CN117384944A - TaRpst9基因敲除突变体在小麦抗条锈病中的应用 - Google Patents
TaRpst9基因敲除突变体在小麦抗条锈病中的应用 Download PDFInfo
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Abstract
本发明属于基因工程技术领域,公开了TaRpst9基因敲除突变体在小麦抗条锈病中的应用。本发明采用反向遗传学方法,构建出用于敲除TaRpst9基因的CRISPR/Cas9基因编辑载体,并通过农杆菌介导法在小麦材料中将TaRpst9基因成功敲除,由此获知TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用。在小麦材料中过表达所述TaRpst9基因,提高小麦对条锈病的抗性。本发明明确了所述TaRpst9基因对小麦抗病性的影响,可用于创制抗条锈病小麦种质材料。
Description
技术领域
本发明属于基因工程技术领域,涉及TaRpst9基因敲除突变体在小麦抗条锈病中的应用。
背景技术
植物在长期的进化过程中,产生了一套独特的免疫系统,这种免疫系统能够识别植物病原菌并激发植物免疫反应,进而抵抗植物病原真菌的侵染。目前观点认为,植物免疫系统由病原相关分子模式触发的免疫(Pattern-Triggered Immunity,PTI)和效应蛋白触发的免疫(Effector-Triggered Immunity,ETI)两层免疫系统构成。
其中,PTI是由位于植物细胞膜上的模式识别受体(Pattern recognitionreceptors,PRRs)识别微生物或病原相关分子模式(microbial-pathogen-associatedmolecular pattern,PAMP)或损伤相关分子模式(damage-associated molecularpatterns,DAMP)而激发的免疫反应。PRRs介导的抗性具有广谱的特点,具备促进植物广谱抗病性的潜力。
PRRs又包括类受体蛋白激酶(receptor-like kinases,RLKs)和类受体蛋白(RLP)。RLKs是植物PTI免疫系统中的重要参与者,在免疫调节过程中发挥着重要作用。RLKs可以结合不同的分子从而识别不同的外界环境,在信号识别后引起自身构象发生改变,最终激活下游信号,导致植物产生活性氧、胼胝质等抗病成分,并引起抗病相关基因表达,从而实现植物对外界病原微生物的免疫防御。
由条形柄锈菌小麦专化形(Puccinia striiformis f.sp.Tritici,Pst)引起的小麦条锈病是小麦生产中的一类重大真菌病害,呈现出分布广、传播快,危害面积大的特点。而传统化学农药的使用加剧了环境污染和病原菌的抗药性,使得农药的应用越来越受到限制。基于此,小麦材料中RLKs的鉴定以及研究RLKs编码基因表达水平对小麦抗病性的影响,将对抗条锈病小麦品种的培育具有重要的贡献意义。
发明内容
一方面,传统农药的应用越来越受到限制;另一方面,调控小麦类受体激酶基因表达对小麦抗病性的影响尚缺乏研究。基于此,本发明从基因工程角度出发,研究小麦类受体激酶基因表达水平对小麦抗病性的影响,这将对抗病品种的培育具有重要的贡献价值。具体地,本发明采用如下技术方案。
本发明提供TaRpst9基因在小麦抗条锈病中的应用,所述TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用。
进一步地,在上述应用中,所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ IDNO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
进一步地,在上述应用中,敲除所述TaRpst9-7A、TaRpst9-7B和TaRpst9-7D中的任一基因,降低小麦材料对条锈病的抗性。
本发明还提供一种小麦类受体激酶在小麦抗条锈病中的应用,所述小麦类受体激酶由TaRpst9基因编码;所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ ID NO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
本发明还进一步提供一种抗条锈病小麦品种的培育方法,所述方法是在小麦材料中过表达TaRpst9基因;所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ ID NO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
与现有技术相比,本发明“TaRpst9基因敲除突变体在小麦抗条锈病中的应用”具有以下有益效果:
本发明基于CRISPR/Cas9基因编辑技术成功构建CRISPR/Cas9基因编辑载体,并在小麦材料中实现了TaRpst9基因移码突变,达到TaRpst9基因敲除目的。
本发明获得了TaRpst9基因敲除小麦突变体L91和L140株系,所述L91和L140株系对条锈菌的抗性降低,获知TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用。
基于TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用,可以将TaRpst9基因用于创制抗条锈病小麦品种。具体方法是:在小麦材料中过表达TaRpst9基因,过表达所述TaRpst9基因的方法不限于农杆菌介导法,基因枪法等。
附图说明
图1是T1代TaRpst9基因敲除小麦突变体Hi-TOM测序靶点的序列比对图。“TaRpst9-target”表示靶点序列,“TaRpst9-7A”、“TaRpst9-7B”、“TaRpst9-7D”表示野生型Fielder小麦品种中TaRpst9基因在7A、7B、7D染色体靶点处的序列;“TaRpst9-KO-91-7A”、“TaRpst9-KO-91-7B”、“TaRpst9-KO-91-7D”分别表示7A、7B、7D染色体上的TaRpst9基因敲除突变体株系L91;“TaRpst9-KO-140-7A”、“TaRpst9-KO-140-7B”、“TaRpst9-KO-140-7D”分别表示7A、7B、7D染色体上的TaRpst9基因敲除突变体株系L140。
图2是T1代TaRpst9基因敲除小麦突变体L91和L140株系接种条锈菌无毒生理小种CYR23的表型结果。“Fielder”表示普通的Fielder小麦植株;“RPst9-KO-91”表示TaRpst9基因敲除小麦突变体L91株系;“RPst9-KO-140”表示TaRpst9基因敲除小麦突变体L140株系。
图3是T1代TaRpst9基因敲除小麦突变体L91和L140株系接种条锈菌无毒生理小种CYR23的相对生物量统计图。“Fielder”表示普通的Fielder小麦植株;“RPst9-KO-91”表示TaRpst9基因敲除小麦突变体L91株系;“RPst9-KO-140”表示TaRpst9基因敲除小麦突变体L140株系。
具体实施方式
下面结合实施例对本发明的技术方案进行清楚、完整地描述,显然,所描述的实施例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。
本发明各实施例中涉及的引物序列如表1所示。
表1,引物序列
。
实施例1
本实施例提供用于靶向敲除TaRpst9基因的重组载体的构建。
本发明所述TaRpst9基因位于小麦基因组第7号染色体,在第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D,核苷酸序列分别如1、2、3所示。用于靶向敲除所述小麦类受体激酶基因TaRpst9的sgRNA的序列如SEQ ID NO:4所示,该序列后三位的序列为PAM序列GGG。
合成TaRpst9基因的sgRNA序列(陕西中科创生物科技有限公司),其中sgRNA的寡核苷酸对直接退火形成双链,体系为:
Rpst9-gR1-F(10μM):1μL,Rpst9-gR1-R(10μM):1μL,1×TE:5μL,ddH2O补足10μL。沸水中煮5min,之后自然冷却至室温。
将退火形成的产物与BtgZ1酶切后的中间载体进行连接,连接反应体系为:
5×ligation Buffer:2μL,sgRNA的寡核苷酸对:4μL,酶切后的中间载体:2μL,T4ligase:1μL,ddH2O补足10μL,16℃过夜进行连接反应。
将连接产物转化大肠杆菌,重组质粒经转化到DH5α感受态细胞,之后在含有卡那霉素的LB平板上培养至单克隆菌株长出后,挑取菌株进行菌液PCR鉴定及测序,测序结果比对正确后摇菌提取质粒。
利用LR反应将中间载体与终载pCas9连接,体系为:
中间载体:2μL,pCas9:0.5μL,LR enzyme:0.5μL,1×TE补足5μL。
之后再转化DH5α感受态细胞,在含有卡那霉素的LB平板上培养至克隆长出后,挑单克隆进行菌液PCR鉴定及测序,测序结果比对正确后摇菌提取质粒,TaRpst9的CRISPR/Cas9基因编辑载体即构建成功。
实施例2
本实施例提供TaRpst9基因敲除小麦突变体的培育和鉴定。
将所述CRISPR/Cas9基因编辑载体转入农杆菌EHA105菌株,以Fielder小麦材料的愈伤组织为转化受体,采用农杆菌介导法将CRISPR/Cas9基因编辑载体导入到Fielder受体小麦材料中,经过愈伤组织卡纳霉素抗性筛选、预再生、再生、生根等步骤产生T0代转基因植株。待幼苗生长发育良好后,采集叶片利用CTAB法提取DNA,再以提取的DNA为模板,以Cas9-F和Cas9-R为引物检测CRISPR/Cas9基因编辑载体是否转化成功,对转化成功的植株加代处理。
TaRpst9基因敲除是否成功需要深度测序来确定,以上述提取的DNA为模板和靶向序列两侧的特异性引物(Rpst9-gR1-F、Rpst9-gR1-R)对检测片段进行扩增,扩增产物进行Hi-TOM测序(浙江省杭州水稻所测序组),结果如图1所示。图1中,“TaRpst9-target”表示靶点序列,“TaRpst9-7A”、“TaRpst9-7B”、“TaRpst9-7D”表示野生型Fielder小麦品种中TaRpst9基因在7A、7B、7D染色体靶点处的序列;“TaRpst9-KO-91-7A”、“TaRpst9-KO-91-7B”、“TaRpst9-KO-91-7D”分别表示7A、7B、7D染色体上的TaRpst9基因敲除突变体株系L91;“TaRpst9-KO-140-7A”、“TaRpst9-KO-140-7B”、“TaRpst9-KO-140-7D”分别表示7A、7B、7D染色体上的TaRpst9基因敲除突变体株系L140。比对图1结果可知,L91和L140两个株系中位于7A、7B、7D染色体的三个拷贝均发生移码突变,证明TaRpst9基因敲除成功。
实施例3
本实施例提供TaRpst9基因敲除突变体小麦植株抗病性实验。
将TaRpst9基因敲除突变体株系L91和L140小麦浸种催芽后播于15×15×12cm规格花盆中,同时播种野生型Fielder小麦材料作为对照,接种条锈菌无毒生理小种CYR23,16℃保湿箱黑暗保湿24h,接着转置于16℃光照16h、14℃黑暗8h光周期的生长培养箱中,14d后观察实验组和对照组的表型变化。
实验结果见图2,“Fielder”表示普通的Fielder小麦植株;“RPst9-KO-91”表示TaRpst9基因敲除小麦突变体L91株系;“RPst9-KO-140”表示TaRpst9基因敲除小麦突变体L140株系。如图2所示,野生型Fielder小麦材料叶片上无明显的条锈菌孢子,L91和L140株系叶片出现明显孢子堆,说明敲除TaRpst9基因,小麦对条锈菌的抗性降低,由此得知TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用。
实施例4
本实施例提供小麦材料接种条锈菌的生物量检测。
采集未接种条锈菌的Fielder小麦叶片和条锈菌夏孢子,按照实施例2中的方法提取DNA,测定DNA的提取浓度,用灭菌的ddH2O将所提取的DNA稀释至相同浓度,再将不同稀释梯度的小麦-条锈菌互作cDNA作为模板,使用TaEF1α(TaEF1α-F、TaEF1α-R)、与PstEF(PstEF-F、PstEF-R)为引物,进行qRT-PCR,并绘制小麦与条锈菌生物量比的标准曲线。
按照实施例2中的方法提取接菌14d后发病叶片的DNA,分别使用TaEF1α、与PstEF为引物,进行qRT-PCR,根据标曲计算生物量,得到条锈菌相对于小麦的相对生物量如图3所示,“Fielder”表示普通的Fielder小麦植株;“RPst9-KO-91”表示TaRpst9基因敲除小麦突变体L91株系;“RPst9-KO-140”表示TaRpst9基因敲除小麦突变体L140株系。从图3可以看出,L91和L140株系接种条锈菌无毒生理小种CYR23后的生物量均显著高于野生型Fielder小麦材料,说明敲除TaRpst9基因,小麦对条锈菌的抗性降低,进一步验证了实施例3中的结论。
以上所述的实施例只是本发明的一部分实施例,而不是全部的实施例。本发明的实施例的详细描述并非旨在限制要求保护的本发明的范围,而是仅仅表示本发明的选定实施例。在依据本发明构思的条件下本领域普通技术人员进行的相关推演和替换,在没有作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。
Claims (5)
1.TaRpst9基因在小麦抗条锈病中的应用,其特征在于,所述TaRpst9基因在小麦抗条锈病免疫反应中起正调控作用。
2.根据权利要求1所述的应用,其特征在于,所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;
所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ ID NO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
3.根据权利要求2所述的应用,其特征在于,敲除所述TaRpst9-7A、TaRpst9-7B和TaRpst9-7D中的任一基因,降低小麦材料对条锈病的抗性。
4.一种小麦类受体激酶在小麦抗条锈病中的应用,其特征在于,
所述小麦类受体激酶由TaRpst9基因编码;
所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;
所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ ID NO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
5.一种抗条锈病小麦品种的培育方法,其特征在于,在小麦材料中过表达TaRpst9基因;
所述TaRpst9基因位于小麦基因组第7号染色体,在所述第7号染色体上有三个拷贝,分别命名为TaRpst9-7A、TaRpst9-7B和TaRpst9-7D;
所述TaRpst9-7A的核苷酸序列如SEQ ID NO:1所示,所述TaRpst9-7B的核苷酸序列如SEQ ID NO:2所示,所述TaRpst9-7D的核苷酸序列如SEQ ID NO:3所示。
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