CN107312793A - Cas9介导的番茄基因编辑载体及其应用 - Google Patents
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Abstract
本发明属于基因工程技术领域，具体公开了一种Cas9介导的番茄基因编辑载体及其应用。发明通过构建CRISPR/Cas9番茄植物表达载体，将Cas9基因导入番茄M82中，对番茄自身DFD基因进行基因编辑。提供一种创建耐储藏转基因番茄的方法，以此得到耐储藏的番茄新品种，提高番茄果实耐储藏性，同时不影响植株的生长与其他品质，可以弥补前人研究方案的不足之处。
Description
技术领域
本发明属于基因工程技术领域，具体公开了一种Cas9介导的番茄基因编辑载体及其应用。
背景技术
储运时间及货架期的限制是园艺产品所面临的主要问题，延长蔬菜和水果储运时间将给供应链上各个环节从农民到批发商到零售商及最终消费者带来巨大的利益，特别是生产及流通渠道效率并不高的中国生鲜产品现状，研究表明，生鲜蔬菜在整个供应链环就的损耗率高达20%-30%，可以通过转基因的技术手段改善而具有巨大的市场潜力。番茄是在世界上栽培极为广泛的蔬菜，也是我国主要的栽培蔬菜之一。
成熟表型是果实发育到末期生物化学和生理学变化的总和，这一表型使器官变得可食用，并依赖动物传播种子，同时使其作为农业产品具有一定的价值。这些变化，虽然在不同的物种间有所不同，但一般包含细胞壁超微结构和质地的改变、淀粉转化为糖、增加果实对采后病原菌的敏感性、改变色素的生物合成和积累、提高芳香物质的含量并释放出香味。这些成熟特性中，有一些趋向于减少果实的货架寿命、增加采收、货运和贮存的成本，尤其是果实硬度的改变和成熟果实全面的降低对微生物侵染的抵抗。据估计，从离开农田到零售，果实和蔬菜产品会损失12一20%。现在常用的降低不期望成熟特征的技术包括成熟前采收、气调贮藏、杀虫剂应用、以及利用化学物质诱导来调控果实适时成熟。不幸的是，这些手段往往增加了生产、货运和加工的费用、同时也降低了果实的品质，是对现有农业生产水平竞争力和可持续发展的挑战。
随着番茄高产、优质、抗逆等多目标育种难度的增加，利用杂种互补效应实现番茄产量、品质、抗性等性状的同步突破，已成为番茄育种研究的一个重要发展方向。因此，为了丰富我国番茄品种的遗传资源，改良番茄的耐储藏性，选育耐储藏强、品味好的品种一直是番茄育种的重要目标。市场上已经存在耐储藏番茄的品种，基本都是通过传统的杂交育种的方式培育而来的，但是这些品种的耐储藏性只能是基本合格，转基因产品耐储藏的时间将相对较长，而且产品可以有效地改善果实采摘后的细菌感染这种在储藏运输中的主要问题。尽管关于转基因技术的争论从诞生之日起就从来没有停止过，但是科技发展的速度是日新月异，伴随着其生物经济的迅速发展，全球转基因作物逐年持续增长，无不显示转基因技术的在育种中的主流特性。
发明内容
本发明的目在于：运用最新的基因编辑技术，提供一种创建耐储藏转基因番茄的方法。该方法采用农杆菌转基因技术，通过构建CRISPR/Cas9植物表达载体和转化番茄，以此得到耐储藏的番茄新品种。
本发明的技术方案：一种通过构建CRISPR/Cas9番茄植物表达载体，所述番茄植物表达载体核苷酸序列如SEQ ID NO.1所示。
番茄植物载体是由CaMV 35S启动子的调控，由AtU6启动子表达，所述的35S启动子引物序列如SEQ ID NO.2所示；AtU6启动子引物序列如SEQ ID NO.3所示。
一种定向突变番茄基因的方法，采用上述的番茄植物表达载体，包括下述步骤：
（1）将CRISPR/Cas9载体与DFD-gRNA片段连接，与35S启动子构建番茄植物表达载体；
（2）将构建的CRISPR/Cas9番茄植物表达载体转化的农杆菌LBA4404感染番茄下胚轴；
（3）感染后的番茄下胚轴置于MS培养基上进行愈伤、出芽、生根培养；
（4）将该生根转化苗移栽入土栽培，PCR鉴定；
（5）田间种植、F1代自交后，形成稳定的转基因自交系，获得耐储藏性好的转基因番茄新品种。
上述方法中PCR鉴定中的上游引物DFD-JCF：acaaacataaagtagtggaccca如SEQ IDNO.4所示；下游引物DFD-JCR：acctctttcggctatttcgtata如SEQ ID NO.5所示。
上述方法中采用PCR鉴定，其20μL反应体系中，
含DFD基因的转基因植株的基因组DNA 1.0μL
PCR Master Mix 10μL
10 pM上游引物 1.0μL
10 pM下游引物 1.0μL
ddH2O 补至20μL。
上述方法中PCR工作程序为：94℃预变性3 min；94℃变性35 s，55℃退火35 s，72℃延伸1 min，35个循环；72℃终延伸10 min，4℃保存。
有益效果：本发明通过构建CRISPR/Cas9植物表达载体，将Cas9基因导入番茄M82中，对番茄自身DFD基因进行基因编辑。提高番茄果实耐储藏性，同时不影响植株的生长与其他品质，可以弥补前人研究方案的不足之处，开创基因编辑运用转基因植物的先河，因而该技术路线具有较强、前沿的创新性。
附图说明
附图1对CRISPR/Cas9参与表达载体的示意图说明：spcas9由CaMV 35S启动子的控制，参与由atu6启动子表达；双划线标记DFD靶位点的靶序列，单划线标记的PAM，下黑点是DFD/DFD基因突变位点；附图2、DFD/DFD基因突变位点：基因中A替换为T编辑；附图3、农杆菌转化番茄下胚轴组织培养再生；附图4、转化再生番茄植物的DFD基因PCR检测；附图5为DFD基因片段测序结果；附图6、转基因番茄对的结果与储藏。
具体实施方式
番茄已成为世界上的主要蔬菜作物，由于番茄生产的季节性和需求的周年均衡性矛盾，导致番茄旺季上市，采后腐烂损失高达50%。本发明利用基因编辑技术与转基因相结合提高番茄过时耐储藏的方法。通过农杆菌介导与番茄下胚轴组培技术，将CRISPR/Cas9植物表达载体转入番茄，利用Cas9蛋白编辑DFD基因，提高了番茄果实的耐储藏性获得了可用于耐储藏番茄新品种选育的特异材料。
实施例、1）、材料
番茄M82种子的直接来源由新疆农业科学院园艺作物研究所培育保存，原始来源是2008年9月由王柏柯从美国番茄遗传中心引进。
PCR所用的高保真聚合酶、DNA回收试剂盒及克隆载体由北京全式金生物技术有限公司购买。限制性内切酶和连接酶购自TaKaRa公司，PCR引物合成与片段测序均委托生工生物工程(上海)股份有限公司完成。
2）、表达载体构建
spCas9载体从中国科学院高彩霞研究员处获得，此载体为公开载体，一般研究人员可通过在线质粒库引进（http://www.addgene.org/），由北京康伟生物公司直接合成设计的靶基因。用于组合的主要载体是新疆农业科学院园艺作物研究所生物技术实验室保存的pCAM1301载体，此载体为公开载体，一般实验室都可引进。DFD基因的序列为公开序列，一般研究人员可通过在线的生物技术信息中心获得（https://www.ncbi.nlm.nih.gov/，登陆号：AY573803）。DFD基因的供体序列使用聚合酶环状延伸克隆构建到pCAM1301载体上。载体上spcas9基因由35S启动子驱动，gRNA由拟南芥U6启动子驱动。构建好的载体通过热激法转化到大肠杆菌DH5a菌株中扩增。测序验证载体序列，再通过冻融法转化农杆菌EHA105菌株，扩增以备侵染利用。
3）、番茄遗传转化及转化体再生
A、播种
挑取健康的番茄种子蒸馏水冲洗数次后，先用70%酒精30s，再用20% NaClO表面消毒20min，最后用无菌水清洗3-4次，播种于1/2MS培养基中。环境要求16h/8h，光强1800Lx，26±1℃。
B、预培养
取14-16d幼苗，在超净工作台中取无菌下胚轴，切去叶片和叶柄，外植体约0.5cm长短，在含有3.0 mg/L 6-BA和0.2 mg/L NAA的MS分化培养基上预培养2d。
C、共培养
将预培养的下胚轴刺破置于OD600=1农杆菌悬浮液中8-12min，用滤纸吸干多余的菌液，置于共培养基上，28℃暗培养2d。
D、分化培养与生根
共培养后的下胚轴置于分化培养基中（含有2.0 mg/L 6-BA+1.0 mg/L IAA+3 mg/L 潮霉素+100mg/L 羧苄青霉素）。待筛选出的抗性再生芽长到2cm高时，转接到MS+0.1 mg/LIAA+ 2 mg/L 潮霉素+80 mg/L 羧苄青霉素的生根培养基中，待生根后移栽。
4）、转基因植株的分子检测
PCR检测：将当代转化植株编号为C0-1、C0-2等。各株系自交后代为C1-1、C1-2、C2-1、C2-2等以此类推。去转化植株嫩叶片0.2g用天根DNA提取试剂盒提取基因组DNA。利用DFD-JCF/DFD-JCR引物扩增转基因植株中DFD基因，20μL体系中，包括MIX 10μL，ddH2O 7μL，引物各1μL。PCR反应条件：94℃预变性3 min；94 ℃变性35 s，55 ℃退火35 s，72 ℃延伸1 min，35个循环；72 ℃终延伸10min，4℃保存。
PCR结束后，于1%琼脂糖凝胶中分别点样进行电泳观察，然后将PCR产物回收转化，菌液送生工生物工程(上海)股份有限公司测序。所得结果DFD基因序列中碱基A变为T，由此证明番茄转基因植株基因编辑成功。
5）、转基因植株性状观察及果实耐储藏测试
通过对转基因植株与野生型植株M82田间性状观察相比较，两者在番茄株高、茎粗、固形物、果肉厚度、耐压力上都没显著性差异。从果实发育周期来看也是相差无几。将两组果实采摘后，在同一环境下放置40d后，野生型M82果实明显出现萎蔫变软，而转基因番茄明显比对照耐储藏，如附图6所示。
SEQUENCE LISTING
<110> 新疆农业科学院园艺作物研究所
<120> Cas9介导的番茄基因编辑载体及其应用
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 4144
<212> DNA
<213> 人工序列
<400> 1
atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tatggataag 60
aagtacagca ttggtctgga catcgggacg aattccgttg gctgggccgt gatcaccatg 120
agtacaaggt cccttccaag aagtttaagg ttctggggaa caccgatcgg cacagcatca 180
agaagaatct cattggagcc ctcctgttcg actcaggcga gaccgccgaa gcaacaaggc 240
taaagaaccg caaggagacg gtatacaaga aggaagaata ggatctgcta cctgcaggag 300
attttcagca acgaaatggc gaaggtggac gattcgttct ttcatagatt ggaagaaagt 360
ttcctcgtgg gaagataaga agcacgagag gcatcctatc tttggcaaca ttgtcgacga 420
ggttgcctat cacgaaaagt accccacaat ctatcatctg cggaagaagc ttgtggactc 480
gactgataag gcggattaga ttgatctacc tcgctctggc acacatgatt aagttcaggg 540
gccattttct gatcgagggg gatcttaacc cggacaatag cgatgtggac aagttgttca 600
tccagctcgt ccaaacctac aaagctcttt gaggaaaacc caattaatgc ttcaggcgtc 660
gacgccaagg cgatcctgtc tgcacgcctt tcaaagtctc gccggcttga gaacttgatc 720
gctcaactcc cgggcgaaaa gaagaacgct gttcgggaat ctcattgcac tttcgttggg 780
gctcacacca aacttcaaga gtaattttga tctcgctgag gacgcaaagc tgcagctttc 840
caaggacact tatgacgatg acctggataa cctttgccca aatcggcgat cagtacgcgg 900
acttgttcct cgccgcgaag aatttgtcgg acgcgatcct cctgagtgat attctccgcg 960
tgaacaccga gattacaaag gccccgctct cggcgagtat atcagcgcta tgacgagcac 1020
catcaggatc tgaccctttt gaaggctttg gtccggcagc aactcccaga gaagtacaag 1080
gaaatcttct ttgatcaatc caagaacggc tacgctggtt atattgcggc gggcatcgca 1140
ggaggaattc tacaagttta tcaagccaat tctggagaag atggatggca cagaggaact 1200
cctggtgaag ctcaataggg aggacctttt gcggaagcaa agaactttcg atacggcgca 1260
tccctcacca gattcatctc ggggagctgc acgccatcct gagaaggcag gaagacttct 1320
acccctttct taaggataac cgggagaaga tcgaaaagat tctgacgttc agaattccta 1380
ctattcggac cactcgcccg gggtaattcc agatttgcgt ggatgaccag aaagagcgag 1440
gaaaccatca caccttggaa cttcgaggaa gtggtcgata agggcgcttc cgcacagagc 1500
ttcatgagcg ctgacaaatt ttgacaagaa cctgcctaat gagaaggtcc ttcccaagca 1560
ttccctcctg tacgagtatt tcactgttta taacgaactc acgaaggtga agtatgtgac 1620
cgagggaatg gcaagccccc ttcctgagcg gcgagcaaaa gaaggcgatc gtggaccttt 1680
tgtttaagac caatcggaag gtcacagtta agcagctcaa ggaggactac ttcaagaaga 1740
ttgaatgctt cgattcgttg agatcgcggc gtggaagaca ggtttaacgc ctcactgggg 1800
acttaccacg atctcctgaa gatcattaag gataaggact tcttggacaa cgaggaaaat 1860
gaggatatcc tcgaagacat tgcctgactc ttcgttgttt gaggataggg aaatgatcga 1920
ggaacgcttg aagacgtatg cccatctctt cgatgacaag gttatgaagc agctcaagag 1980
aagaagatac accggatggg gaaggctgcc cgcaagcttt caatggcatt agagacaagc 2040
aatcagggaa gacaatcctt gactttttga agtctgatgg cttcgcgaac aggaatttta 2100
tgcagctgat tcacgatgac tcacttactt tcaagaggat atccagaggc tcaagtgtcg 2160
ggacaaggtg acagtctgca cgagcatatc gccaaccttg cgggatctcc tgcaatcaag 2220
aagggtattc tgcagacagt caaggttgtg gatgagcttg gaaggtcatg ggaggcataa 2280
gcccgagaac atcgttattg agatggccag agaaaatcag accacacaaa agggtcagaa 2340
gaactcgagg gagcgcatga agcgcatcga ggaaggcatt aaggagtggg gagtcagatc 2400
ttaaggagca cccggtggaa aacacgcagt tgcaaaatga gaagctctat ctgtactatc 2460
tgcaaaatgg cagggatatg tatgtggacc aggagttgga tattaaccgc cttcggatta 2520
cgacgtcgat catatcgttc ctcagtcctt ccttaaggat gacagcattg acaataaggt 2580
tctcaccagg tccgacaaga accgcgggaa gtccgataat gtgcccagcg aggaagtcgt 2640
aagaagatga agaactactg gaggcaactt ttgaatgcca agttgatcac acagaggaag 2700
tttgataacc tcactaaggc cgagcgcggg gtctcagcga actggacaag gcgggcttca 2760
ttaagggcaa ctggttgaga ctagacagat cacgaagcac gtggcgcaga ttctcgattc 2820
acgcatgaac acgaagtacg atgagaatga caagctatcc gggaagtgaa ggtcatcacc 2880
ttgaagtcaa actcgtttct gacttcagga aggatttcca attttataag gtgcgcgaga 2940
tcaacaatta tcaccatgct catgacgcat acctcaacgc tgtgtcggaa cagcattgat 3000
taagaagtac ccgaagccga gtccgaattc gtgtacggtg actataaggt ttacgatgtg 3060
cgcaagatga tcgccaagtc agagcaggaa attggcaagg ccactgcgaa tatttctttt 3120
actctaacat tatgaatttc tttagactga gatcacgctg gctaatggcg aaatccggaa 3180
gagaccactt attgagacca acggcgagac aggggaaatc gtgtgggaca aggggaggat 3240
ttcgccacag tccgcaaggt tctctctatc ctcaagtgaa tattgtcaag aagactgaag 3300
tccagacggg cgggttctca aaggaatcta ttctgcccaa gcggaactcg gataagctta 3360
tcgcagaaag aaggactggg atccgaagaa gtatgaggtt tcgactcacc aacggtggct 3420
tactctgtcc tggttgtggc aaaggtggag aagggaaagt caaagaagct caagtctgtc 3480
aaggagctcc tggtatcacc attatggaga ggtccagctt caaaagaatc cgatcgattt 3540
tctcgaggcg aagggatata aggaagtgaa gaaggacctg atcattaagc ttccaaagta 3600
cagtcttttc gagttggaaa cggcaggaag cgcatgttgg cttccgcgga gagctccaga 3660
agggtaacga gcttgctttg ccgtccaagt atgtgaactt cctctatctg gcatcccact 3720
acgagaagct caagggcagc ccagagataa cgaacagaag caactgtttg tgggcaacac 3780
aagcattatc ttgacgagat cattgaacag atttcggagt tcagtaagcg cgtcatcctc 3840
gccgacgcga atttggataa ggttctctca gctacaacaa gcaccgggac aagcctatcg 3900
agagcaggcg gaaaatatca ttcatctctt caccctgaca aaccttgggg ctcccgctgc 3960
attcaagtat tttgacacta cgattgatcg gaagagataa cttctacgaa ggaggtgctg 4020
gatgcaccct tatccaccaa tcgattactg gcctctacga gacgcggatc gacttgagtc 4080
agctcggtgg cgataagaga cccgcagcaa ccaagaaggc agggcagcaa agaagaagaa 4140
gtga 4144
<210> 2
<211> 485
<212> DNA
<213> 人工序列
<400> 2
ggatcctcta gagtcccccg tgttctctcc aaatgaaatg aacttcctta tatagaggaa 60
gggtcttgcg aaggatagtg ggattgtgcg tcatccctta cgtcagtgga gattccagat 120
aggcctaacg cttgtccaag atctattcag gatatcacat caatccactt gctttgaaga 180
cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg 240
accactgtcg gcagaggcat cttcaacgat ggcctttcct ttatcgcaat gatggcattt 300
gtaggagcca ccttcctttt ccactatctt cacaataaag tgacagatag ctgggcaatg 360
gaatccgagg aggtttccgg atattaccct ttgttgaaaa gtctcaattg ccctttggtc 420
ttctgagact gtatctttga tatttttgga gtagacaagt gtgtcgtgct ccaccatgtt 480
gacga 485
<210> 3
<211> 313
<212> DNA
<213> 人工序列
<400> 3
ttccgtggga gaaatctcaa aattccggca gaacaatttt gaatctcgat ccgtagaaac 60
gagacggtca ttgttttagt tccaccacga ttatatttga aatttacgtg agtgtgagtg 120
agacttgcat aagaaaataa aatctttagt tgggaaaaaa ttcaataata taaatgggct 180
tgagaaggaa gcgagggata ggcctttttc taaaataggc ccatttaagc tattaacaat 240
cttcaaaagt accacagcgc ttaggtaaag aaagcagctg agtttatata tggttagaga 300
cgaagtagtg att 313
<210> 4
<211> 23
<212> DNA
<213> 人工序列
<400> 4
acaaacataa agtagtggac cca 23
<210> 5
<211> 23
<212> DNA
<213> 人工序列
<400> 5
acctctttcg gctatttcgt ata 23
Claims (6)
1.一种通过构建CRISPR/Cas9番茄植物表达载体，其特征在于：所述番茄植物表达载体核苷酸序列如SEQ ID NO.1所示。
2.如权利要求1所示番茄植物载体是由CaMV 35S启动子的调控，由AtU6启动子表达，所述的35S启动子引物序列如SEQ ID NO.2所示；AtU6启动子引物序列如SEQ ID NO.3所示。
3.一种定向突变番茄基因的方法，采用权利要求1所述的番茄植物表达载体，其特征在于，包括下述步骤：
（1）将CRISPR/Cas9载体与DFD-gRNA片段连接，与35S启动子构建番茄植物表达载体；
（2）将构建的CRISPR/Cas9番茄植物表达载体转化的农杆菌LBA4404感染番茄下胚轴；
（3）感染后的番茄下胚轴置于MS培养基上进行愈伤、出芽、生根培养；
（4）将该生根转化苗移栽入土栽培，PCR鉴定；
（5）田间种植、F1代自交后，形成稳定的转基因自交系，获得耐储藏性好的转基因番茄新品种。
4.如权利要求3所述的一种定向突变番茄基因的方法，其特征在于，所述PCR鉴定中的上游引物DFD-JCF：acaaacataaagtagtggaccca如SEQ ID NO.4所示；下游引物DFD-JCR：acctctttcggctatttcgtata如SEQ ID NO.5所示。
5.如权利要求3所述的一种定向突变番茄基因的方法，其特征在于，采用PCR鉴定，其20μL反应体系中，
含DFD基因的转基因植株的基因组DNA 1.0μL
PCR Master Mix 10μL
10 pM上游引物 1.0μL
10 pM下游引物 1.0μL
ddH2O 补至20μL。
6.如权利要求5所述的一种定向突变番茄基因的方法，其特征在于，PCR工作程序为：94℃预变性3 min；94℃变性35 s，55℃退火35 s，72℃延伸1 min，35个循环；72℃终延伸10min，4℃保存。
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