctcctc 120
ttcaccctct ccaccctctg cttctgcctc tgattagcct ttttgttgtt gttgttgttg 180
ctgctgtttt ttgcgtgtcg gtgcgcaggc gagatcccgg cgggcacggc ggaggacgtg 240
gacgcggcgg tggcggcggc gcgggaggcg ctgaagagga accggggccg cgactgggcg 300
cgcgcgccgg gcgccgtccg ggccaagtac ctccgcgcaa tcgcggccaa ggtagggtgg 360
tgactacccc cacccccccc ccccccccca acgcgacccg cgtgcgtgtg ttccgtacag 420
ggggaggagc tccgcgtggc tctccagtag gtttttgagc cccaaatcga tcgatatgct 480
ctagttttaa gtttgctgct taaattcctc aagggtttag tttgcaacca aatccttatt 540
ttagcttcgg tataagcccc ccatatgatg tgcgtgcgtc ggcatcggaa gtgcgtatcc 600
tctgttctgg actaggaatt ggccataggt tgatcgacag ttcgagtatt ctgcttctgt 660
ttggaataag ttggaagcat ggctgattgt gtatctggat gctgtttttg tggtgattcg 720
tttcaagctc ttgttaattg atgggttcaa gcggagaggg tgcgcaacaa caagtgtata 780
tggctcacgg ccatgggtgt gcacatttga ttggtgcgca acaacaagtg tatattgttt 840
gtgtgcttcg ttagttggca ggtcctagtc actaaatcac tattggattg gtactagtta 900
cttttgtgcc ttgacgatgg gactggatta ctagcctttt ggttgccttt gtggtattcc 960
gttgttatgg gcctgttgat ggatggatcc ctttaatttc tagtgccaaa tgcatgctag 1020
atttctcaca gtttttctct tcaggttata tttctcgtat ttccttttcc taaaggattg 1080
ctttttcatg tattttctgg catatatagg ttattattat tattattctc cagaacaaga 1140
ttacccatat tatggatcac tagtgtacac ttttttggat gaaaaaccta cttactgaaa 1200
gtaaaacagt gaccagtgca cactttactt gaactgtcaa accatcaatt ttctagcaaa 1260
gcaggggatg ctagccttcc agtctaaatg acagtaaact actatacttt tgtccgtagg 1320
tttggaaata tgctaatttc tatcataaaa attttcatgg catatgcgag cattttatga 1380
tcaccttttc cctttttctt cagataatcg agaggaaatc tgagctggct agactagaga 1440
cgcttgattg tgggaagcct cttgatgaag cagcatggga catggtatgt ggccagttat 1500
ccactgtatg aatatgtagt tgcctacaca gcaatctttc ctgaacatga atcctgatgt 1560
atgatattcc atttgtcagg acgatgttgc tggatgcttt gagtactttg cagatcttgc 1620
agaatccttg gacaaaaggc aaaatgcacc tgtctctctt ccaatggaaa actttaaatg 1680
ctatcttcgg aaagagccta tcggtgtagt tgggttgatc acaccttggt atttcacatt 1740
tttctctcat cctgcgctta tatttattta tgacccaagc atggtactaa atagtactag 1800
taacatgcat atactgaatg agtttacaac tttacatgat ttttttgaac tatgaaagtt 1860
gaagacattt gagattttat tcctcttctc ttgtgcaaac atattattgt ctcacaaatt 1920
gtacctagca gctactctct ccgtttcata ttataagtcg tttgactttt ttcctagtca 1980
aaatgtgtta agtttgacca agtttataga aaaatttagc aacatctaaa atatcaaagt 2040
catgttttag tgttttttca ggctctcatg taagcaattt tgatgtgccc tctcctttct 2100
tcttaatata atgatacaca gctcttgtgt attcaaagga aaatatatat atatataatg 2160
atacacacct ctcctccgtg ttaatgcagc tcatttgttc tgtcccggtt caaatatcta 2220
tttttctcat atgttgtcag catgattcac ttaatttagt atatagaaga tgccattatt 2280
tatgtctgga atcttactgc agaagggaaa acaattgata acggaattga ttgcattcta 2340
atttgttgtt tctttgttat gttcttatcg acaattacaa atttgattct gagaatcatg 2400
ttcgggatgt gtatttctac tgcaggaact atcctctcct gatggcaaca tggaaggtag 2460
ctcctgccct ggctgctggc tgtacagctg tactaaaacc atctgaattg gcttccgtgt 2520
aagtttaaca tgttaacttg ttaatgtcat acccatgcta gttgcaatga catttgattt 2580
taaaatgttg tggcatgtcc atgctgcaag caatgtaatt tgaaatctct ctctatcatt 2640
aattaccagg acttgtttgg agcttgctga tgtgtgtaaa gaggttggtc ttccttcagg 2700
tgtgctaaac atagtgactg gattaggttc tgaagccggt gctcctttgt catcacaccc 2760
tggtgtagac aaggtacagc tattcctcct gtaatcatgt ataccccatc aatggaaatg 2820
atattcctct caatacatgg tttatgtttt ctgttaggtt gcatttactg ggagttatga 2880
aactggtaaa aagattatgg cttcagctgc tcctatggtt aaggtttgtt tccaaatttc 2940
tgtggatatt ttttgttctc tttctactaa ctctctatta tcaattctca atgttgtcct 3000
tttcttttaa ctcctttact ttttagaatt gtgatcaaga cactttgagc atcattctag 3060
tagccagttc tatcctgttt cttacctttt tatggttcgt cttttcttga cagcctgttt 3120
cactggaact tggtggaaaa agtcctatag tggtgtttga tgatgttgat gttgaaaaag 3180
gtacatgcca cttgctatga ttaactaatt ctgaagtgcg ggactttgta aagcacttaa 3240
ctgagctgga tgctagaccc ccaaaagccc tttttggtgt cttgggcttg ttgcagaaat 3300
actggtccca gacgagcagg atgcaagaaa attaactact tttgccactg attagtattt 3360
cttagaagtt acacctcaag gattagcaat actttcttaa aatgtgctat tgattaaaaa 3420
gatgtcctgt attattttga gcagatcttg tactggttga tcggcttgca tgaaaatatt 3480
gttgaggatt ataatgccat gccaactgag taaagaaaag agttgtaaaa tatgttatgc 3540
aacatgaata tatatgtgat ttcatttttc ctttttcttt tcgtggcaag gaaggcagtt 3600
aggaaggact gatgtgaaaa gcacaagtac tattcttagt tctggaaaac tgtgttcttt 3660
attttcctaa ctacaattca ccttgattag tcagtaactt gatattggca attctagctg 3720
attatgaatt ctgtttatat ttcactaatt ttgaatcttt aattacattt tatggttgaa 3780
atttaacgtt ttgtctggtt atggactctg tttgtattca ctcaatttgg atcttccatt 3840
agatttcatt gttggtcctt cttcttgtac agctgttgag tggactctct ttggttgctt 3900
ttggaccaat ggccagattt gcagtgcaac atcgcgtctt attcttcatg taagcattga 3960
atatatccgt caatcataat ctattgttgt acttgatttt ttttctgatc aactcctgag 4020
ttcagattat tatatgatgc cattactatt gcacagagcg aataaaattg tatttatgca 4080
cagcatgtat tttgagtaat atatgcattg cctattattt aatatataga ttgtagcact 4140
taattttgtg tccatgtctc tatgatgttt attactttat tattgccggc atgaagcaac 4200
tttgaactct atgttgatct tgaactaaaa ttgaaattaa ttggcttatt gctattaatg 4260
atatagcttt cagcttcttg ctcctgacca tgaaagtttt gcagaaaaaa atcgctaaag 4320
aatttcaaga aaggatggtt gcatgggcca aaaatattaa ggtgtcagat ccacttgaag 4380
agggttgcag gcttgggccc gttgttagtg aaggacaggt accacatgta aactttttct 4440
aaattcaaaa aagaaatgcc actgatcaat ggtaggtcct tccaagcctt attgctggat 4500
tgttgcactg ttttgtcaat tttgtgtaat atagttctga atgaattagt cggtgtatgc 4560
tcttgctagt tgctagtatg tggtacaggg tcttcctact ttgagcaaat tcgtgttaaa 4620
atgcattgat gaaaaggcca cctttccgta ggtttatctt gtcataattt aaaccccaat 4680
aaaattttaa ttttttgttt tgaccccatg gcactttaat gaaatcactt agccatgagc 4740
ttttgtatat attttcaaag caccagaatg tttagatggt ttgttggaaa tcttacacat 4800
cctattgcct tgtgtcagta tgagaagatt aagcaatttg tatctaccgc caaaagccaa 4860
ggtgctacca ttctgactgg tggggttaga cccaaggtaa taatctacta cacggttgta 4920
tatataggta cccacatatc attatgaagt agaaataatc ttgtatgttt ttgtcagcat 4980
ctggagaaag gtttctatat tgaacccaca atcattactg atgtcgatac atcaatgcaa 5040
atttggaggg aagaagtttt tggtccagtg ctctgtgtga aagaatttag cactgaagaa 5100
gaagccattg aattggccaa cgatactcag tgagtttttt ttttaataca gttcattgtc 5160
ctgttcaatc ttgcagcata tgtatatact ctgtggcata tgaacttatt ctgctactac 5220
tacttttgat agttatggtc tggctggtgc tgtgctttcc ggtgaccgcg agcgatgcca 5280
gagattaact gaggtatatc caagtgaagg gggttggcat tgtttgattc atatgacatg 5340
gttgcatcaa gctgatattc aagaatctca tttattactt gcattctatg catctccagt 5400
tcttccctgg actccggtca atgttaatat agtttgtttg ctagtagtat gctactccaa 5460
ttaagttgct cttcacttcc acatcatctg atccatgact ttatatttga cccctttttt 5520
ttgcaaaaga aagggaaata cttaacgaaa atttcctact gcaggagatc gatgccggaa 5580
ttatctgggt gaactgctcg caaccctgct tctgccaagc tccatggggc gggaacaagc 5640
gcagcggctt tggacgcgag ctcggagaag ggtgggtagc acacaacaat ctcactttaa 5700
aacaccattt cgatcgtctg atgatctcga cctgacatca tgcctttggt attttcattc 5760
acttttcagg ggcattgaca actacctaag cgtcaagcaa gtgacggagt acgcctccga 5820
tgagccgtgg ggatggtaca aatccccttc caagctgtaa 5860
<210> 2
<211> 20
<212> DNA
<213> designed
<400> 2
gactagagac gcttgattgt 20
<210> 3
<211> 20
<212> DNA
<213> designed
<400> 3
aagagcctat cggtgtagtt 20
Claims (6)
1.一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，利用CRISPR/Cas9系统对普通水稻中的BADH2基因进行定点敲除，所述BADH2基因DNA序列如SEQID No.1所示，或为与SEQ ID No.1所示序列存在90％以上序列相似性的序列。
2.如权利要求1所述的一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，所述CRISPR/Cas9系统采用的sgRNA引导序列为SEQ ID No.2或SEQ ID No.3所示的任一序列。
3.如权利要求1所述的一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，所述BADH2基因转化子如SEQ ID No.2或SEQ ID No.3所示序列位点的突变，采用如下引物检测：
U3/U6-F：5’-AGCCTTCCAGTCTAAATGACAGTAA-3’
U3/U6-R：5’-GCTGCTAGGTACAATTTGTGAGACA-3’
4.如权利要求1所述的一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，包括如下步骤：
(1)设计基于BADH2基因的sgRNA序列；
(2)设计并合成sgRNA引物；
(3)引物退火成双链；
(4)连接；
(5)转化大肠杆菌；
(6)提取质粒；
(7)凝胶电泳及测序；
(8)转化农杆菌；
(9)转化愈伤组织；
(10)对转化子中BADH2基因的敲除效果进行检测；
(11)突变判定；
(12)香味物质2-AP相对定量分析；
(13)观察突变体农艺性状。
5.如权利要求4所述的一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，所述步骤(10)对转化子中BADH2基因的敲除效果进行检测：提取T0代叶片DNA，利用引物U3/U6-F(AGCCTTCCAGTCTAAATGACAGTAA)和U3/U6-R(GCTGCTAGGTACAATTTGTGAGACA)对转化子BADH2基因DNA中SEQ ID No.2和SEQ ID No.3所在位点序列进行PCR扩增，将获得的PCR产物目标片段克隆至pMD-18-T载体，转化大肠杆菌，挑取单克隆测序，测10个单克隆以上，保证获得5个以上阳性克隆。
6.如权利要求5所述的一种利用CRISPR/Cas9系统敲除BADH2基因创制香稻的方法，其特征在于，PCR扩增程序为：95℃预变性3min；94℃变性30s、60℃退火30s、72℃延伸1min，共32个循环；最后72℃延伸2min。
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