CN113943740A - 一种可调控烟叶钾含量的NtCHA1基因及其应用 - Google Patents
一种可调控烟叶钾含量的NtCHA1基因及其应用 Download PDFInfo
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Abstract
本发明涉及基因工程技术领域,涉及调控烟叶钾含量的基因,具体涉及一种可调控烟叶钾含量的NtCHA1基因及其应用。本发明提供一种调控烟叶钾含量的NtCHA1基因,其核苷酸序列如SEQ ID NO.1所示。还提供一种基于所述调控烟叶钾含量的NtCHA1基因的应用,在烟草中过表达NtCHA1基因可以显著提高烟叶的钾含量,通过CRISPR/cas9敲除该基因后烟叶钾含量与对照相比显著降低,可见NtCHA1基因参与了烟草钾含量的调控。本发明明确了NtCHA1基因对烟叶钾含量的调控功能,为解析烟草钾离子含量的分子调控提机制供了新的证据,也为通过过表达该基因提高烟叶钾含量的应用提供了依据。
Description
技术领域
本发明涉及基因工程技术领域,涉及调控烟叶钾含量的基因,具体涉及一种可调控烟叶钾含量的NtCHA1基因及其应用。
背景技术
烟草是我国重要的叶用性经济作物,烟叶钾含量的高低严重影响着烟叶经济价值的大小。烟叶中的钾离子参与烟叶的生理生化反应,与烟株的抗逆性关系密切,对于烟草农业来说,它还影响着烟叶的内在品质和工业可用性,因此通常把钾含量作为衡量烟叶品质的重要指标。钾对烟叶成熟度、香吃味、燃烧性、安全性等方面均有重要影响,烟叶钾含量提高可以改善烟叶的组织结构,使烟叶结构细腻,而且还能提高烟叶外观色泽,使烟叶呈深橘黄色,香气足,吃味好,富有弹性和韧性,填充性增强;另外钾还可以增强烟叶糖类、色素类、芳香类物质的合成积累,因而钾含量越高,往往烟叶的质量越优。
针对我国烟叶钾含量偏低问题,传统的栽培施肥措施效果并不理想。有研究认为:高钾施用量增加了上部叶中的烟碱含量,如文献胡国松,王志彬,王凌,韩锦峰,穆琳.烤烟烟碱累积特点及部分营养元素对烟碱含量的影响[J].河南农业科学,1999(01):10-14.。也有研究认为:施钾对烟草农艺性状和烟碱含量的影响,结果发现,施钾可提高烟叶中钾含量,促进植株生长,降低烟叶中烟碱含量,如文献舒海燕,杨铁钊,曹刚强,凌华,田保明.烟叶钾含量与烟株农艺性状和烟碱含量的相关分析[J].中国农学通报,2007(02):275-278.。因此,单独依靠栽培施肥措施并不能有效的控制烟叶中的钾含量,因而目前的研究中,着重通过发掘烟草中与钾吸收转运相关基因,达到提高烟叶钾含量的目的。
CHA(Cation/H+antiporter)是一类位于植物细胞质膜或液泡膜上的阳离子和H+反向转运体。CHA的主要功能是选择性转运Ca2+等阳离子,如文献Harbin,Tokyo U O,Tokyo.Characteristics of Cation/H~+Antiporters in Plants[J].Genomics andApplied Biology,2012.。其主要介绍了阳离子/H+逆向转运蛋白(CAXs)是一组蛋白质,CAXs的研究在调节植物生长、增加作物养分吸收和减少土壤污染物方面发挥着重要作用。虽然类似于上述研究的现有技术中揭示了CHA的主要功能是选择性转运Ca2+等阳离子,可以调节植物生长、增加作物养分吸收,但对钾离子的转运报道甚少。如文献Bao,Ai-Ke;Du,Bao-Qiang;Touil,Leila;Kang,Peng;Wang,Qiang-Long;Wang,Suo-Min(2016).Co-expression of tonoplast Cation/H+antiporter and H+-pyrophosphatase fromxerophyte Zygophyllum xanthoxylum improves alfalfa plant growth undersalinity,drought and field conditions.Plant Biotechnology Journal,14(3),964–975.doi:10.1111/pbi.12451,其主要介绍了Cation/H+antiporter和来自旱生植物的H+-pyrophosphatase的共表达改善了盐度、干旱和田间条件下的苜蓿植物生长,转基因苜蓿植株的生长性能与叶和根中更多的Na+、K+和Ca2+积累有关,结果表明,来自旱生植物的液泡膜NHX和H+-PPase基因的共表达显着改善了苜蓿的生长,并增强了其对高盐度和干旱的耐受性。虽然上述现有技术中公开了共表达生物基因改善了盐度、干旱和田间条件下的苜蓿植物生长中有关Na+、K+和Ca2+的积累,并且即便在缺水环境下苜蓿植物由于上述积累也可以获得更好的生长。
虽然,烟草领域中也Cation/H+antiporter基因的克隆的报道,如李佳皓,吕承承,罗银,鲁黎明,李立芹(2019).烟草NtKEA4基因的克隆、生物信息学及表达分析.分子植物育种,17(18):5946-5952,主要介绍了烟草NtKEA4基因的克隆及其表达收到低钾、高盐、脱落酸(ABA)、低温等非生物胁迫诱导。但是,烟草中还未见报道鉴定钾离子调控功能的Cation/H+antiporter的相关报道。
发明内容
为弥补上述领域存在的不足及空白,本发明为调控烟叶钾离子含量并结合CHA提供了烟草阳离子/氢离子反向转运体NtCHA1的候选基因,并提供了一种调控烟叶钾含量的NtCHA1基因的应用。
本发明请求保护的技术方案如下:
一种可调控烟叶钾含量的NtCHA1基因,其核苷酸序列为SEQ ID NO:1所示。
一种可调控烟叶钾含量的蛋白,其特征在于,其氨基酸序列为SEQ ID NO:2所示,或根据所述的NtCHA1基因编码为SEQ ID NO:2所示的氨基酸序列的蛋白。
一种可调控烟叶钾含量的NtCHA1基因的应用,其特征在于,使用所述的NtCHA1基因或所述的蛋白在调控烟草钾离子含量中的应用。
优选的,利用所述NtCHA1基因构建CRISPR/cas9载体,包括以下过程:
(1)根据所述NtCHA1基因的基因组序列设计靶位点,所述靶位点为:
sgRNA:CTCAAATGGAGTATTCCAAG
(2)根据步骤(1)中的所述靶位点设计引物,得到靶位点引物,所述靶位点引物序列为:
P1:ATTGCTCAAATGGAGTATTCCAAG;
P2:AAACCTTGGAATACTCCATTTGAG;
(3)根据步骤(1)中的靶位点,在靶位点两侧设计编辑材料的检测引物,得到检测引物,所述检测引物序列为:
NtCHA1-SdF:CACGGGTTCAATTAGTCTCGCA;
NtCHA1-SdR:GAGACCTTGGATCTAACTCGAG;
(4)制备dsDNA:根据步骤(2)中所述的靶位点引物通过退火形成互补DNA oligo,得到dsDNA;
(5)酶切pHSE401载体得到酶切产物,将所述酶切产物与步骤(4)中得到的dsDNA进行连接,得到连接产物;酶切后对酶切产物进行电泳检测分析,回收11520bp的酶切产物备用;
(6)测序验证:将步骤(5)中得到的连接产物转化为大肠杆菌,筛选阳性克隆并进行菌落PCR检测,经PCR检测验证正确的阳性克隆菌株培养扩增后进一步进行测序分析,得到所述CRISPR/cas9载体;所述菌落PCR检测时,所用引物序列为:
U6-26p-F:TGTCCCAGGATTAGAATGATTAGGC;
U6-26p-R:AAACCGATTCATCGCAACCAATTC。
一种利用所述NtCHA1基因构建的提高烟叶钾含量的转基因烟草材料。
优选的,利用所述NtCHA1基因构建的提高烟叶钾含量的转基因烟草材料包括以下步骤:
A构建NtCHA1过表达载体:
a1.克隆NtCHA1基因并连接TOPO载体:利用NtCHA1基因特异引物进行扩增,得到NtCHA1基因片段,并纯化回收,将回收的所述基因片段进行TOPO克隆,连接到TOPO载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为2418bp的质粒提取DNA,构建得到TOPO载体;
a2.植物过表达载体的构建:利用BamH I/EcoR V酶切所述TOPO载体和pENTRTM2B,得到目的基因片段NtCHA1和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;挑取转化DH5α后的克隆,提取质粒DNA,构建得到入门克隆载体;将所述入门克隆载体和表达载体通过LR反应后转化大肠杆菌DH5α感受态细胞,得到重组表达载体。
B:烟草的遗传转化:
b1.表达载体转化农杆菌,将农杆菌感受态细胞溶解后加入重组表达载体进行农杆菌转化,在LB固体培养基中培养后,得到含有目标载体的农杆菌克隆;
b2.烟草转化,挑取含有目标载体的农杆菌克隆,培养得到含目标载体的农杆菌悬浮菌液;将清洗后的烟草叶片,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,放入含目标载体的无菌MS液体培养基农杆菌悬浮菌液中,静置15-20min;取出烟草叶片,于MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,温室条件下分化培养,分化出芽后,将长至3-5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净移植于灭菌的营养土中,为转化后的烟草植株。
优选的,所述NtCHA1基因特异引物,其核苷酸序列为:
CHA1-F:GGATCCATGGCTTCAACTTTACCTATGAAATGT;
CHA1-R:GATATCCTATCTAGTATTTTTAGTTAGAATCGTT。
优选的,所述LR反应体系为:加入构建成功的所述入门克隆载体(50-150ng)1-7μL,以及0.5μL表达载体,TE Buffer至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LRCloneaseTMⅡ enzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min。
优选的,所述LB固体培养基含有壮观霉素100mg/L和利福平25mg/L;所述MS培养基含有0.02mg/L 6-BA、2mg/L NAA。
优选的,所述表达载体为Destination Vector,即pK2GW7载体;所述分化培养基为含有0.5mg/L 6-BA、0.1mg/L NAA、100mg/L卡那霉素、500mg/L头孢霉素的MS培养基。
本发明通过系统研究,首次证明了烟草NtCHA1基因在调控烟叶钾含量中的功能。利用CRISPR/cas9敲除NtCHA1基因降低烟叶钾含量14%左右。本发明通过系统研究,通过构建NtCHA1过表达载体,农杆菌转化、烟草转化,获得NtCHA1基因过表达烟草植株,其烟叶钾含量比对照显著提高。利用YC/T 173-2003火焰光度法检测烟叶钾含量编辑后的NtCHA1-OV与对照组VC相比,烟叶中的钾含量显著升高。综上,本发明为提高烟叶钾含量提供了新的基因资源和材料。
附图说明
图1为引物对U6-26p-F/U6-26p-R扩增阳性克隆电泳图。图中,泳道从左到右分别是:(1)DL2000 DNA Marker(Takara),(2)以ddH2O为模板的PCR产物,(3-4)以质粒DNA为模板的PCR产物。DL2000 DNA Marker(Takara)条带从上到下分别是2000bp,1000bp,750bp,500bp,250bp,100bp;扩增产物大小400bp左右。
图2为引物对NtCHA1-SdF/NtCHA1-SdR扩增编辑材料检测突变位点电泳图。图中,泳道从左到右分别是:(1)是DL2000 DNA Marker(Takara)、(2-3)是以提取DNA为模板的PCR产物。DL2000 DNA Marker(Takara)条带从上到下分别是2000bp,1000bp,750bp,500bp,250bp,100bp;扩增大小为651bp。
图3为CRISPR/cas9编辑材料突变信息,突变体缺失5个碱基。
图4为CRISPR/cas9编辑材料测序峰图。
图5为CRISPR/cas9编辑材料与对照烟叶钾离子含量,NtCHA1-CP为编辑材料,CK为对照。
图6为构建的pTOPO-NtCHA1载体中NtCHA1基因扩增图。图中泳道从左到右依次为:(1)M:1kb DNA Ladder,(2)以ddH2O为模板的PCR产物,(3-4)以质粒DNA为模板的的酶切产物;M:1kb DNA Ladder条带从上到下依次为10000bp,8000bp,6000bp,5000bp,4000bp,3000bp,2000bp,1000bp;其中,扩增产物大小为2418bp。
图7为pK2GW7-NtCHA1载体酶切鉴定。图中泳道从左到右依次为:(1)M:1kb DNALadder,(2)以ddH2O为模板的阴性对照产物,(3-4)以质粒DNA为模板的酶切产物,构建不成功的克隆。(5)空泳道,(6-7)以质粒DNA为模板的酶切产物,为正确的克隆;M:1kb DNALadder条带从上到下依次为10000bp,8000bp,6000bp,5000bp,4000bp,3000bp,2000bp,1000bp。
图8为过表达植株NtCHA1基因表达分析,NtCHA1-OV为过表达材料,VC为转空载体对照。
图9为过表达材料与对照烟叶钾离子含量,NtCHA1-OV为过表达材料,VC为转空载体对照。
具体实施方式
下面结合实施例进一步描述本发明,需要理解的是,下述实施例仅作为对本发明的解释和说明,不以任何方式限制本发明的范围。
若未特别说明,以下实施例中使用的试剂均为本领域常规试剂,可商购获得或按照本领域常规方法配制而得,规格为实验室纯级即可;使用的实验方法和实验条件均为本领域常规的实验方法和实验条件,可参考相关实验手册(例如《分子克隆实验指南》)、公知文献或厂商说明书。除非另有定义,本文使用的所有科学技术用语的含义与本发明所属领域普通技术人员通常理解的含义相同。
本发明根据烟草基因组信息,分离到NtCHA1基因,其核苷酸序列如SEQ ID NO.1所示,序列长度为2418bp,编码的氨基酸序列如序列SEQ ID NO.2所示,长度为806氨基酸。
本发明利用CRISPR/cas9技术敲除NtCHA1基因后,可显著降低烟叶钾离子含量。
本发明利用基因工程手段过表达NtCHA1基因,可显著提高烟叶钾离子含量。
本发明的烟草NtCHA1基因是烟草钾离子含量正调控因子,通过基因工程技术过表达该基因可达到提高烟叶钾离子含量的效果,从而在提高烟叶钾离子含量中发挥重要作用。
因此,本发明的第一个目的是提供烟草NtCHA1基因,其核苷酸序列如SEQ ID NO.1所示。
本发明的第二个目的是提供所述的烟草NtCHA1基因编码的蛋白,其氨基酸序列如SEQ ID NO.2所示。
本发明的第三个目的是提供了所述的烟草NtCHA1基因在调控烟叶钾含量方面的功能,即敲除NtCHA1基因可以降低烟叶钾含量,而过表达NtCHA1基因可以提高烟叶钾含量。
本发明还提供了一种能提高烟叶钾含量的方法,所述方法为使植物过表达权利要求1所述的基因NtCHA1,其中所述植物为烟草。
实施例1利用CRISPR/cas9敲除NtCHA1基因降低烟叶钾含量
1.1实验材料
1.1.1供试菌株
大肠杆菌(Escherichia coli)DH5α。农杆菌(Agrobacterium)感受态细胞C58C1。以上生物材料本实验室亦有保存,申请人声明可自申请日起二十年内向公众发放用于验证实验。
1.1.2供试烟草品种
烟草:野生型烟草云烟87,本实验室保存,申请人声明可自申请日起二十年内向公众发放用于验证实验。
1.1.3供试试剂
pHSE401载体、pK2GW7载体、Trans-T1感受态细胞、质粒提取试剂盒、PCR产物回收试剂盒、胶回收试剂盒、植物RNA提取试剂盒、植物基因组提取试剂盒均购白北京全式金生物技术有限公司;LBA4404感受态细胞、CRISPR/Cas9植物表达载体南本实验室保存;LRClonaseTMⅡ enzyme Mix、反转录酶M.MLV、RNA酶抑制剂购自美国Promega公司;T4 DNA连接酶、Bsa I限制性内切酶购自纽英伦生物技术(北京)有限公司;DNeasy Plant Mini Kit购自德国QIAGEN公司;SYBR~Premix Ex Taq II购白宝生物工程(大连)有限公司;Annealing Buffer for DNA Oligos(10x)购自上海碧云天生物技术有限公司;MS粉购自美国PHytoTechnology Laboratories公司;蔗糖、琼脂购自重庆鼎国生物技术有限公司;氨苄青霉素(Ampicillin)、卡那霉素(Kanamycin)、利福平(Rifampicin)等抗生素购自北京索莱宝科技有限公司;引物合成和测序均由北京华大基因科技股份有限公司完成。
1.1.4供试培养基
LB液体培养基:含有卡那霉素100mg/L和利福平25mg/L。所述MS培养基含有6-BA/6-苄氨基嘌呤/α-萘乙酸(0.02mg/L)、NAA(2mg/L)。分化培养基:含有6-BA/6-苄氨基嘌呤(0.5mg/L)、NAA/α-萘乙酸(0.1mg/L)、潮霉素(20mg/L)、头孢霉素(500mg/L)的MS培养基。以上培养基都在121℃,20min条件下湿热灭菌。
1.1.5实验仪器
致微(厦门)仪器有限公司GI-54DS自动压力蒸汽灭菌器。上海精宏实验设备有限公司DHG-9240A电热恒温鼓风干燥箱,DNP-90-52电热恒温培养箱。台苏州安泰空气技术有限公司SW-CJ-2FD超净工作台。北京赛多利斯科学仪器有限公司SQP电子分析天平。BackmanOptima L-XP制备型超速离心机。北京鼎昊源科技有限公司HR220 MiniSmart迷你离心机。Eppendorf Centrifuge-54188冷冻离心机。MX-S可调式和固定式混匀仪。卡尤迪生物科技H203-100C加热制冷型金属浴。BIO-RAD Thermal Cycler PCR仪。Milli-Q超纯水系统Millipore。上海医用分析仪器厂TGL-16G制冰机。北京大龙兴创实验仪器有限公司,Bandelin Sonopuls HD 2070超声破碎仪。北京六一仪器厂DYY-12电泳仪。Alliance4.7Chroma Uvitec凝胶成像仪。上海知信ZX-S22双孔不锈钢恒温水浴锅。
1.2实验方法
1.2.1构建CRISPR/cas9载体
(1)根据NtCHA1基因组序列设计靶位点,如下;
sgRNA:CTCAAATGGAGTATTCCAAG;
具体的,根据NtCHA1基因序列,利用在线工具ZiFiTTargeter Version4.2纠选择合适的靶位点,筛选要求为:①靶位点主要包括20个碱基,并且这20个碱基后面是NGG(N为任意碱基)3个碱基的PAM区(Protospacer adjacent motif,PAM);②靶位点尽量选择在基因编码区的前端。
(2)根据步骤(1)中的靶位点设计引物,得到靶位点引物,所述靶位点引物为:
SEQ ID NO: | 名称 | 序列(5’→3’) |
3 | P1 | ATTGCTCAAATGGAGTATTCCAAG |
4 | P2 | AAACCTTGGAATACTCCATTTGAG |
(3)根据步骤(1)中的靶位点,在靶位点两侧设计编辑材料的检测引物,得到检测引物,所述检测引物为:
SEQ ID NO: | 名称 | 序列(5’→3’) |
5 | NtCHA1-SdF | CACGGGTTCAATTAGTCTCGCA |
6 | NtCHA1-SdR | GAGACCTTGGATCTAACTCGAG |
扩增长度为651bp。
(4)制备dsDNA:根据步骤(2)中的得到的靶位点引物通过退火形成互补DNAoligo,得到dsDNA;具体反应体系为:反应体系50μL,包括P1 20μL,P2 20μL,10×Annealingbuffer5μL,灭菌双蒸水5μL。退火程序为:95℃,5min;90℃,1min;80℃,1min;70℃,1min;60℃,1min;50℃,1min;40℃,1min;30℃,1min;20℃,1min;10℃,1min。
(5)酶切pHSE401载体得到酶切产物,将所述酶切产物与步骤(4)中得到的dsDNA进行连接,得到连接产物;
具体步骤为:利用BsaI酶对pHSE401载体进行酶切,酶切体系50μL,包括:质粒5μL,10×buffer 5μL,Bsa I 2μL,灭菌双蒸水38μL,37℃酶切1h;
酶切后对酶切产物进行电泳检测分析,可见1200bp和11520bp两个条带,回收11520bp的酶切产物备用。
利用T4DNA连接酶将所回收的大片段酶切产物与步骤(4)所制备dsDNA进行连接,连接体系20μL:所回收载体酶切产物3μL,退火所形成dsDNA产物10μL,T4 DNA buffer 2μL,T4 DNA连接酶1μL,灭菌双蒸水4μL,16℃过夜连接,得到连接产物。
(6)测序验证:将步骤(5)中得到的连接产物转化为大肠杆菌,筛选阳性克隆(pHSE401载体抗性为卡那霉素)并进行菌落PCR检测;所述菌落PCR检测时,所用引物设计为:
SEQ ID NO: | 名称 | 序列(5’→3’) |
7 | U6-26p-F | TGTCCCAGGATTAGAATGATTAGGC |
8 | U6-26p-R | AAACCGATTCATCGCAACCAATTC |
PCR体系如下:
PCR程序如下:
其中,退火温度根据引物自身温度调整。然后将PCR产物取出,用2%琼脂糖凝胶电泳检测扩增结果。
如图1所示,利用引物对U6-26p-F/U6-26p-R扩增阳性克隆后进行琼脂糖凝胶电泳,M:DL2000,经PCR检测验证正确的阳性克隆菌株培养扩增后进一步进行测序分析,得到pHSE401-CHA1载体;测序时所用引物为上述U6-26p-F。
1.2.2农杆菌转化
将农杆菌感受态细胞C58C1溶解后加入上述步骤(6)中得到的载体pHSE401-CHA1进行农杆菌转化,得到含有目标载体的农杆菌克隆。具体为:从-80℃冰箱中取出农杆菌感受态细胞(C58C1),放置冰上溶解后加入载体pHSE401-CHA1 4μL;液氮速冻1分钟,转入37℃水浴5分钟,再冰浴2分钟,向混合物中加入1mL LB液体培养基,28℃、220rpm培养3~4小时;培养物涂布于含有卡那霉素100mg/L和利福平25mg/L的LB固体培养基上,28℃倒置培养2~3天,可见含有目标载体的农杆菌克隆。
1.2.3烟草转化
将得到的含有目标载体的农杆菌克隆经划线接种后,在含有卡那霉素和利福平的LB培养基中进行扩繁,得到含目标载体的农杆菌LB液体培养基悬浮菌液。具体为:挑取含有目标载体的农杆菌克隆,在含有卡那霉素和利福平的LB平板上划线,28℃培养2-3天;刮取划线菌斑接菌于含有卡那霉素和利福平的LB培养基中,28℃,220rpm震荡培养,菌液浓度达到OD=0.5~0.8时进行侵染。
取野生型烟草叶片利用乙醇和HgCl2处理后用无菌水进行冲洗,并吸去烟草叶片表面液体,得到无菌野生型烟草叶片;具体为:将野生型烟草叶片置于500mL广口瓶中,加入适量75%乙醇,漂洗1min;弃乙醇,加入0.1%的HgCl2溶液,置摇床上室温振荡15~30分钟;弃溶液,用无菌水冲洗6遍。
将得到的无菌野生型烟草叶片切成小片后,放入含目标载体的农杆菌LB液体培养基悬浮菌液中进行培养后,将烟草叶片转入分化培养基中进行培养,直至烟草叶片切口处逐渐形成愈伤组织并分化出芽。具体为:将得到的无菌野生型烟草叶片取出,用无菌吸水纸洗去表面液体,取无菌叶片用剪刀切成1cm×1cm的小片,将切成小片的烟草叶片放入含目标载体的农杆菌LB液体培养基悬浮菌液中,静置15~20min;取出烟草叶片,用无菌滤纸吸去多余菌液,于含有6-BA(0.02mg/L)、NAA(2mg/L)的MS培养基中25℃暗培养两天;将烟草叶片转入分化培养基中,切口接触培养基,分化培养基为含有6-BA(0.5mg/L)、NAA(0.1mg/L)、潮霉素(20mg/L)、头孢霉素(500mg/L)的MS培养基,每2~3周继代一次,切口处逐渐形成愈伤组织,最后分化出芽。待芽长至3~5cm时,切取芽,将切取的芽诱导生根,生根后移植于灭菌的营养土中,得到多株T0代转基因烟草幼苗;具体为;将长至3~5cm的芽切下,转入MS培养基诱导生根,生根后的转基因植株由生根培养基中取出,用自来水洗净培养基,移植于灭菌的营养土中。
1.2.4测序筛选编辑材料
待转基因植株在营养土中的T0代转基因烟草幼苗生长1周后,选取叶片提取DNA,利用检测引物NtCHA1-SdF/NtCHA1-SdR扩增后得到扩增产物,如图2所示,检测引物扩增编辑材料,M:DL2000,扩增大小为651bp,经琼脂糖凝胶电泳检测后利用正向引物测序,经对测序结果进行分析,获得一株NtCHA1基因在编辑位点缺失5个碱基TTCCA的编辑材料;种植该编辑材料得T1代植株,通过测序筛选纯合突变单株并收种,获得具有NtCHA1纯合突变的T2代烟草种子;
具体为:待T0代转基因苗生长1周左右,选取20株烟苗取叶片并利用DNeasy PlantMini Kit(QIAGEN)提取DNA,利用1.2.1构建CRISPR/cas9载体中的步骤(3)中设计的引物NtCHA1-SdF/SdR进行扩增,扩增产物纯化后利用正向引物测序。如图3所示,对测序结果分析,获得一株NtCHA1基因缺失5个碱基TTCCA的编辑材料。种植该编辑材料T1代植株,通过测序筛选纯合突变单株,如图4所示为CRISPR/cas9编辑材料测序峰图,并收种获得T2代种子。
对得到的具有NtCHA1纯合突变的T2代烟草种子在温室进行种植,得到T2代烟草株系,利用YC/T 173-2003的方法检测植株烟叶钾含量;具体为:在温室种植得到的突变体株系与野生型烟草植株对照,在烟株开花期,取9-11叶位叶片(从下部计算),杀青烘干,利用YC/T 173-2003的方法检测烟叶钾含量。编辑材料的T2代烟草相比含有SEQ ID NO.1序列的烟草叶片钾含量显著降低;如图5所示,其中NtCHA1-CP为编辑材料,CK为对照;与CK对照组相比,NtCHA1-CP编辑材料中的钾含量降低14%左右。
实施例2利用过表达NtCHA1提高烟叶钾离子含量
2.1实验材料
2.1.1供试菌株,同实施例中的1.1.1中记载的供试菌株。
2.1.2供试烟草品种
烟草:烟草云烟87,本实验室保存,申请人声明可自申请日起二十年内向公众发放用于验证实验。
2.1.3供试试剂
PCR-Blunt II-TOPO克隆载体、pK2GW7载体、质粒提取试剂盒、PCR产物回收试剂盒、胶回收试剂盒、植物RNA提取试剂盒、植物基因组提取试剂盒均购白北京全式金生物技术有限公司;6-BA 6-苄氨基嘌呤;CRISPR/Cas9植物表达载体南本实验室保存;LRClonaseTMⅡenzyme Mix、反转录酶M.MLV、RNA酶抑制剂购自美国Promega公司;T4 DNA连接酶、Bsa I限制性内切酶购自纽英伦生物技术(北京)有限公司;Proteinase k购自美国AbMole品牌公司;PrimeScriptTM RT reagent Kit购自TaKaRa公司;其他,同实施例中的1.1.3中记载的供试试剂。
2.1.4供试培养基,同实施例中的1.1.4中记载的供试培养基。
2.1.5实验仪器,同实施例中的1.1.5中记载的实验仪器。
2.2实验方法
2.2.1构建NtCHA1过表达载体
利用Gateway技术构建NtCHA1基因过表达载体。
(1)克隆NtCHA1基因并连接TOPO载体
以烟草品种云烟87的叶片cDNA为模板,利用NtCHA1基因特异引物进行扩增(如下),得到大小约为2418bp的基因片段,纯化回收;
SEQ ID | 名称 | 序列(5’→3’) | 酶切位点 |
9 | CHA1-F | <u>GGATCC</u>ATGGCTTCAACTTTACCTATGAAATGT | BamHI |
10 | CHA1-R | <u>GATATC</u>CTATCTAGTATTTTTAGTTAGAATCGTT | EcoRV |
PCR体系如下:
PCR程序如下:
其中,退火温度根据引物自身温度调整。然后将PCR产物取出,用2%琼脂糖凝胶电泳检测扩增结果。
将回收的NtCHA1基因片段进行TOPO克隆,连接到(3.5kb)载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为2418bp的质粒提取DNA,构建的载体命名为pTOPO-NtCHA1;如图6所示,为构建的pTOPO-NtCHA1载体中NtCHA1基因扩增图,M:1kb DNA Ladder,扩增产物大小为2418bp。
(2)植物过表达载体的构建
a、入门克隆pENTRTM2B-NtCHA1的构建
BamH I/EcoR V酶切pTOPO-NtCHA1和pENTRTM2B,得到目的基因片段NtCHA1和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;
挑取转化DH5α后的克隆,提取质粒DNA,BamH I/EcoR V酶切,酶切结果为3.8kb的载体片段和2.4kb左右的片段为正确的克隆,正确的克隆命名为pENTRTM2B-NtCHA1;
b、通过LR反应得到植物表达载体
入门克隆pENTRTM2B-NtCHA1和表达载体pK2GW7 LR反应后转化大肠杆菌DH5α感受态细胞,得到植物重组表达载体pK2GW7-NtCHA1。利用BamH I/EcoR V酶切鉴定pK2GW7-NtCHA1,正确的克隆可以切出两条片段,大小约2.4kb和11kb。如图7所示,pK2GW7-NtCHA1载体酶切鉴定,M:1kb DNA Ladder,泳道6和7为正确的克隆,大小约2.4kb和11kb。
上述LR反应体系:构建成功的入门载体pENTRTM2B-NtCHA1(50-150ng)1-7μL,0.5μLDestination Vector(即pK2GW7载体),TE Buffer(缓冲液)至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LR ClonaseTMⅡenzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min。
2.2.2烟草的遗传转化
表达载体转化农杆菌:将农杆菌感受态细胞C58C1溶解后加入重组表达载体pK2GW7-NtCHA1进行农杆菌转化,在LB固体培养基中培养后,得到含有目标载体的农杆菌克隆。具体为:从-80℃冰箱中取出农杆菌感受态细胞,放置冰上溶解后加入4μL重组表达载体pK2GW7-NtCHA1;液氮速冻1分钟,转入37℃水浴5分钟,再冰浴2分钟,向混合物中加入1mLLB液体培养基,28℃、220rpm培养3-4小时;培养物涂布于含有壮观霉素100mg/L和利福平25mg/L的LB固体培养基上,28℃倒置培养2-3天,可见含有目的载体的农杆菌克隆;
烟草转化:
a、挑取含有目标载体的农杆菌克隆,在含有壮观霉素和利福平的LB平板上划线,28℃培养2-3天;刮取划线菌斑接菌于含有壮观霉素和利福平的MS培养基中,28℃,220rpm振荡培养,菌液浓度达到OD=0.5-0.8时6,000rpm离心5分钟富集菌体,弃上清,再用20mL液体MS培养基重悬菌体,得到含目标载体的农杆菌悬浮菌液;
b、将烟草叶片置于500mL广口瓶中,加入适量75%乙醇,漂洗1min;弃乙醇,加入0.1%的HgCl2溶液,置摇床上室温振荡15-30分钟;弃HgCl2溶液,用无菌水冲洗6遍;
c、将烟草叶片取出,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,将切成小片的烟草叶片放入含目标载体的无菌MS液体培养基悬浮菌液中,静置15-20min;取出烟草叶片,使用无菌滤纸吸去多余菌液,于含有6-BA(0.02mg/L)、NAA(2mg/L)的MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,切口接触培养基,于温室条件下分化培养;分化培养基为含有6-BA(0.5mg/L)、NAA(0.1mg/L)、卡那霉素(100mg/L)、头孢霉素(500mg/L)的MS培养基,每2-3周继代培养1次,切口处逐渐长出愈伤组织,最终分化出芽;
d、将长至3-5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净培养基,移植于灭菌的营养土中,为转化后的烟草植株。
2.2.3转基因材料中NtCHA1基因表达分析
根据NtMYB330基因序列设计qRT-PCR引物:
SEQ ID NO: | 名称 | 序列(5’→3’) |
11 | NtCHA1-QF | GGCTGCTTGGATTCTACTTGCT |
12 | NtCHA1-QR | CCTGAGAGCAACGTTTAGCCAT |
将温室种植T1代转基因株系和转空载体对照,在开花期,提取叶片总RNA,利用TaKaRa公司PrimeScriptTM RT reagent Kit反转录试剂盒合成cDNA作为模板进行实时荧光定量PCR分析,分析过表达株系中NtCHA1基因表达情况,如图8所示,转基因材料中NtCHA1基因的表达水平(左侧)比对照(右侧)显著提高。
PCR体系如下:
PCR程序如下:
其中,退火温度根据引物自身温度调整。然后将PCR产物取出,用2%琼脂糖凝胶电泳检测扩增结果。
2.2.4转基因植株中钾含量的分析
将得到的NtCHA1表达量显著提高的转基因株系NtCHA1-OV和转空载体作为对照在开花期取9-11叶位叶片(从下部计算),杀青烘干,利用YC/T 173-2003火焰光度法检测烟叶钾含量。YC/T 173-2003是指烟草及烟草制品中钾的测定,也称火焰光度法。
结果,如图9所示,左侧的NtCHA1-OV为转基因过表达材料中钾含量,右侧VC为转空载体作为对照烟叶中钾含量,明显可以看出NtCHA1-OV对比VC钾含量显著升高。
除上述具体实施过程外,这里需要注意的是,本发明应当还包括一种利用CRISPR/cas9技术敲除所述NtCHA1基因编辑后的烟草,其烟叶钾含量显著降低;当然还可以获得通过过表达所述NtCHA1基因编辑后的烟草。
以上描述了本发明的基本原理、主要特征和本发明的优点。本行业的技术人员应该了解,本发明不受上述实施例的限制,上述实施例和说明书中描述的只是说明本发明的原理,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。
序列表
<110> 云南省烟草农业科学研究院
<120> 一种可调控烟叶钾含量的NtCHA1基因及其应用
<130> P210778-YCN
<160> 12
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2418
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atggcttcaa ctttacctat gaaatgtcca ccacctatga aagcaacctc aaatggagta 60
ttccaagggg ataatccatt ggactatgca cttcctcttg ccattgtaca aatatgcttg 120
gtgcttgtac tcactcgagt cctcgcctat cttcttcgcc ctttaagaca accccgcgtc 180
attgctgaga ttgttggagg agttatactt ggtccgtctg ctcttggccg gaacgctaag 240
tatttgcacg caatatttcc atcaaggagt ctcacagtat tagatacctt agccaacttt 300
ggcctcctct tctttctttt cttagttggg ctcgagttag atccaaggtc tcttcgtcgg 360
acaggaaaga aagctcttag tattgcacta gctggaatta gtgtcccttt tgcattagga 420
atagggacat cctttgttct cagaggaact attggtaaag gagttagtca aggacctttt 480
ctagtattca tgggagtagc cctttctatt acagccttcc ccgtcttggc tcgtatccta 540
gctgaactca agcttttaac gacagatgtt ggtcgaatgg caatgtctgc tgcagcagtt 600
aatgatgtgg ctgcttggat tctacttgct cttgctattg ccctttcagg taccggtcat 660
tcgccccttg tttcactatg ggtacttttg tgtgggactg gttttgtcct actttgcata 720
ttcatctgtc ctcctatatt caaatggatg gctaaacgtt gctctcaggg cgagccagtg 780
aatgagttat atatctgtgc tacattagca gctgttttag ctgcaggatt tgttactgat 840
actattggta ttcatgcctt atttggagcg tttgtgctcg gagttcttgt accaaaggaa 900
gggccatttt ctggtgctct agtggaaaaa gtcgaggatc ttgtctccgg tctattcctt 960
ccactttact tcgtctccag tggattaaag acgaacgtag ctactattca aggcgcacaa 1020
tcatggggtc ttcttgttct agtcatattt acagcatgtt ttgggaaaat tgttggcact 1080
attttggtct ctctcttgtg taagatgccg gttcaggagg ctgttacgct tggtttcttg 1140
atgaatacta aaggtttagt ggagctcatt gtccttaaca ttggaaaaga tagaggggta 1200
ttgaatgatc aaacatttgc aatcatggtt ttgatggcgc tcttcacaac attcatcacg 1260
acacctatag tggtatcagt atataagcca gctaaactgg ctataaccga atacaagaac 1320
agaacgattg agaggaaaga cacgagtaaa caactccgaa tcttgacctg tttccatagc 1380
acaaagaaca ttcccacaat gatcaatctc atcgaggctt ctcgtggtac tgagaagaaa 1440
ggactctgcg tctatgcaat gcatcttatg gaactttccg aaagatcttc agctattcta 1500
atggtgcaca aggctagaaa aaatggactt cccttttgga aaaagggaga agtttcagat 1560
tctaatcaaa ttgttgttgc ttttgagact tttgagcaac tcagtaaggt ctctatccgg 1620
cctacaactg caatctctcc tatgaatagc atgcacgagg acatcatcgc tagtgcagag 1680
agaaagaggg tcgcgatgat aattctcccg ttccataaac atcaacgaat cgatggacat 1740
ttggaaacaa caagagctga tcttaggcat gtaaaccgga gagtacttca gcacgcgcct 1800
tgttcagttg gtatattagt agaccgagga ctaggtggtg catctcatgt atccgctagc 1860
aatgttgact ttaaagtaac cgtcttgttc tttggaggct atgatgatcg cgaagctctt 1920
gcttatggta cgcgtatagc agagcatcct ggcattaact tagtcgtggt tcgttttgta 1980
ctagaccctg aggttgttgg aaaaagtgtt aagttagata tggaacaaac ttatagtcct 2040
gaggcaaatt ccaaggatga agagttactt atcgacttaa aacacaaaat ttccaagaat 2100
ggttcagtca aatatgaaga gaagacagta aaggatgttg cagggactat agaatcgatt 2160
cgttcatata gtcgatgcaa tctgtttctt gttggaagaa tgtctgaggg tcaagtagta 2220
gcagcattgg ataaaaagag tgattgtcca gaattagggc cattaggtaa cttgttaact 2280
tgtccagaat tttcaactac agcatcagtt ttggtggtgc aacaatatcg aagcgagtta 2340
tctcaagatt caatcaattc attgaaggat ggagagttaa cagaagcgat aacgattcta 2400
actaaaaata ctagatag 2418
<210> 2
<211> 805
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ala Ser Thr Leu Pro Met Lys Cys Pro Pro Pro Met Lys Ala Thr
1 5 10 15
Ser Asn Gly Val Phe Gln Gly Asp Asn Pro Leu Asp Tyr Ala Leu Pro
20 25 30
Leu Ala Ile Val Gln Ile Cys Leu Val Leu Val Leu Thr Arg Val Leu
35 40 45
Ala Tyr Leu Leu Arg Pro Leu Arg Gln Pro Arg Val Ile Ala Glu Ile
50 55 60
Val Gly Gly Val Ile Leu Gly Pro Ser Ala Leu Gly Arg Asn Ala Lys
65 70 75 80
Tyr Leu His Ala Ile Phe Pro Ser Arg Ser Leu Thr Val Leu Asp Thr
85 90 95
Leu Ala Asn Phe Gly Leu Leu Phe Phe Leu Phe Leu Val Gly Leu Glu
100 105 110
Leu Asp Pro Arg Ser Leu Arg Arg Thr Gly Lys Lys Ala Leu Ser Ile
115 120 125
Ala Leu Ala Gly Ile Ser Val Pro Phe Ala Leu Gly Ile Gly Thr Ser
130 135 140
Phe Val Leu Arg Gly Thr Ile Gly Lys Gly Val Ser Gln Gly Pro Phe
145 150 155 160
Leu Val Phe Met Gly Val Ala Leu Ser Ile Thr Ala Phe Pro Val Leu
165 170 175
Ala Arg Ile Leu Ala Glu Leu Lys Leu Leu Thr Thr Asp Val Gly Arg
180 185 190
Met Ala Met Ser Ala Ala Ala Val Asn Asp Val Ala Ala Trp Ile Leu
195 200 205
Leu Ala Leu Ala Ile Ala Leu Ser Gly Thr Gly His Ser Pro Leu Val
210 215 220
Ser Leu Trp Val Leu Leu Cys Gly Thr Gly Phe Val Leu Leu Cys Ile
225 230 235 240
Phe Ile Cys Pro Pro Ile Phe Lys Trp Met Ala Lys Arg Cys Ser Gln
245 250 255
Gly Glu Pro Val Asn Glu Leu Tyr Ile Cys Ala Thr Leu Ala Ala Val
260 265 270
Leu Ala Ala Gly Phe Val Thr Asp Thr Ile Gly Ile His Ala Leu Phe
275 280 285
Gly Ala Phe Val Leu Gly Val Leu Val Pro Lys Glu Gly Pro Phe Ser
290 295 300
Gly Ala Leu Val Glu Lys Val Glu Asp Leu Val Ser Gly Leu Phe Leu
305 310 315 320
Pro Leu Tyr Phe Val Ser Ser Gly Leu Lys Thr Asn Val Ala Thr Ile
325 330 335
Gln Gly Ala Gln Ser Trp Gly Leu Leu Val Leu Val Ile Phe Thr Ala
340 345 350
Cys Phe Gly Lys Ile Val Gly Thr Ile Leu Val Ser Leu Leu Cys Lys
355 360 365
Met Pro Val Gln Glu Ala Val Thr Leu Gly Phe Leu Met Asn Thr Lys
370 375 380
Gly Leu Val Glu Leu Ile Val Leu Asn Ile Gly Lys Asp Arg Gly Val
385 390 395 400
Leu Asn Asp Gln Thr Phe Ala Ile Met Val Leu Met Ala Leu Phe Thr
405 410 415
Thr Phe Ile Thr Thr Pro Ile Val Val Ser Val Tyr Lys Pro Ala Lys
420 425 430
Leu Ala Ile Thr Glu Tyr Lys Asn Arg Thr Ile Glu Arg Lys Asp Thr
435 440 445
Ser Lys Gln Leu Arg Ile Leu Thr Cys Phe His Ser Thr Lys Asn Ile
450 455 460
Pro Thr Met Ile Asn Leu Ile Glu Ala Ser Arg Gly Thr Glu Lys Lys
465 470 475 480
Gly Leu Cys Val Tyr Ala Met His Leu Met Glu Leu Ser Glu Arg Ser
485 490 495
Ser Ala Ile Leu Met Val His Lys Ala Arg Lys Asn Gly Leu Pro Phe
500 505 510
Trp Lys Lys Gly Glu Val Ser Asp Ser Asn Gln Ile Val Val Ala Phe
515 520 525
Glu Thr Phe Glu Gln Leu Ser Lys Val Ser Ile Arg Pro Thr Thr Ala
530 535 540
Ile Ser Pro Met Asn Ser Met His Glu Asp Ile Ile Ala Ser Ala Glu
545 550 555 560
Arg Lys Arg Val Ala Met Ile Ile Leu Pro Phe His Lys His Gln Arg
565 570 575
Ile Asp Gly His Leu Glu Thr Thr Arg Ala Asp Leu Arg His Val Asn
580 585 590
Arg Arg Val Leu Gln His Ala Pro Cys Ser Val Gly Ile Leu Val Asp
595 600 605
Arg Gly Leu Gly Gly Ala Ser His Val Ser Ala Ser Asn Val Asp Phe
610 615 620
Lys Val Thr Val Leu Phe Phe Gly Gly Tyr Asp Asp Arg Glu Ala Leu
625 630 635 640
Ala Tyr Gly Thr Arg Ile Ala Glu His Pro Gly Ile Asn Leu Val Val
645 650 655
Val Arg Phe Val Leu Asp Pro Glu Val Val Gly Lys Ser Val Lys Leu
660 665 670
Asp Met Glu Gln Thr Tyr Ser Pro Glu Ala Asn Ser Lys Asp Glu Glu
675 680 685
Leu Leu Ile Asp Leu Lys His Lys Ile Ser Lys Asn Gly Ser Val Lys
690 695 700
Tyr Glu Glu Lys Thr Val Lys Asp Val Ala Gly Thr Ile Glu Ser Ile
705 710 715 720
Arg Ser Tyr Ser Arg Cys Asn Leu Phe Leu Val Gly Arg Met Ser Glu
725 730 735
Gly Gln Val Val Ala Ala Leu Asp Lys Lys Ser Asp Cys Pro Glu Leu
740 745 750
Gly Pro Leu Gly Asn Leu Leu Thr Cys Pro Glu Phe Ser Thr Thr Ala
755 760 765
Ser Val Leu Val Val Gln Gln Tyr Arg Ser Glu Leu Ser Gln Asp Ser
770 775 780
Ile Asn Ser Leu Lys Asp Gly Glu Leu Thr Glu Ala Ile Thr Ile Leu
785 790 795 800
Thr Lys Asn Thr Arg
805
<210> 3
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
attgctcaaa tggagtattc caag 24
<210> 4
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
aaaccttgga atactccatt tgag 24
<210> 5
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
cacgggttca attagtctcg ca 22
<210> 6
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gagaccttgg atctaactcg ag 22
<210> 7
<211> 25
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
tgtcccagga ttagaatgat taggc 25
<210> 8
<211> 24
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aaaccgattc atcgcaacca attc 24
<210> 9
<211> 33
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
ggatccatgg cttcaacttt acctatgaaa tgt 33
<210> 10
<211> 34
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
gatatcctat ctagtatttt tagttagaat cgtt 34
<210> 11
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
ggctgcttgg attctacttg ct 22
<210> 12
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cctgagagca acgtttagcc at 22
Claims (10)
1.一种可调控烟叶钾含量的NtCHA1基因,其核苷酸序列为SEQ ID NO:1所示。
2.一种可调控烟叶钾含量的蛋白,其特征在于,其氨基酸序列为SEQ ID NO:2所示,或根据权利要求1所述的NtCHA1基因编码为SEQ ID NO:2所示的氨基酸序列的蛋白。
3.一种可调控烟叶钾含量的NtCHA1基因的应用,其特征在于,使用权利要求1所述的NtCHA1基因或权利要求2所述的蛋白在调控烟草钾离子含量中的应用。
4.根据权利要求3所述的应用,其特征在于,利用所述NtCHA1基因构建CRISPR/cas9载体,包括以下过程:
(1)根据所述NtCHA1基因的基因组序列设计靶位点,所述靶位点为:
sgRNA:CTCAAATGGAGTATTCCAAG
(2)根据步骤(1)中的所述靶位点设计引物,得到靶位点引物,所述靶位点引物序列为:
P1:ATTGCTCAAATGGAGTATTCCAAG;
P2:AAACCTTGGAATACTCCATTTGAG;
(3)根据步骤(1)中的靶位点,在靶位点两侧设计编辑材料的检测引物,得到检测引物,所述检测引物序列为:
NtCHA1-SdF:CACGGGTTCAATTAGTCTCGCA;
NtCHA1-SdR:GAGACCTTGGATCTAACTCGAG;
(4)制备dsDNA:根据步骤(2)中所述的靶位点引物通过退火形成互补DNA oligo,得到dsDNA;
(5)酶切pHSE401载体得到酶切产物,将所述酶切产物与步骤(4)中得到的dsDNA进行连接,得到连接产物;酶切后对酶切产物进行电泳检测分析,回收11520bp的酶切产物备用;
(6)测序验证:将步骤(5)中得到的连接产物转化为大肠杆菌,筛选阳性克隆并进行菌落PCR检测,经PCR检测验证正确的阳性克隆菌株培养扩增后进一步进行测序分析,得到所述CRISPR/cas9载体;所述菌落PCR检测时,所用引物序列为:
U6-26p-F:TGTCCCAGGATTAGAATGATTAGGC;
U6-26p-R:AAACCGATTCATCGCAACCAATTC。
5.根据权利要求3所述的应用,其特征在于,利用所述NtCHA1基因构建的提高烟叶钾含量的转基因烟草材料。
6.根据权利要求4所述的应用,其特征在于,利用所述NtCHA1基因构建的提高烟叶钾含量的转基因烟草材料包括以下步骤:
A构建NtCHA1过表达载体:
a1.克隆NtCHA1基因并连接TOPO载体:利用NtCHA1基因特异引物进行扩增,得到NtCHA1基因片段,并纯化回收,将回收的所述基因片段进行TOPO克隆,连接到TOPO载体后转化大肠杆菌DH5α感受态细胞,提取质粒进行PCR检测,挑选扩增产物大小约为2418bp的质粒提取DNA,构建得到TOPO载体;
a2.植物过表达载体的构建:利用BamH I/EcoR V酶切所述TOPO载体和pENTRTM2B,得到目的基因片段NtCHA1和载体pENTRTM2B线性化片段,胶回收后进行连接、转化感受态细胞DH5α;挑取转化DH5α后的克隆,提取质粒DNA,构建得到入门克隆载体;将所述入门克隆载体和表达载体通过LR反应后转化大肠杆菌DH5α感受态细胞,得到重组表达载体;
B:烟草的遗传转化:
b1.表达载体转化农杆菌,将农杆菌感受态细胞溶解后加入重组表达载体进行农杆菌转化,在LB固体培养基中培养后,得到含有目标载体的农杆菌克隆;
b2.烟草转化,挑取含有目标载体的农杆菌克隆,培养得到含目标载体的农杆菌悬浮菌液;将清洗后的烟草叶片,用无菌吸水纸吸去表面液体,使用剪刀将无菌叶片切成约1cm×1cm的小片,放入含目标载体的无菌MS液体培养基农杆菌悬浮菌液中,静置15-20min;取出烟草叶片,于MS培养基中25℃暗培养两天;随后,把烟草叶片转入分化培养基中,温室条件下分化培养,分化出芽后,将长至3-5cm的芽切下,转入MS培养基诱导生根,取出生根后的转基因植株用自来水洗净移植于灭菌的营养土中,为转化后的烟草植株。
7.根据权利要求6所述的应用,其特征在于,所述NtCHA1基因特异引物,其核苷酸序列为:
CHA1-F:GGATCCATGGCTTCAACTTTACCTATGAAATGT;
CHA1-R:GATATCCTATCTAGTATTTTTAGTTAGAATCGTT。
8.根据权利要求6所述的应用,其特征在于,所述LR反应体系为:加入构建成功的所述入门克隆载体(50-150ng)1-7μL,以及0.5μL表达载体,TE Buffer至总体积8μL;混匀,冰浴2min,轻弹2次;加入2μL LR CloneaseTMⅡenzyme Mix,轻弹,混匀,离心,25℃水浴1h;然后加入1μL Proteinase K轻弹,混匀,37℃水浴10min。
9.根据权利要求6所述的应用,其特征在于,
所述LB固体培养基含有壮观霉素100mg/L和利福平25mg/L;
所述MS培养基含有0.02mg/L 6-BA、2mg/L NAA。
10.根据权利要求6所述的应用,其特征在于,所述表达载体为Destination Vector,即pK2GW7载体;所述分化培养基为含有0.5mg/L 6-BA、0.1mg/L NAA、100mg/L卡那霉素、500mg/L头孢霉素的MS培养基。
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