CN112391454A - 烟草疫霉纤维素合酶v1109l突变位点检测方法 - Google Patents
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Abstract
本申请属于烟草基因工程技术领域,具体涉及烟草疫霉纤维素合酶CesA3中V1109L突变位点检测方法专利申请事宜。与野生型相比,烟草疫霉纤维素合酶CesA3基因的cDNA序列自5’端起第3325位核苷酸由G突变为C后,烟草疫霉对烯酰吗啉抗性可由敏感型突变为高抗型;对应地,烟草疫霉纤维素合酶CesA3自N端起第1109位氨基酸由野生型的缬氨酸突变为亮氨酸。发明人基于AS‑PCR技术原理,设计了相关引物,以便对突变后具有烯酰吗啉抗性的烟草疫霉进行快速检测和鉴定,进而可为烟草黑胫病防治策略调整、以及延缓和控制抗药性的进一步发展和相关新的化学药物的研发奠定一定技术基础。
Description
技术领域
本申请属于烟草基因工程技术领域,具体涉及烟草疫霉纤维素合酶CesA3中V1109L突变位点检测方法专利申请事宜。
背景技术
烟草黑胫病(Tobacco black shank)是一种世界范围的毁灭性土传病害,其病原菌为疫霉菌烟草变种(Phytophthora parasitica var nicotianae (Breda de Hann)Tuker,即,烟草疫霉)。近年来,随着烤烟连作种植面积的扩大,田间病残生物量不断积累,烟草黑胫病已成为我国烟草的主要根茎部病害,造成经济损失巨大。据不完全统计,烟草黑胫病在河南主要烟区的发病率为8%~15%,每年因烟草黑胫病引起的产值损失达2.0~3.0亿元。烟草疫霉在烟株苗期至成株期均可侵染,主要侵染烟草茎秆,也可侵染叶片。典型症状为茎基形成水渍状黑斑,皮层组织变黑凹陷,并沿茎部上下扩展,髓部黑褐色坏死并干缩呈碟片状。
化学防治是烟草黑胫病防控的重要措施,羧酸酰胺类杀菌剂主要通过抑制病原菌休止孢和孢子囊的萌发、芽管伸长以及菌丝生长来抑制病原菌,是目前用于卵菌病害防治的主要杀菌剂。烯酰吗啉是一种上世纪80年代由美国氰氨公司研发的肉桂酰胺类杀菌剂,对由霜霉属、疫霉属等引起的卵菌病害具有优异的保护和治疗效果,通过抑制病原菌纤维素合成,从而干扰细胞壁的正常形成。已有研究表明,不同病原卵菌对羧酸酰胺类杀菌剂产生抗性的原因主要为纤维素合酶CesA3的单核苷酸点突变,导致药剂与突变后氨基酸结合能力减弱,例如:致病疫霉CesA3 1105位甘氨酸突变为缬氨酸或丙氨酸(Aoki, Y.,Furuya, S., Suzuki, S., 2011. Method for rapid detection of the PvCesA3 geneallele conferring resistance to mandipropamid, a carboxylic acid amidefungicide, in Plasmopara viticola populations. Pest Manag. Sci. 67, 1557-1561)、葡萄霜霉病菌1105位甘氨酸突变为丝氨酸(Blum, M., Waldner, M., Gisi, U.,2010. A single point mutation in the novel PvCesA3 gene confers resistance tothe carboxylic acid amide fungicide mandipropamid in Plasmopara viticola.Fungal Genet. Biol. 47, 499-510)、黄瓜霜霉病菌1105位甘氨酸突变为缬氨酸或色氨酸(Blum, M., Waldner, M., Olaya, G., Cohen, Y., Gisi, U., Sierotzki, H., 2011.Resistance mechanism to carboxylic acid amide fungicides in the cucurbitdowny mildew pathogen Pseudoperonospora cubensis. Pest Manag.Sci. 67, 1211-1214)、瓜类疫霉病菌1109位缬氨酸突变为亮氨酸(Chen, L., Zhu, S., Lu, X., Pang,Z., Cai, M., Liu, X., 2012. Assessing the risk that Phytophthora melonis candevelop a point mutation (V1109L) in CesA3 conferring resistance tocarboxylic acid amide fungicides. PLoS One. 7)、辣椒疫霉1077位谷氨酰胺突变为赖氨酸等(Pang, Z., Shao, J., Chen, L., Lu, X., Hu, J. , Qin, Z., Liu, X., 2013.Resistance to the novel fungicide pyrimorph in Phytophthora capsici: riskassessment and detection of point mutations in CesA3 that confer resistance.PLoS One. 8(2))等。但对于烟草疫霉纤维素合酶CesA3是否存在类似突变、以及对杀菌剂抗性影响如何,尚未见到较为详细报道。
发明内容
在对野生型烟草疫霉菌株和烟草疫霉对烯酰吗啉抗性菌株的纤维素合酶CesA3基因序列比对基础上,发明人发现烟草疫霉纤维素合酶基因CesA3的特定位点发生了突变,并导致了相关抗性变化,为此,本申请的主要目的在于提供相关检测引物及检测方法,从而为相关抗性鉴定、以及最终烟草黑胫病的防治奠定一定技术基础。
本申请所采取的技术方案详述如下。
烟草疫霉纤维素合酶中Q1077H突变位点检测用引物,针对烟草疫霉纤维素合酶CesA3 的Q1077H突变位点,设计PCR检测用引物序列(如SEQ ID No.1~2所示)如下:
Pn3231-F1:5’-GGCTTCTTCGTCATGAGCCAT-3’,
PnA3-R:5’-CTACGTCGACGCCGTCGAAC-3’;
烟草疫霉纤维素合酶中V1109L突变位点检测用引物,针对烟草疫霉纤维素合酶CesA3的V1109L突变位点,设计PCR检测用引物序列(如SEQ ID No.3所示)如下:
Pn3325-F1:5’- GTGTTCGGCTCGTTGCTGC-3’,
PnA3-R:5’-CTACGTCGACGCCGTCGAAC-3’;
所述烟草疫霉纤维素合酶基因CesA3,与野生型(野生型的ncbi登录号MF662568)相比,其cDNA序列自5’端起第3231位核苷酸由G突变为T后,烟草疫霉对烯酰吗啉抗性可由敏感型突变为高抗型;对应地,烟草疫霉纤维素合酶CesA3自N端起第1077位氨基酸由野生型的谷氨酰胺(Q)突变为组氨酸(H);
与野生型相比,其cDNA序列自5’端起第3325位核苷酸由G突变为C后,烟草疫霉对烯酰吗啉抗性可由敏感型突变为高抗型;对应地,烟草疫霉纤维素合酶CesA3自N端起第1109位氨基酸由野生型的缬氨酸(V)突变为亮氨酸(L)。
利用所述检测用引物的突变位点PCR检测方法,包括如下步骤:
(一)提取DNA模板
对待测样品,提取其基因组RNA,并反转录制备cDNA备用;
(二)PCR扩增
以步骤(一)中所提取制备的cDNA为模板,利用所设计引物进行PCR扩增;
PCR扩增时,50μL扩增体系设计如下:
模板cDNA,1μL (30-50ng);
F引物, 1μL(10μM);
R引物,1μL(10μM);
Premix TaqTM (大连TaKaRa公司产品), 25 μl;
ddH2O,22μL;
反应程序为:95℃预变性10 min;95℃变性30 s,70℃退火30s,72℃延伸45s,35个反应循环;72℃最后延伸10 min;PCR扩增产物4℃保存;
(三)检测判定
将步骤(二)中PCR扩增产物进行2%琼脂糖凝胶电泳检测;
若PCR扩增产物为218bp的片段,则表明待测烟草疫霉样品的纤维素合酶基因CesA3存在G3231T(Q1077H)位点突变;
若PCR扩增产物为122bp的片段,则表明待测烟草疫霉样品的纤维素合酶基因CesA3存在G3325C(V1109L)位点突变。
烟草疫霉纤维素合酶CesA3 的Q1077H突变位点在烯酰吗啉抗性中的应用,当野生型烟草疫霉纤维素合酶CesA3第3231位核苷酸由G突变为T后,对应地,烟草疫霉纤维素合酶CesA3第1077位氨基酸由野生型的谷氨酰胺(Q)突变为组氨酸(H)后,烟草疫霉对烯酰吗啉抗性由敏感型突变为高抗型,即Q1077H位点突变后,烟草疫霉对烯酰吗啉具有抗性。
烟草疫霉纤维素合酶CesA3 的V1109L突变位点在烯酰吗啉抗性中的应用,当野生型烟草疫霉纤维素合酶CesA3编码基因第3325位核苷酸由G突变为C后,对应地,烟草疫霉纤维素合酶CesA3第1109位氨基酸由野生型的缬氨酸(V)突变为亮氨酸(L)后,烟草疫霉对烯酰吗啉抗性由敏感型突变为高抗型,即V1109L位点突变后,烟草疫霉对烯酰吗啉具有抗性。
病原菌的抗药性是相关疾病防控过程中必须考虑的因素,而随着化学药剂的普遍应用,烟草疫霉对化学药剂抗性问题也日益严重。微生物的化学药剂抗性检测判定中,室内生物学评价方法(如菌丝生长速率法、孢子萌发法、菌丝干重法、水平扩散法等)是最为基础的方法,而随着基因工程研究的深入,针对特定碱基突变所导致的抗性变化,利用分子生物学手段进行快速检测判定得到了日益重视。
本申请中,在对烟草疫霉烯酰吗啉抗性菌株和野生型(即敏感型)菌株基因序列对比基础上,发明人认为由于烟草疫霉纤维素合酶CesA3相关位点的突变导致了烟草疫霉对烯酰吗啉抗性的变化。在此基础上,发明人基于AS-PCR(Allele-Specific PCR,等位特异PCR)技术原理,设计了相关引物,以便对突变后具有烯酰吗啉抗性的烟草疫苗进行快速检测和鉴定,而通过有效监测和预警田间烟草疫霉对烯酰吗啉抗性发生发展动态,进而可为烟草黑胫病防治策略调整、以及延缓和控制抗药性的进一步发展和相关新的化学药物的研发奠定一定技术基础。
附图说明
图1为对烯酰吗啉表现敏感和抗性的烟草疫霉菌株纤维素合酶相关基因CesA3全序列比对后,发现与抗性相关的3231位碱基突变位点;其中,LY1和SS10为敏感菌株,SS10-17为抗性菌株;
图2为对烯酰吗啉表现抗性和敏感的烟草疫霉菌株纤维素合酶相关蛋白CesA3全序列比对后,发现与抗性相关的1077位氨基酸突变位点;其中,LY1和SS10为敏感菌株,SS10-17为抗性菌株;
图3为AS-PCR引物对进行温度梯度PCR扩增电泳图;Sensitive:敏感菌株SS10;HighResistant:高抗菌株SS10-17;
图4为采用特异性引物对Pn3231F1/PnA3R扩增对烯酰吗啉表现敏感和抗性的烟草疫霉菌株的AS-PCR电泳图;M:DL2000 DNA Marker;CK为阴性对照;SS10-17为抗性菌株;SS10和LY1为敏感菌株;
图5为对烯酰吗啉表现敏感和抗性的烟草疫霉菌株纤维素合酶相关基因CesA3全序列比对后,发现与抗性相关的3325位碱基突变位点;其中,LY1和SS10为敏感菌株,SS10-2、SS10-4、SS10-8为抗性菌株;
图6为对烯酰吗啉表现抗性和敏感的烟草疫霉菌株纤维素合酶相关蛋白CesA3全序列比对后,发现与抗性相关的1109位氨基酸突变位点;其中,LY1和SS10为敏感菌株,SS10-2、SS10-4、SS10-8为抗性菌株;
图7为AS-PCR引物对进行温度梯度PCR扩增电泳图;Sensitive:敏感菌株SS10;HighResistant:高抗菌株SS10-2;
图8为采用特异性引物对Pn3325F1/PnA3R扩增对烯酰吗啉表现敏感和抗性的烟草疫霉菌株的AS-PCR电泳图;M:DL2000 DNA Marker;CK为阴性对照;SS10-2、SS10-4、SS10-8为抗性菌株;SS10和LY1为敏感菌株。
具体实施方式
下面结合实施例对本申请做进一步的解释说明。在介绍具体实施例前,就下述实施例中部分实验背景情况简要介绍说明如下。
实验菌株
需要解释的是,为便于描述,本文中,“抗性菌株”指对烯酰吗啉具有抗性的烟草疫霉菌株;“敏感菌株”均指对烯酰吗啉敏感的烟草疫霉菌株(也即野生型菌株);
前期研究过程中,发明人从河南地区相关烟田土壤中采集获得了野生型的烟草疫霉菌株,并将其中两株对烯酰吗啉敏感、且活力较好菌株命名为烟草疫霉SS10、烟草疫霉LY1。进一步地,以烟草疫霉SS10为亲本,通过在室内采用烯酰吗啉药剂驯化法获得了抗烯酰吗啉的抗性菌株。具体药剂驯化法进行突变体诱导过程为:将敏感菌株SS10的菌饼接种在0.5 μg/mL的烯酰吗啉含药平板上,培养4 d后挑取快速生长的菌落转接到药剂浓度逐步提高的含药平板上进行继代驯化(烯酰吗啉药剂浓度逐渐从为1调整至5、10、50、100 μg/mL)。驯化过程中及驯化最后,将筛选所得具有抗药性的菌株在无药平板上培养并测定其敏感性变化,最后,对抗性稳定遗传的菌株进行单孢分离保存,并将4株抗性稳定遗传的菌株命名为SS10-17、SS10-2、SS10-4、SS10-8,以便进行分析和进一步验证。
各个菌株对烯酰吗啉的敏感性(或者抗药性),采用菌落(菌丝)生长速率法检测验证,具体验证过程及结果简介如下。
微生物培养用培养基:
燕麦培养基,具体制作方法为:
将燕麦粒30g煮沸20min,双层纱布过滤,去离子水定容至1000ml,再加入18 g琼脂、15g蔗糖搅拌均匀;121℃高压蒸汽灭菌20 min。
具体检测验证过程为:
将烯酰吗啉用二甲基亚砜配置成105μg/mL的母液,将此母液用二甲基亚砜稀释成系列浓度,待培养基冷却到45℃左右,按照1‰(v/v)比例等量加入燕麦培养基中,制成所需浓度的含药平板;以只加二甲基亚砜的燕麦培养基作为空白对照;
敏感菌株的烯酰吗啉药剂终浓度设计为:CK、0.1、0.2、0.3、0.5、0.7 μg/ml;
抗性菌株的烯酰吗啉药剂终浓度设计为:CK、50、100 μg/ml;
在已经预培养好的菌落边缘的同圆周上,用灭菌打孔器制取直径5mm的菌饼,菌丝面朝下接种于各个浓度的培养皿中央(每个浓度平板3次重复);25℃恒温培养;
待空白对照的菌落直径达到6~8cm时,采用十字交叉法测定菌落直径,依照如下公式计算药剂对病原菌的菌丝生长抑制率:
以药剂浓度的对数作为X轴,抑制率的机率值为Y轴,根据两者间的线性关系,计算出毒力回归曲线方程y=bx+a及其相关系数r,求出烯酰吗啉对烟草疫霉的有效抑制中浓度EC50;
抗性倍数=突变体的EC50/亲本菌株的EC50。
具体实验结果如下表1所示。
表1 烟草疫霉对烯酰吗啉敏感和抗性菌株的敏感性
统计计算结果表明,敏感菌株的EC50均小于0.5µg/ml,而抗性菌株的EC50大于100µg/ml,明显高于敏感菌株的EC50,抗性倍数大于220倍。
实施例1
基于相关菌株抗性的明显差异和现有其他物种中相关基因突变原因推测,发明人认为有必要就烟草疫霉中纤维素合酶相关基因CesA3在敏感和突变菌株中是否存在类似突变位点进行进一步研究,本实施例就相关突变位点的确定过程简介如下。
(一)设计引物
根据现有病原菌(大豆疫霉、致病疫霉、橡树疫霉)的纤维素合酶基因和烟草黑胫病菌基因组序列,结合同源比对结果,根据保守序列片段设计PCR扩增获得烟草疫霉纤维素合酶相关基因CesA3引物序列如下:
A3F11:5’-ATGGGGCTCACCGGC-3’,
A3R11:5’-CTACGTCGACGCCGTCGAACCACCAGAG-3’;
(二)提取基因组
参考试剂盒说明书(购自生工生物工程(上海)股份有限公司的UNlQ-10 柱式真菌基因组 DNA 抽提试剂盒(B511375)、柱式真菌总 RNA 抽提纯化试剂盒(B518659);购自宝生生物工程(大连)有限公司的反转录试剂盒PrimeScriptTM1st Strand cDNA Synthesis Kit,分别提取敏感和抗性菌株的基因组DNA和RNA,并反转录为cDNA备用。
(三)PCR扩增获得烟草黑胫病菌纤维素合酶基因CesA3
以步骤(二)所提取DNA和cDNA为模板,
PCR扩增时(采用TaKaRa公司的Premix TaqTM(LA TaqTM Version 2.0 plus dye)),50µL反应体系设计如下:
10×EasyTaq buffer,5 µL;
2.5 mM dNTPs,4 µL;
EasyTaq DNA Plymerase (5U/ µL),1 µL;
Forward Primer (10 μM),1 µL;
Reverse Primer (10 μM),1 µL;
Template DNA(或cDNA),1 µL;
ddH2O,37 µL;
PCR 反应条件如下:95°C、5 min;95°C、30 s,60°C、30s,72°C、3 min,35个循环;72°C、10 min;PCR扩增产物4°C保存。
(四)测序及分析
对步骤(三)中的PCR扩增产物进行1.5%的琼脂糖凝胶电泳检测,并进行测序分析。
结果表明,抗性菌株的CesA3的cDNA基因全长3529 bp,含有1个内含子,编码1142个氨基酸。与敏感菌株的CesA3基因对比后发现(如图1、图2、图5、图6所示),抗性突变体SS10-17在CesA3基因cDNA序列3231位发生由G到T的碱基突变,导致1077位的谷氨酰胺(Gln,Q)突变为组氨酸(His,H)。抗性突变体SS10-2、SS10-4、SS10-8在CesA3基因cDNA序列3325位发生由G到C的碱基突变,导致1109位的缬氨酸(Val,V)突变为亮氨酸(Leu,L)。
实施例2
基于实施例1单位点突变结果发现,结合AS-PCR方法需要,发明人进一步设计了相关引物,以便对相关突变位点进行检测和判定。具体过程简介如下。
(一)引物设计
基于前述单碱基突变序列差异和AS-PCR的方法需要,针对Q1077H突变位点,发明人设计引物序列如下:
Pn3231-F1:5’-GGCTTCTTCGTCATGAGCCAT-3’,
PnA3-R:5’-CTACGTCGACGCCGTCGAAC-3’;
需要解释说明的是,引物设计时,为提高引物特异性,将Pn3231-F1的3’末端倒数第二个碱基与靶标序列进行了错配,同时配合后续PCR扩增程序的调整,以便检测G3231T这一突变位点;
针对V1109L突变位点,发明人设计引物序列如下:
Pn3325-F1:5’- GTGTTCGGCTCGTTGCTGC-3’,
PnA3-R:5’-CTACGTCGACGCCGTCGAAC-3’;
需要解释说明的是,引物设计时,为提高引物特异性,将Pn3325-F1的3’末端倒数第二个碱基与靶标序列进行了错配,同时配合后续PCR扩增程序的调整,以便检测G3325C这一突变位点。
(二)PCR扩增
分别提取并制备敏感和抗性菌株cDNA,以此分别作为模板,并分别利用步骤(一)中引物进行PCR扩增;
PCR扩增体系参考实施例1即可;
PCR反应程序如下:95℃预变性10 min;95℃变性30 s,62~70℃梯度PCR,退火30 s,72℃延伸45 s,35个反应循环;72℃最后延伸10 min;4℃保存。
结果表明:在62~68℃的退火温度下,敏感菌株有隐约条带可见,而在70℃时敏感菌株无条带,同时抗性菌株则在各个退火温度下均有清晰条带(如图3、图7所示)。因此,实际鉴定区分时,选择70℃作为敏感和两种抗性菌株AS-PCR的退火温度。
进一步地,为验证上述退火温度的正确性,参考上述操作,发明人分别以敏感菌株SS10、LY1和抗性菌株SS10-17基因组DNA为模板,利用引物对Pn3231F1/PnA3R,以70℃为退火温度进行PCR扩增。结果如图4、图8所示。
可以看出,仅能从抗性菌株中扩增出目标片段,而不能从敏感菌株中扩增出片段。也即,若能够从菌株基因组DNA中扩增获得218bp或122bp片段,可判定所述菌株为对烯酰吗啉的抗性菌株;若不能扩增获得片段,则判定所检测的烟草疫霉为对烯酰吗啉的敏感菌株。
特定3231位点突变后,其碱基序列长度为3429bp,具体如下:
ATGGGGCTCACCGGCGCGGGCATCATCGCCTCCGTCGTGGGCATCCTGGGCGGCGTGTCGCTGTCCTGCGGCGGCTGGTCCTCGCTGTCCCTCGGCGCTCGCTCGCTCTTCGTCACCACGCAGTTCCTCTCGGCCTTCGCCATGGGGTTTGTGGTAGCTTTTTCCGCCATCGTCTCCCTCTCGGACACGAATGAGTGGGTTGCCGTGGCCGCCGGTGGCGGCGCAGGCTTCGTGATCGCGCTCATCGTGGGTTTCATGACGATTTTCGGCCCGTACATCCTGATCCTGGTCACAGGCGGCATTATCGCCTGCTATTTGCTGCTAGTGGACGCGTACGATGGCGTCAACGTGTTCCCCGAGGACAACCAGCTGGCTCGCCAGGAGTTCGTGATCGCTTTCATGATCATCTTCGAGCTCGTGTGCTGCTCGTCGTCCAAGACGTCGGAGCTGGAGAACCACCGCTTCAAGTACATCATCTTCTCGGCCATCACTGGTGGCTGGATGGCTGCAGACGGCGTCTCTCGTCTCATCGACTCAAGTGCCGTGCTCTCCACTGTGGCCTACACGTCGATGCAGGACGGTGGCAAGGCTGCACTGGACGGCATCGACTCTAGCGCCCAGACGGTCATGTTCTTGGTCTGGGGCGCTGTCTTCGTGGTCGGCGGACTCAACCAGCTGTCGATGCGTTGGGGTCTCATGTGCTACAACCGCGTGGGCGCTCACGCTCAGCTTGGCCCCGTGGAGGAGCAGATGCCTGAGCTCCCCACTGGCGCCACGCTACCGGCTCAGACTATGACGGAACGTGTCCGTCTGGTGTGTGAGAACTGCTTTGCCACTGTCCCCGCTGGCACTGCCTTCTGTACTGAATGTGGTGAGGCTATGCCCTCGGAAGACGCCAACCCGGACGTGAGCATCTCTCAGGCTCAAATGCCGTCAGTCACGATGAACAACAAGAGCCAAGTGCCCGACCGCTGGCAGCAAGTGCCTCACCGTACGTACCTCAGCACCACGTCGTTTGTGGACCCGAAACACGCCAAGGAGGGCGGAGTGAGCATGAAGGACAACAGTCGTAGCATCCGCTTCATGGACTCTGGCGTCCAGGGGCCGGACGGCAAGATGAGCCAGTACAACGACTCGATCGCTGGCGTTCGCAACTATTACGAGCCGTCGTTCCGCTCATTTGCCATGTCCACGTACTCGATCGCTAACCGTGCGGCTGAACCGGTGGAGACGCCCAACATCCGTAAGTACAAGATGTCGGGCAGCGGCATGTTCCACGTCTTCTACTTCGGTACTGCTGCTACCGGTATCTTCTGGCTGTACTACTTGACTACGATGTACCCGCAACAGTATTTCTGCGACCACGCCCGTCCCACGCTTCCTTGCAGTGAACTCCCGTCCAGTGAGACCTCGGGCTGCTACAGTTCGACTGTCAACTTTGACGCTGACTCGGGTGAAGGCTACTGCATCCAGGACGTGCCGTTCATGTCGTGGCTCATGTACGCGATGATGATCTTCAGCGAGTTCCTCAACTACTTCCTGGGTCTGCTTTTCAACTTCAGTATGTGGCGTCCGATCCGTCGTGGTGCTCGCTACATGAACGACTTCAAGCCGCCTATCCCGAAGGAGCAATGGCCCACAGTCGACATCTTCTTGTGTCACTACATGGAACCGGTGACGGACTCGATGCAGACGCTCAAGAACTGTCTGGCCATGCAGTATCCCCCTGAGTTACTGCATATCTTTATTCTGGATGATGGTTACACCAAGTCGGTGTGGGACGCCAACAACCACTTCAAGGTGACGGTTAACACTAAGGTTATTGAGATCGCTGGTGATCTGCGCGGAGATCTCGCTCGTCTCATGCACGAGCGTGTGGTTGGCCCCGTCCAGGACGACCAGAGCTTGAAGGCGTGGCGTCGTCAACACAGTTCGGTCCGTGAGCTCCGTAAGGAAGGCGGCAAGGGAGTTCAGCGTCGTGACTGTGCTGTGGGCTCGCTGTCTGACGACTACGACTACCGTGACCGTGGTATCCCTCGCGTGACGTTCATTGGACGTATGAAGCCTGAGACGCATCACTCGAAGGCTGGTAACATCAACAACGCGCTGTTCAACGAAGGCGCAGACGGCAAGTACCTGTTGATTCTGGATAACGATATGAAGCCGCATCCCAAGTTCCTGCTGGCTGTGCTGCCGTTCTTCTTCTCGGAGGGCGAGGCTGTTGACGGCGGAGGTCGTCAATACAGTGACGATATCTCGTGGAACCAGGTGTCGTATGTGCAGACCCCGCAGTACTTCGAAGACACCCCGCAATTGACAATCATGGGAGACCCCTGTGGACACAAGAACACCATTTTCTTCGACGCTGTGCAGTGTGGACGTGATGGTTTCGACTCGGCCGCTTTCGCCGGTACGAACGCTGTTTTCCGTCGTCAAGCGTTCGACTCTATTGGTGGAATTCAGTACGGTACACAGACAGAAGATGCGTTCACGGGTAACGTGCTGCACACGTCTGGATGGGACTCGGTGTACTTCCGTAAGGACTTTGAAGGTGATGCCAAGGACCGTATCCGTCTGTGTGAAGGTGCCGTGCCCGAGACAGTCGCTGCTGCTATGGGTCAGAAGAAACGTTGGGCCAAGGGTGCCGTCCAGATTCTGTTGATGAAGAATGAAAGCGAAGTCGACCCGGACTGGCGTCCGCCGCGTGTGCCTGCCCCCGACCCGAAGCCGTCGCTCGCGTTCCCGCGTAAGATGTTCTTCTATGACTCGGTGCTGTACCCGTTCGGTTCGATCCCTGCCTTGTGTTACGTGTCGATCGCCGTCTACTACCTGTGCACGGGTGACGCTCCCATTTACGCTCGTGGTACCAAGTTCCTGTACTCTTTCTTGCCCGTGACGTTCTGCCGTTGGGTGCTCAACCTGCTCGCTAACCGCGCTGTCGACAACAACGACGTGTGGCGTGCACAGCAGACGTGGTTCTCATTCTCGTTCATCACAATGATGGCTATTGTGGAGGCTATTCAGGCGCGTGTGACGGGCAAGGACAAGTCGTGGGCTAACACTGGTGCCGGTCAGAAGACGTCATGGACGGAAATCCCCAACGTGCTGTTCTTCTTCACGCTGCTGTTCAGTCAAGTGGTGGCGCTGGTGCGATTCTTCGAGTATGAGAACGCCACGAACCCGTGGAACTACGTGTCTGCCATGTTCTTTGGCTTCTTCGTCATGAGCCATTTCTACCCCATGGTGAAGATGAGTATCACGGAATACTGTGGTTGGGACCACACGGCTGCTACCTTCACGGCCAACGTGTTCGGCTCGTTGCTGGTGGTGTACGTGGTGGTGTTCGTGCAGCTGTGGCAAGTGTACTACGAGGGCAACTTGCAGGTGGCCCAGGGCACAGACTCTGGTGGTTCGACGGCGTCGACGTAG。
特定3231位点突变后,突变后具体氨基酸序列(1142个氨基酸)如下:
MGLTGAGIIASVVGILGGVSLSCGGWSSLSLGARSLFVTTQFLSAFAMGFVVAFSAIVSLSDTNEWVAVAAGGGAGFVIALIVGFMTIFGPYILILVTGGIIACYLLLVDAYDGVNVFPEDNQLARQEFV IAFMIIFELVCCSSSKTSEL ENHRFKYIIF SAITGGWMAA DGVSRLIDSS
AVLSTVAYTSMQDGGKAALDGIDSSAQTVMFLVWGAVFVVGGLNQLSMRWGLMCYNRVGAHAQLGPVEEQMPELPTGATLPAQTMTERVRLVCENCFATVPAGTAFCTECGEAMPSEDANPDVSISQAQMPSVTMNNKSQVPDRWQQVPHRTYLSTTSFVDPKHAKEGGVSMKDNSRSIRFMDSGVQGPDGKMSQYNDSIAGVRNYYEPSFRSFAMSTYSIANRAAEPVETPNIRKYKMSGSGMFHVFYFGTAATGIFWLYYLTTMYPQQYFCDHARPTLPCSELPSSETSGCYSSTVNFDADSGEGYCIQDVPFMSWLMYAMMIFSEFLNYFLGLLFNFSMWRPIRRGARYMNDFKPPIPKEQWPTVDIFLCHYMEPVTDSMQTLKNCLAMQYPPELLHIFILDDGYTKSVWDANNHFKVTVNTKVIEIAGDLRGDLARLMHERVVGPVQDDQSLKAWRRQHSSVRELRKEGGKGVQRRDCAVGSLSDDYDYRDRGIPRVTFIGRMKPETHHSKAGNINNALFNEGADGKYLLILDNDMKPHPKFLLAVLPFFFSEGEAVDGGGRQYSDDISWNQVSYVQTPQYFEDTPQLTIMGDPCGHKNTIFFDAVQCGRDGFDSAAFAGTNAVFRRQAFDSIGGIQYGTQTEDAFTGNVLHTSGWDSVYFRKDFEGDAKDRIRLCEGAVPETVAAAMGQKKRWAKGAVQILLMKNESEVDPDWRPPRVPAPDPKPSLAFPRKMFFYDSVLYPFGSIPALCYVSIAVYYLCTGDAPIYARGTKFLYSFLPVTFCRWVLNLLANRAVDNNDVWRAQQTWFSFSFITMMAIVEAIQARVTGKDKSWANTGAGQKTSWTEIPNVLFFFTLLFSQVVALVRFFEYENATNPWNYVSAMFFGFFVMSHFYPMVKMSITEYCGWDHTAATFTANVFGSLLVVYVVVFVQLWQVYYEGNLQVAQGTDSGGSTAST。
特定3325位突变后,核苷酸序列(3429bp)如下:
ATGGGGCTCACCGGCGCGGGCATCATCGCCTCCGTCGTGGGCATCCTGGGCGGCGTGTCGCTGTCCTGCGGCGGCTGGTCCTCGCTGTCCCTCGGCGCTCGCTCGCTCTTCGTCACCACGCAGTTCCTCTCGGCCTTCGCCATGGGGTTTGTGGTAGCTTTTTCCGCCATCGTCTCCCTCTCGGACACGAATGAGTGGGTTGCCGTGGCCGCCGGTGGCGGCGCAGGCTTCGTGATCGCGCTCATCGTGGGTTTCATGACGATTTTCGGCCCGTACATCCTGATCCTGGTCACAGGCGGCATTATCGCCTGCTATTTGCTGCTAGTGGACGCGTACGATGGCGTCAACGTGTTCCCCGAGGACAACCAGCTGGCTCGCCAGGAGTTCGTGATCGCTTTCATGATCATCTTCGAGCTCGTGTGCTGCTCGTCGTCCAAGACGTCGGAGCTGGAGAACCACCGCTTCAAGTACATCATCTTCTCGGCCATCACTGGTGGCTGGATGGCTGCAGACGGCGTCTCTCGTCTCATCGACTCAAGTGCCGTGCTCTCCACTGTGGCCTACACGTCGATGCAGGACGGTGGCAAGGCTGCACTGGACGGCATCGACTCTAGCGCCCAGACGGTCATGTTCTTGGTCTGGGGCGCTGTCTTCGTGGTCGGCGGACTCAACCAGCTGTCGATGCGTTGGGGTCTCATGTGCTACAACCGCGTGGGCGCTCACGCTCAGCTTGGCCCCGTGGAGGAGCAGATGCCTGAGCTCCCCACTGGCGCCACGCTACCAGCTCAGACTATGACGGAACGTGTCCGTCTGGTGTGTGAGAACTGCTTTGCCACTGTCCCCGCTGGCACTGCCTTCTGTACTGAATGTGGTGAGGCTATGCCCTCGGAAGACGCCAACCCGGACGTGAGCATCTCTCAGGCTCAAATGCCGTCAGTCACGATGAACAACAAGAGCCAAGTGCCCGACCGCTGGCAGCAAGTGCCTCACCGTACGTACCTCAGCACCACGTCGTTTGTGGACCCGAAACACGCCAAGGAGGGCGGAGTGAGCATGAAGGACAACAGTCGTAGCATCCGCTTCATGGACTCTGGCGTCCAGGGGCCGGACGGCAAGATGAGCCAGTACAACGACTCGATCGCTGGCGTTCGCAACTATTACGAGCCGTCGTTCCGCTCATTTGCCATGTCCACGTACTCGATCGCTAACCGTGCGGCTGAACCGGTGGAGACGCCCAACATCCGTAAGTACAAGATGTCGGGCAGCGGCATGTTCCACGTCTTCTACTTCGGTACTGCTGCTACCGGTATCTTCTGGCTGTACTACTTGACTACGATGTACCCGCAACAGTATTTCTGCGACCACGCCCGTCCCACGCTTCCTTGCAGTGAACTCCCGTCCAGTGAGACCTCGGGCTGCTACAGTTCGACTGTCAACTTTGACGCTGACTCGGGTGAAGGCTACTGCATCCAGGACGTGCCGTTCATGTCGTGGCTCATGTACGCGATGATGATCTTCAGCGAGTTCCTCAACTACTTCCTGGGTCTGCTTTTCAACTTCAGTATGTGGCGTCCGATCCGTCGTGGTGCTCGCTACATGAACGACTTCAAGCCGCCTATCCCGAAGGAGCAATGGCCCACAGTCGACATCTTCTTGTGTCACTACATGGAACCGGTGACGGACTCGATGCAGACGCTCAAGAACTGTCTGGCCATGCAGTATCCCCCTGAGTTACTGCATATCTTTATTCTGGATGATGGTTACACCAAGTCGGTGTGGGACGCCAACAACCACTTCAAGGTGACGGTTAACACTAAGGTTATTGAGATCGCTGGTGATCTGCGCGGAGATCTCGCTCGTCTCATGCACGAGCGTGTGGTTGGCCCCGTCCAGGACGACCAGAGCTTGAAGGCGTGGCGTCGTCAACACAGTTCGGTCCGTGAGCTCCGTAAGGAAGGCGGCAAGGGAGTTCAGCGTCGTGACTGTGCTGTGGGCTCGCTGTCTGACGACTACGACTACCGTGACCGTGGTATCCCTCGCGTGACGTTCATTGGACGTATGAAGCCTGAGACGCATCACTCGAAGGCTGGTAACATCAACAACGCGCTGTTCAACGAAGGCGCAGACGGCAAGTACCTGTTGATTCTGGATAACGATATGAAGCCGCATCCCAAGTTTCTGCTGGCTGTGCTGCCGTTCTTCTTCTCGGAGGGCGAGGCTGTAGACGGCGGAGGTCGTCAATACAGTGACGATATCTCGTGGAACCAGGTGTCGTATGTGCAGACCCCGCAGTACTTCGAAGACACCCCGCAATTGACAATCATGGGAGACCCCTGTGGACACAAGAACACCATTTTCTTCGACGCTGTGCAGTGTGGACGTGATGGTTTCGACTCGGCCGCTTTCGCCGGTACGAACGCTGTTTTCCGTCGTCAAGCGTTCGACTCTATTGGTGGAATTCAGTACGGTACACAGACAGAAGATGCGTTCACGGGTAACGTGCTGCACACGTCTGGATGGGACTCGGTGTACTTCCGTAAGGACTTTGAAGGTGATGCCAAGGACCGTATCCGTCTGTGTGAAGGTGCCGTGCCCGAGACAGTCGCTGCTGCTATGGGTCAGAAGAAACGTTGGGCCAAGGGTGCCGTCCAGATTCTGTTGATGAAGAATGAAAGCGAAGTCGACCCGGACTGGCGTCCGCCGCGTGTGCCTGCCCCCGACCCGAAGCCGTCGCTCGCGTTCCCGCGTAAGATGTTCTTCTATGACTCGGTGCTGTACCCGTTCGGTTCGATCCCTGCCTTGTGTTACGTGTCGATCGCCGTCTACTACCTGTGCACGGGTGACGCTCCCATTTACGCTCGTGGTACCAAGTTCCTGTACTCTTTCTTGCCCGTGACGTTCTGCCGTTGGGTGCTCAACCTGCTCGCTAACCGCGCTGTCGACAACAACGACGTGTGGCGTGCACAGCAGACGTGGTTCTCATTCTCGTTCATCACAATGATGGCTATTGTGGAGGCTATTCAGGCGCGTGTGACGGGCAAGGACAAGTCGTGGGCTAACACTGGTGCCGGTCAGAAGACGTCATGGACGGAAATCCCCAACGTGCTGTTCTTCTTCACGCTGCTGTTCAGTCAAGTGGTGGCGCTGGTGCGATTCTTCGAGTATGAGAACGCCACGAACCCGTGGAACTACGTGTCTGCCATGTTCTTTGGCTTCTTCGTCATGAGCCAGTTCTACCCCATGGTGAAGATGAGTATCACGGAATACTGTGGTTGGGACCACACGGCTGCCACCTTCACGGCCAACGTGTTCGGCTCGTTGCTGCTGGTGTACGTGGTGGTGTTCGTGCAGCTGTGGCAAGTGTACTACGAGGGCAACTTGCAGGTGGCCCAGGGCACAGACTCTGGTGGTTCGACGGCGTCGACGTAG。
特定3325位点突变后,编码氨基酸(1142个)序列如下
MGLTGAGIIASVVGILGGVSLSCGGWSSLSLGARSLFVTTQFLSAFAMGFVVAFSAIVSLSDTNEWVAVAAGGGAGFVIALIVGFMTIFGPYILILVTGGIIACYLLLVDAYDGVNVFPEDNQLARQEFVIAFMIIFELVCCSSSKTSELENHRFKYIIFSAITGGWMAADGVSRLIDSSAVLSTVAYTSMQDGGKAALDGIDSSAQTVMFLVWGAVFVVGGLNQLSMRWGLMCYNRVGAHAQLGPVEEQMPELPTGATLPAQTMTERVRLVCENCFATVPAGTAFCTECGEAMPSEDANPDVSISQAQMPSVTMNNKSQVPDRWQQVPHRTYLSTTSFVDPKHAKEGGVSMKDNSRSIRFMDSGVQGPDGKMSQYNDSIAGVRNYYEPSFRSFAMSTYSIANRAAEPVETPNIRKYKMSGSGMFHVFYFGTAATGIFWLYYLTTMYPQQYFCDHARPTLPCSELPSSETSGCYSSTVNFDADSGEGYCIQDVPFMSWLMYAMMIFSEFLNYFLGLLFNFSMWRPIRRGARYMNDFKPPIPKEQWPTVDIFLCHYMEPVTDSMQTLKNCLAMQYPPELLHIFILDDGYTKSVWDANNHFKVTVNTKVIEIAGDLRGDLARLMHERVVGPVQDDQSLKAWRRQHSSVRELRKEGGKGVQRRDCAVGSLSDDYDYRDRGIPRVTFIGRMKPETHHSKAGNINNALFNEGADGKYLLILDNDMKPHPKFLLAVLPFFFSEGEAVDGGGRQYSDDISWNQVSYVQTPQYFEDTPQLTIMGDPCGHKNTIFFDAVQCGRDGFDSAAFAGTNAVFRRQAFDSIGGIQYGTQTEDAFTGNVLHTSGWDSVYFRKDFEGDAKDRIRLCEGAVPETVAAAMGQKKRWAKGAVQILLMKNESEVDPDWRPPRVPAPDPKPSLAFPRKMFFYDSVLYPFGSIPALCYVSIAVYYLCTGDAPIYARGTKFLYSFLPVTFCRWVLNLLANRAVDNNDVWRAQQTWFSFSFITMMAIVEAIQARVTGKDKSWANTGAGQKTSWTEIPNVLFFFTLLFSQVVALVRFFEYENATNPWNYVSAMFFGFFVMSQFYPMVKMSITEYCGWDHTAATFTANVFGSLLLVYVVVFVQLWQVYYEGNLQVAQGTDSGGSTAST。
SEQUENCE LISTING
<110> 中国烟草总公司郑州烟草研究院
江西中烟工业有限责任公司
<120> 烟草疫霉纤维素合酶V1109L突变位点检测方法
<130> none
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 21
<212> DNA
<213> 人工设计
<400> 1
ggcttcttcg tcatgagcca t 21
<210> 2
<211> 20
<212> DNA
<213> 人工设计
<400> 2
ctacgtcgac gccgtcgaac 20
<210> 3
<211> 19
<212> DNA
<213> 人工设计
<400> 3
gtgttcggct cgttgctgc 19
Claims (5)
1.烟草疫霉纤维素合酶中V1109L突变位点检测用引物,其特征在于,针对烟草疫霉纤维素合酶CesA3 的V1109L突变位点,设计PCR检测用引物序列如下:
Pn3325-F1:5’- GTGTTCGGCTCGTTGCTGC-3’,
PnA3-R:5’-CTACGTCGACGCCGTCGAAC-3’。
2.权利要求1所述V1109L突变位点检测用引物在烟草疫霉抗性中的应用,其特征在于,用于判定烟草疫霉对烯酰吗啉抗性。
3.烟草疫霉纤维素合酶中V1109L突变位点在烟草疫霉抗性中的应用,其特征在于,与野生型相比,烟草疫霉纤维素合酶CesA3的cDNA序列自5’端起第3325位核苷酸由G突变为C后,烟草疫霉对烯酰吗啉抗性可由敏感型突变为高抗型;对应地,烟草疫霉纤维素合酶CesA3自N端起第1109位氨基酸由野生型的缬氨酸突变为亮氨酸。
4.利用权利要求1所述检测用引物的V1109L突变位点的PCR检测方法,其特征在于,包括如下步骤:
(一)提取DNA模板
对待测样品,提取其基因组RNA,并反转录制备cDNA;
(二)PCR扩增
以步骤(一)中所提取制备的cDNA为模板,利用所设计引物进行PCR扩增;
(三)检测判定
将步骤(二)中PCR扩增产物进行琼脂糖凝胶电泳检测;
若PCR扩增产物为122bp的片段,则表明待测烟草疫霉样品的纤维素合酶基因CesA3存在V1109L位点突变。
5.如权利要求4所述V1109L突变位点的PCR检测方法,其特征在于,步骤(二)中,PCR扩增时,50μL扩增体系设计如下:
模板cDNA,1μL;
F引物, 1μL;
R引物,1μL;
Premix TaqTM,25 μl;
ddH2O,22μL;
反应程序为:95℃预变性10 min;95℃变性30 s,70℃退火30s,72℃延伸45s,35个反应循环;72℃最后延伸10 min。
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Citations (2)
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CN105861655A (zh) * | 2016-04-11 | 2016-08-17 | 中国农业大学 | 一种快速鉴定大豆疫霉对烯酰吗啉抗药性的方法及专用引物对 |
JP2019142920A (ja) * | 2019-04-18 | 2019-08-29 | 住友化学株式会社 | 植物病原性微生物防除組成物及び植物病原性微生物の防除方法 |
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CN105861655A (zh) * | 2016-04-11 | 2016-08-17 | 中国农业大学 | 一种快速鉴定大豆疫霉对烯酰吗啉抗药性的方法及专用引物对 |
JP2019142920A (ja) * | 2019-04-18 | 2019-08-29 | 住友化学株式会社 | 植物病原性微生物防除組成物及び植物病原性微生物の防除方法 |
Non-Patent Citations (3)
Title |
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LEI CHEN ET AL.: "Assessing the Risk That Phytophthora melonis Can Develop a Point Mutation (V1109L) in CesA3 Conferring Resistance to Carboxylic Acid Amide Fungicides", 《PLOS ONE》, vol. 7, no. 7, 27 July 2012 (2012-07-27), pages 2 - 3 * |
MATHIAS BLUM ET AL.: "The cellulose synthase 3 ( CesA3 ) gene of oomycetes: structure,phylogeny and influence on sensitivity to carboxylic acid amide (CAA) fungicides", 《FUNGAL BIOLOGY》, vol. 116, 13 February 2012 (2012-02-13), pages 533 - 534 * |
MU,W.: "Phytophthora nicotianae cellulose synthase 3 (CesA3) gene, complete cds:MF662568.1", 《GENBANK》, 28 July 2018 (2018-07-28), pages 1 - 2 * |
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