CN117384917A - 一种烟草果胶甲酯酶抑制子基因NtPMEI及其应用和方法 - Google Patents
一种烟草果胶甲酯酶抑制子基因NtPMEI及其应用和方法 Download PDFInfo
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Abstract
本发明属于分子生物学和基因工程领域,且公开了烟草果胶甲酯酶抑制子基因NtPMEI及其在调节烟草对赤星病菌抗性中的应用,应用方法如下:从栽培烟草中克隆得到NtPMEI基因,并构建了含有NtPMEI基因的过表达载体及含有NtPMEI基因靶位点的敲除载体;在赤星病抗病品种净叶黄(JYH)中创制NtPMEI过表达转基因材料,感病品种NC82中创制NtPMEI敲除材料。通过对转基因材料和野生型材料的抗病性鉴定发现,在净叶黄中,过表达材料病斑直径显著高于野生型;而在NC82中,敲除材料的病斑明显减小,表明NtPMEI基因负向调控烟草对赤星病菌的抗性。敲除感病品种NC82中NtPMEI基因使得烟草对烟草赤星病菌的抗性增强。本发明证实了NtPMEI基因在调节烟草赤星病抗性中的新用途。
Description
技术领域
本发明属于分子生物学和基因工程技术领域,具体为烟草果胶甲酯酶抑制子基因NtPMEI及其在调节烟草对赤星病菌抗性中的应用。
背景技术
烟草赤星病是由链格孢菌侵染引起的真菌性病害,主要危害烟草叶片,一般发生于烟株打顶后和下部烟叶进入成熟采收的阶段。目前赤星病的防治主要有栽培防治、生物防治、药剂防治及培育抗病新品种等方法。在栽培过程中加强管理是防治赤星病菌的有效手段,通过合理施肥、密植、轮作、田间管理等措施可以在一定程度上减少病害的发生,但面临病害大规模发生等紧急情况时,效果甚微;生物防治具有高效、绿色、健康的特点,具有较好的应用前景,但大规模应用于烟田成本较高;药剂防治是目前生产中运用最为广泛的防治方式,见效快、方便快捷、效果好,在实践中也取得不错的防治效果,但存在药剂残留、影响烟叶品质以及病菌抗药性等缺点;培育抗病优质新品种是最为经济有效的措施,可以在一定程度上控制烟草赤星病的大规模发生。但目前对烟草赤星病抗性基因的报道还相对有限,因此提出一种烟草果胶甲酯酶抑制子基因NtPMEI及其在调节烟草对赤星病菌抗性中的应用。
发明内容
本发明的目的在于提供烟草果胶甲酯酶抑制子基因NtPMEI及其在调节烟草对赤星病菌抗性中的应用,以解决上述背景技术中提出的问题。
为了实现上述目的,本发明提供如下技术方案:
烟草果胶甲酯酶抑制子基因NtPMEI全长591bp,有1个外显子,开放阅读框591bp,编码196个氨基酸,核苷酸序列、氨基酸序列如SEQ ID NO.1、SEQ ID NO.2所示。
从栽培烟草(Nicotiana tabacum)中克隆得到NtPMEI基因,并构建了含有NtPMEI基因的过表达载体及含有NtPMEI基因靶位点的敲除载体;
在赤星病抗病品种净叶黄(JYH)中创制过表达转基因材料,感病品种NC82中创制NtPMEI敲除材料,并对NtPMEI过表达和敲除株系进行检测;
对转基因材料和野生型材料进行抗病性鉴定,发现在净叶黄中,过表达材料病斑直径显著高于野生型;而在NC82中,敲除材料的病斑明显减小,表明NtPMEI基因负向调控烟草对赤星病菌的抗性。
优选地,所述克隆得到NtPMEI基因的操作步骤如下:
提取烟草总RNA,进行cDNA反转录,根据NtPMEI基因的序列设计出完整编码框的上下游特异性引物,进行PCR扩增反应;所述特异性引物为PMEI-KLF:ACATATGCCCGTCGACATGA,PMEI-KLR:TGCTCACCATGAATTCCTATGA;
PCR反应结束后,制备1.2%琼脂糖凝胶,150V电压下电泳15min,检测扩增片段特异性,并测序验证;对验证正确的扩增片段进行DNA纯化。
优选地,所述敲除载体的构建操作步骤如下:
根据NtPMEI基因序列,利用在线工具设计合适的靶位点,由北京六合华大基因科技有限公司合成为引物,将合成完毕的单链Oligo DNA退火形成DNA双链,退火反应体系及程序如下:
95℃,2min;-0.1℃/8sec,降至25℃;4℃保存;
使用限制性内切酶BsaI酶切表达载体CRISPR/Cas9,37℃,反应15min后回收酶切产物并进行纯化;
将靶点用T4连接酶连接到线性化载体上,4℃过夜连接;
连接产物转化大肠杆菌感受态细胞DH5α后挑取单克隆菌落进行检测,用载体上的引物JC-F和靶位点的下游引物PCR扩增,目的条带约为450bp,选择阳性克隆菌液测序;
对测序正确的菌落扩大培养,提取质粒,PCR及测序鉴定正确后,得到植物敲除表达载体,命名为pORE-Cas9-NtPMEI;
采用热激法转化至农杆菌感受态细胞EHA105,获得含有目的质粒的农杆菌菌株用于后续实验。
优选地,所述过表达载体的构建操作步骤如下:
使用限制性内切酶EcoRⅠ和SalⅠ酶切表达载体PRI101-eGFP;
根据NtPMEI基因序列及EcoRⅠ和SalⅠ酶切位点,设计同源重组引物CZ-F、CZ-R并进行PCR扩增;
将扩增产物与线性化PRI101-eGFP载体进行同源重组,37℃连接30min;将连接产物转化大肠杆菌,提取阳性克隆的质粒,测序正确后获得NtPMEI过表达载体,命名为PRI101-NtPMEI;转化农杆菌用于后续实验。
优选地,所述转基因烟草植株的获得步骤如下:
消毒后的种子播种于MS培养基中,人工气候室培养至5-6周大小,备用;将含有目的基因载体的农杆菌接种于含有抗生素(Kan、Rif均为50mg/L)的液体LB培养基中,28℃,220rpm,培养约24h,至OD600为0.5-0.6;将菌液分装到1.5mL离心管中,12000rpm离心1min;用100μL无菌水重悬,然后加入终浓度为20mg/L的乙酰丁香酮,用于侵染;
取无菌烟苗叶片,剪成1.0×1.0cm的小块,并用镊子将其放入农杆菌侵染液侵染10min;将侵染后的叶片捞出,平铺在无菌滤纸上吸干叶片上的农杆菌菌液,然后叶面向下平铺到共培养培养基上,26℃黑暗培养3d;
共培养完成后,叶正面朝上,平铺于MS选择培养基(MS+6-BA 1mg/L+NAA 0.1mg/L+Cef 500mg/L+Kan 80mg/L)中,放置于人工气候室培养2-3周;
将分化出的幼苗切割转移至生根培养基中(MS+Cef 500mg/L+Kan 80mg/L);
利用无菌水清洗根部,转移至基质中长大。
优选地,所述NtPMEI过表达和突变烟草植株的检测包括转NtPMEI烟草DNA水平的鉴定、转NtPMEI烟草RNA水平的鉴定和NtPMEI敲除烟草DNA水平的鉴定:
所述转NtPMEI烟草DNA水平的鉴定,使用2×CTAB缓冲液提取T0代转NtPMEI烟草叶片的基因组DNA,进行PCR检测,所用引物为PMEI-F1、PMEI-R1,用构建好的载体作阳性对照,用非转基因植株净叶黄作为阴性对照,结果显示,转基因植物中和阳性对照可以扩增出片段大小为350bp左右的片段,而阴性对照净叶黄野生型中没有扩增出,表明得到了净叶黄转基因阳性植株;
所述转NtPMEI烟草RNA水平的鉴定,提取T0代阳性苗的总RNA,cDNA反转录,以烟草管家基因(NTU60495)为内参进行表达量检测,每个样品设三次重复,通过ABI 7500FastReal-TimePCR System的SDSShell.exe分析系统分析NtPMEI基因的相对表达量,非转化烟草的NtPMEI基因的表达水平低,而T0代转NtPMEI烟草中,PMEI-OE-1、PMEI-OE-2均过量表达,表达量提高倍数约为5-7倍;
所述NtPMEI敲除烟草DNA水平的鉴定,选取NC82 NtPMEI基因敲除植株进行基因测序,敲除材料测序比对所用引物为PMEI-JCF1、PMEI-JCR1,在感性品种NC82中得到了转基因敲除纯合植株,相比于NC82野生型,NC82敲除株系PMEI-KO-1在目标靶位点插入了一个A碱基,PMEI-KO-2在目标靶位点插入了一个T碱基。
优选地,所述应用方法中赤星病抗性鉴定是采用离体叶片接种的方法使转基因烟草感染烟草赤星病菌,分别在在接种3d、5d、7d后观察叶片,使用Image J软件计算坏死斑面积,每个叶片上的坏死斑取平均值,单位为cm2,其接种方法下:
(1)待烟草植株生长至8周,取生长状态健康一致且处于相同叶位的叶片;
(2)用燕麦培养基培养链格孢菌,28℃恒温培养约15d左右,此时菌快长满整个培养基,菌体呈黑色,边缘颜色较浅;
(3)配置孢子悬浮液。向培养基中加入10mL无菌水配置的1%葡萄糖溶液,用刷子轻轻刮落菌丝,使孢子脱落到1%葡萄糖溶液中,随后将葡萄糖溶液用纱布过滤,留下滤液,即为孢子悬浮液。将孢子悬浮液置于血球计数器下测量孢子的浓度,并稀释到3×105个/mL备用;
(4)将离体叶片背面朝上放置入盛有水的海绵托盘上,在其叶柄处覆盖蘸水的脱脂棉保湿,将80μL的孢子悬浮液接种于叶片背面左右两侧对称的4处后用灭过菌的牙签在接种处划出大小、深浅一致的伤口;
(5)用保鲜膜封口,28℃恒温,16h光照/8h黑暗进行培养,持续观察。
本发明的有益效果如下:
本发明通过从栽培烟草中克隆得到NtPMEI基因,并构建了含有NtPMEI基因的过表达载体及含有NtPMEI基因靶位点的敲除载体,在赤星病抗病品种净叶黄(JYH)中创制NtPMEI过表达转基因材料,感病品种NC82中创制NtPMEI敲除材料。通过对转基因材料和野生型材料的抗病性鉴定发现,在净叶黄中,过表达材料病斑直径显著高于野生型;而在NC82中,敲除材料的病斑明显减小,表明NtPMEI基因负向调控烟草对赤星病菌的抗性,该设计提供了烟草果胶甲酯酶抑制子基因NtPMEI以及利用该基因调节烟草对赤星病菌抗性的用途。本发明通过在抗病品种净叶黄中过表达NtPMEI基因使得烟草对烟草赤星病菌的抗性减弱,敲除感病品种NC82中NtPMEI基因使得烟草对烟草赤星病菌的抗性增强。本发明证实了NtPMEI基因在调节烟草赤星病抗性中的新用途。
附图说明
图1为本发明T0代过表达转基因烟草DNA水平检测结果示意图;
图2为本发明T0代过表达转基因阳性烟草植株RNA水平检测结果示意图;
图3为本发明T0代敲除烟草植株DNA水平检测结果示意图;
图4为本发明净叶黄NtPMEI过表达材料与野生型在接种链格孢菌后3d、5d、7d的表型及接种链格孢菌后5d的病斑直径大小示意图;
图5为本发明NC82 NtPMEI敲除材料与野生型在接种链格孢菌后3d、5d、7d的表型及接种链格孢菌后5d的病斑直径大小示意图。
具体实施方式
下面将结合本发明实施例中的附图,对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
如图1至图5所示,本发明实施例提供了烟草果胶甲酯酶抑制子基因NtPMEI在调节烟草对赤星病菌抗性中的应用,应用方法如下:
S1:从栽培烟草中克隆得到NtPMEI基因,并构建了含有NtPMEI基因的过表达载体及含有NtPMEI基因靶位点的敲除载体;
S2:在赤星病抗病品种净叶黄(JYH)中创制NtPMEI过表达转基因材料,感病品种NC82中创制NtPMEI敲除材料,通过对转基因材料和野生型材料的抗病性鉴定发现,在净叶黄中,过表达材料病斑直径显著高于野生型;而在NC82中,敲除材料的病斑明显减小,表明NtPMEI基因负向调控烟草对赤星病菌的抗性。
上述技术方案的工作原理和有益效果为:通过实验证明,过表达NtPMEI烟草中烟草对赤星病菌抗性下降,而NtPMEI功能丧失后可以提高烟草对烟草赤星病菌的抗性,因此,发现了调节烟草对赤星病菌抗性的方法,同时提供了相应的转基因材料。
如图所示,在一个实施例中,应用方法S1中栽培烟草中克隆得到NtPMEI基因的操作步骤如下:
A1:按照FastPure Plant Total RNA Isolation Kit植物总RNA提取(Vazyme)试剂盒说明书提取烟草总RNA,取幼嫩的新鲜叶片约50-100mg于2mL离心管中,加入钢珠,打磨至粉末状;加入500μL Buffer PRLPlus,涡旋震荡30-60s,12,000rpm 4℃离心5min;吸取上清至吸附柱Ⅱ中,12,000rpm 4℃离心2min;向滤液中加入0.5倍滤液积的无水乙醇,吹打混匀,转移至吸附柱Ⅳ中,12,000rpm 4℃离心2min,弃滤液;加入700μL Buffer PRW1,室温放置1min,12,000rpm 4℃离心30sec,弃滤液;加入500μL Buffer PRW2,12,000rpm 4℃离心30sec,弃滤液,重复两次;12,000rpm 4℃离心2min,开盖放置2-5min;转移吸附柱至1.5mL离心管中,加入RNase-free ddH2O,室温放置2min,12,000rpm 4℃离心1min,检测后-80℃保存;
A2:按照EasyScriptAll-in-One First-Strand cDNA Synthesis SuperMix forqPCR(TransGen Biotech)说明书将提取的RNA反转录为cDNA,在RNase-free离心管中配制混合液:
混匀后42℃孵育15min,85℃加热5sec,-20℃保存;
A3:根据NtPMEI基因的序列设计出完整编码框的上下游特异性引物,进行PCR扩增反应:
所述特异性引物如下:(如SEQ ID NO.3、SEQ ID NO.4所示)
PMEI-KLF:ACATATGCCCGTCGACATGA
PMEI-KLR:TGCTCACCATGAATTCCTATGA
反应条件为95℃预变性5min,95℃变性30sec,根据所使用的引物的Tm值确定退火温度,退火时间30sec,72℃延伸,延伸时间根据目的片段的长度和所用PCR反应的酶的活性确定,循环数设置为35cycles,72℃延伸10min,最后4℃保存;
PCR反应结束后,制备1.2%琼脂糖凝胶,150V电压下电泳15min,检测扩增片段特异性,并测序验证;
A4:通过琼脂糖凝胶电泳和测序验证正确后按照FastPure Gel DNA ExtractionMini Kit DNA(Vazyme)试剂盒说明书对PCR产物进行纯化回收:
在紫外灯照射条件下切下含有目的片段的凝胶置于2mL离心管中;加入等倍体积的Buffer GDP,50℃水浴直至凝胶块完全溶解,将溶胶液转移至吸附柱(置于收集管上)中,12,000rpm离心30-60sec;弃滤液,加入300μL Buffer GDP,静止1min。12,000rpm离心30-60sec,弃滤液,加入700μL Buffer GW,12,000rpm离心30-60sec,重复2次;弃滤液,把吸附柱置回收集管中,12,000rpm离心2min;将吸附柱置于灭菌的2mL离心管中,加入20-30μLElution Buffer于吸附柱中央,室温静置2min,12,000rpm离心1min得到纯化后的DNA。
上述技术方案的工作原理和有益效果为:该设计采用基因克隆方法从烟草中获得序列正确的烟草果胶甲酯酶抑制子NtPMEI基因。
如图所示,在一个实施例中,应用方法S1中敲除载体的构建操作步骤如下:
B1:根据NtPMEI基因序列,利用在线工具http://www.multicrispr.net/index.htmL设计合适的靶位点,由北京六合华大基因科技有限公司合成为引物,将合成完毕的单链Oligo DNA退火形成DNA双链,退火反应体系如下及程序:
95℃,2min;-0.1℃/8sec,降至25℃;4℃保存;
B2:使用限制性内切酶BsaI(NEB)酶切表达载体CRISPR/Cas9:
37℃,反应15min;回收酶切产物并进行纯化,DNA纯化方法如上。将靶点用T4连接酶(Vazyme)连接到线性化载体上,4℃过夜连接;
B3:将连接产物转化大肠杆菌感受态细胞DH5α:
冰上解冻感受态细胞,加入连接产物,轻弹管壁混匀,冰上静置30min;42℃热激30sec,迅速置于冰上2min;向离心管中加入900μL不含抗生素的LB液体培养基,37℃,220rpm,孵育1h;5,000rpm离心5min,弃900μL上清,用剩余培养基重悬菌体,涂布在含有Kan抗性的LB平板上;37℃培养箱中倒置培养12-16h;
挑取单克隆菌落进行检测,用载体上的引物JC-F(如SEQ ID NO.5所示)和靶位点的下游引物PCR扩增,目的条带约为450bp;选择阳性克隆菌液进行测序;测序引物为CX-F(如SEQ ID NO.6所示);
B4:对测序正确的菌落扩大培养,按照FastPure Plasmid Mini Kit(Vazyme)试剂盒说明书提取质粒:
取1-5mL过夜培养的菌液,10,000rpm离心1min,弃培养基;向留有菌体沉淀的离心管中加入250μL Buffer P1,混匀后加入250μL Buffer P2,温和地上下颠倒混匀8-10次;再加入350μL Buffer P3,立即温和地上下颠倒混匀8-10次,12,000rpm离心10min;将上清液转移至吸附柱中,12,000rpm离心30-60sec,弃废液;向吸附柱加入600μL Buffer PW2,12,000rpm离心30-60sec,弃废液,重复2次;12,000rpm离心1min;把吸附柱置于新的灭菌的1.5mL离心管中,加入30-100μL 55℃的Elution Buffer至吸附柱膜中央,室温静置2min;12,000rpm离心1min得到质粒溶液。质粒PCR鉴定正确后送生物公司测序,结果显示,该质粒为将NtPMEI基因的靶位点插入pORE-Cas9载体中BsaI酶切位点间得到的植物表达载体,命名为pORE-Cas9-NtPMEI;
B5:采用热激法转化至农杆菌感受态细胞EHA105:
解冻感受态细胞,加入质粒DNA,轻弹管壁混匀,依次于冰上静置5min、液氮5min、28℃水浴5min、冰浴5min;加入700μL不含抗生素的LB液体培养基,28℃振荡培养2-3h;6,000rpm离心1min收菌,留取100μL左右上清轻轻吹打重悬菌块,涂布于含Kan和Rif抗性的LB平板上,倒置放于28℃培养箱培养2-3d;
挑取单克隆菌落进行菌落PCR,使用JC-F和靶点下游的引物,阳性克隆目的条带约为450bp,选取条带正确单克隆进行测序。成功获得含有目的质粒的农杆菌菌株用于后续实验。
上述技术方案的工作原理和有益效果为:该设计构建了NtPMEI基因的敲除载体pORE-Cas9-NtPMEI,并将该载体转化EHA105农杆菌感受态细胞,获得用于转化烟草的沉默NtPMEI基因的编辑载体的农杆菌菌株。
如图所示,在一个实施例中,应用方法S1中过表达载体的构建为表达载体酶切、目的基因扩增、DNA纯化、连接产物转化及质粒提取方法同上,阳性菌落鉴定、测序所使用的引物为35S-F和M13-R通用引物,其操作步骤如下:
C1:使用限制性内切酶EcoRⅠ(NEB)和SalⅠ(NEB)酶切表达载体PRI101-eGFP;
C2:根据NtPMEI基因序列及EcoRⅠ和SalⅠ酶切位点,设计同源重组引物CZ-F、CZ-R(如SEQ ID NO.7、SEQ ID NO.8所示)并进行PCR扩增;
C3:按照ClonExpress II One Step Cloning Kit(Vazyme)说明书将扩增产物与线性化PRI101-eGFP载体进行同源重组,37℃连接30min;
C4:将连接产物转化大肠杆菌,提取阳性克隆的质粒,测序正确后获得NtPMEI基因植物过表达载体,命名为PRI101-NtPMEI。转化农杆菌,将经检测后成功获得含有目的质粒的农杆菌菌株用于后续实验。
上述技术方案的工作原理和有益效果为:该设计构建了NtPMEI基因的过表达载体PRI101-NtPMEI,并将NtPMEI过表达载体转化EHA105农杆菌感受态细胞,获得用于转化烟草的含NtPMEI基因过表达载体的农杆菌菌株。
如图所示,在一个实施例中,应用方法S2中转基因烟草植株的获得步骤如下:
D1:消毒后的种子,播种于MS培养基中,人工气候室培养至5-6周大小,备用;
D2:将含有目的基因载体的农杆菌接种于含有抗生素(Kan、Rif均为50mg/L)的液体LB培养基中,28℃,220rpm,培养约24h,至OD600为0.5-0.6;将菌液分装到1.5mL离心管中,12000rpm离心1min;用100μL无菌水重悬,然后加入终浓度为20mg/L的乙酰丁香酮,准备用于侵染;
D3:取无菌烟苗叶片,剪成1.0×1.0cm的小块,并用镊子将其放入农杆菌侵染液侵染10min;将侵染后的叶片捞出,平铺在无菌滤纸上,吸干叶片上的农杆菌菌液,然后叶面向下平铺到共培养培养基上,26℃黑暗培养3d;
D4:共培养完成后,叶正面朝上,平铺于MS选择培养基(MS+6-BA 1mg/L+NAA0.1mg/L+Cef 500mg/L+Kan 80mg/L)中,放置于人工气候室培养2-3周;
D5:将分化出的幼苗切割转移至生根培养基中(MS+Cef 500mg/L+Kan80mg/L);
D6:利用无菌水清洗根部,转移至基质中长大;
D7:转基因植物的检测。
上述技术方案的工作原理和有益效果为:该设计利用所构建的含有表达载体的农杆菌菌株转化烟草,经检测获得转基因烟草植株。
如图所示,在一个实施例中,转基因烟草植株的获得步骤D7中转基因植物的检测包括有转NtPMEI烟草DNA水平的鉴定、转NtPMEI烟草RNA水平的鉴定和NtPMEI敲除烟草DNA水平的鉴定;
转NtPMEI烟草DNA水平的鉴定,使用CTAB提取T0代转NtPMEI烟草叶片的基因组DNA:取幼嫩的新鲜叶片约50-100mg于2mL离心管中,加入钢珠,打磨至粉末状;加入600μL 2×CTAB缓冲液(Solarbio),摇匀;加入600μL苯酚:氯仿:异戊醇(25:24:1)DNA抽提液(Acmec),摇匀;12,000rpm 4℃离心10min;吸取上清至新的灭菌的1.5ml离心管中,加入等体积预冷的异丙醇,轻轻上下颠倒,混匀;12,000rpm 4℃离心5min,弃上清;用75%乙醇清洗2次,无水乙醇清洗1次,待乙醇挥发完后,加入ddH2O溶解DNA,-20℃保存,用PCR检测得到的转基因植物,所用引物为PMEI-F1、PMEI-R1,用构建好的载体作阳性对照,用非转基因植株净叶黄作为阴性对照,结果显示,转基因植物中和阳性对照可以扩增出片段大小为350bp左右的片段,而阴性对照净叶黄野生型中没有扩增出,表明得到了净叶黄转基因阳性植株;
转NtPMEI烟草RNA水平的鉴定,提取T0代阳性苗的总RNA,cDNA反转录,使用ChamQSYBR Color qPCR Master Mix(Vazyme),以烟草管家基因(NTU60495)为内参进行表达量检测,每个样品设三次重复,通过ABI 7500Fast Real-Time PCR System的SDSShell.exe分析系统分析NtPMEI基因的相对表达量(图2),图2表达量检测结果表明,非转化烟草的NtPMEI基因的表达水平低,而T0代转NtPMEI烟草中,PMEI-OE-1、PMEI-OE-2均过量表达,表达量提高倍数约为5-7倍;
NtPMEI敲除烟草DNA水平的鉴定,选取NC82基因敲除植株进行基因测序,敲除材料测序比对所用引物为PMEI-JCF1、PMEI-JCR1(如SEQ ID NO.11、SEQ ID NO.12所示),结果显示(图3)在感性品种NC82中得到了转基因敲除纯合植株,相比于NC82野生型,NC82敲除株系PMEI-KO-1在目标靶位点插入了一个A碱基,PMEI-KO-2在目标靶位点插入了一个T碱基。本实施例利用所构建的含有表达载体的农杆菌菌株转化烟草,经检测获得转基因烟草植株。
如图所示,在一个实施例中,应用方法S2中赤星病抗性鉴定是采用离体叶片接种的方法使转基因烟草感染烟草赤星病菌,分别在在接种3d、5d、7d后观察叶片,使用Image J软件计算坏死斑面积,每个叶片上的坏死斑取平均值,单位为cm2,其步接种方法下:
(1)待烟草植株生长至8周,取生长状态健康一致且处于相同叶位的叶片;
(2)用燕麦培养基培养链格孢菌,28℃恒温培养约15d左右,此时菌快长满整个培养基,菌体呈黑色,边缘颜色较浅;
(3)配置孢子悬浮液。向培养基中加入10mL无菌水配置的1%葡萄糖溶液,用刷子轻轻刮落菌丝,使孢子脱落到1%葡萄糖溶液中,随后将葡萄糖溶液用纱布过滤,留下滤液,即为孢子悬浮液。将孢子悬浮液置于血球计数器下测量孢子的浓度,并稀释到3×105个/mL备用;
(4)将离体叶片背面朝上放置入盛有水的海绵托盘上,在其叶柄处覆盖蘸水的脱脂棉保湿,将80μL的孢子悬浮液接种于叶片背面左右两侧对称的4处后用灭过菌的牙签在接种处划出大小、深浅一致的伤口;
(5)用保鲜膜封口,28℃恒温,16h光照/8h黑暗进行培养,持续观察。
上述技术方案的工作原理和有益效果为:通过转基因烟草叶片离体接种赤星病菌后发现,对于抗病材料净叶黄,过表达材料的抗病性要弱于野生型;对于感病材料NC82,敲除材料的抗病性要强于野生型,过表达NtPMEI基因使烟草赤星病抗性降低,而敲除NtPMEI基因使烟草赤星病抗性提高,说明NtPMEI基因负向调控烟草对赤星病的抗性。
如图所示,在一个实施例中,烟草果胶甲酯酶抑制子基因NtPMEI,全长591bp,有1个外显子,开放阅读框591bp,编码196个氨基酸。
需要说明的是,在本文中,诸如第一和第二等之类的关系术语仅仅用来将一个实体或者操作与另一个实体或操作区分开来,而不一定要求或者暗示这些实体或操作之间存在任何这种实际的关系或者顺序。而且,术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含,从而使得包括一系列要素的过程、方法、物品或者设备不仅包括那些要素,而且还包括没有明确列出的其他要素,或者是还包括为这种过程、方法、物品或者设备所固有的要素。
尽管已经示出和描述了本发明的实施例,对于本领域的普通技术人员而言,可以理解在不脱离本发明的原理和精神的情况下可以对这些实施例进行多种变化、修改、替换和变型,本发明的范围由所附权利要求及其等同物限定。
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Claims (9)
1.一种烟草果胶甲酯酶抑制子基因NtPMEI,其特征在于,所述果胶甲酯酶抑制子基因NtPMEI的核苷酸序列如SEQ ID No.1所示,编码的氨基酸序列如SEQ ID NO.2所示。
2.根据权利要求1所述的烟草果胶甲酯酶抑制子基因NtPMEI,其特征在于:全长591bp,有1个外显子,开放阅读框591bp,编码196个氨基酸。
3.权利要求1或2任一所述烟草果胶甲酯酶抑制子基因NtPMEI在调节烟草对赤星病菌抗性中的应用。
4.权利要求3所述应用的具体应用方法,其特征在于包括以下步骤:
S1:从栽培烟草中克隆得到NtPMEI基因,并构建了含有NtPMEI基因的过表达载体及含有NtPMEI基因靶位点的敲除载体,获得了烟草NtPMEI过表达和敲除株系;
S2:在赤星病抗病品种净叶黄(JYH)中创制NtPMEI过表达材料,感病品种NC82中创制NtPMEI敲除材料,通过对转基因材料和野生型材料的抗病性鉴定发现,在净叶黄中,过表达材料病斑直径显著高于野生型;而在NC82中,敲除材料的病斑明显减小,表明NtPMEI基因负向调控烟草对赤星病菌的抗性。
5.根据权利要求4所述的应用方法,其特征在于:所述步骤S1的操作步骤如下:
选取栽培烟草(Nicotiana tabacum)进行NtPMEI基因的克隆鉴定;将NtPMEI基因分别与植物过表达载体PRI 101-eGFP、敲除载体CRISPR/Cas9进行连接,构建烟草表达载体;将烟草表达载体质粒用热激法转化至农杆菌感受态细胞EHA105中,利用叶盘法获得烟草NtPMEI过表达和敲除株系,采集叶片提取DNA和RNA进行分子水平鉴定。
6.根据权利要求4所述的应用方法,其特征在于:所述步骤S2中赤星病抗性鉴定是采用离体叶片接种的方法使烟草赤星病菌侵染烟草叶片,分别在接种3d、5d、7d后观察叶片,使用Image J软件计算坏死斑面积,每个叶片上的坏死斑取平均值,单位为cm2,其接种方法下:
(1)待转基因株系生长至8周,取生长状态健康一致且处于相同叶位的叶片;
(2)用燕麦培养基培养链格孢菌,28℃恒温培养约15d左右,此时菌快长满整个培养基,菌体呈黑色,边缘颜色较浅;
(3)配置孢子悬浮液。向培养基中加入10mL无菌水配置的1%葡萄糖溶液,用刷子轻轻刮落菌丝,使孢子脱落到1%葡萄糖溶液中,随后将葡萄糖溶液用纱布过滤,留下滤液,即为孢子悬浮液。将孢子悬浮液置于血球计数器下测量孢子的浓度,并稀释到3×105个/mL备用;
(4)将离体叶片背面朝上放置入盛有水的海绵托盘上,在其叶柄处覆盖蘸水的脱脂棉保湿,将80μL的孢子悬浮液接种于叶片背面左右两侧对称的4处后用灭过菌的牙签在接种处划出大小、深浅一致的伤口;
(5)用保鲜膜封口,28℃恒温,16h光照/8h黑暗进行培养,持续观察。
7.根据权利要求4所述的应用方法,其特征在于,用于扩增烟草果胶甲酯酶抑制子基因NtPMEI的引物序列如下:
PMEI-KLF:ACATATGCCCGTCGACATGA
PMEI-KLR:TGCTCACCATGAATTCCTATGA。
8.含有权利要求1或2任一所述基因的生物材料,其特征在于,所述生物材料包括质粒载体、噬菌体载体、病毒载体或工程菌。
9.含有权利要求1或2任一所述基因的基因片段、重组载体、重组菌或蛋白在调节烟草对赤星病菌抗性的用途。
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