CN1928120A - 鉴别芥菜类蔬菜抗芜菁花叶病毒的分子标记方法 - Google Patents
鉴别芥菜类蔬菜抗芜菁花叶病毒的分子标记方法 Download PDFInfo
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Abstract
本发明的鉴别芥菜类蔬菜抗芜菁花叶病毒基因分子标记方法,包括以下步骤:提取芥菜类蔬菜不同种质的基因组DNA,以其为模板,根据已克隆的芥菜类蔬菜抗芜菁花叶病毒基因BjRT设计引物,进行链式聚合酶反应,扩增产物采用琼脂糖凝胶电泳,在芥菜类蔬菜检测抗芜菁花叶病毒特征带。在高抗TuMV的种质中检测到抗TuMV标记基因的特征带,而在不抗TuMV的种质中未能检测到该特征带。本发明首次提出采用人工合成的核苷酸分子标记在芥菜类蔬菜种质中鉴别抗TuMV种质,为鉴别芥菜类蔬菜中抗TuMV种质资源奠定基础。
Description
技术领域
本发明涉及一种植物分子标记技术,确切地说是一种鉴别芥菜类蔬菜抗芜菁花叶病毒的分子标记方法,属于分子生物学技术领域。
背景技术
随着分子生物学的发展,分子标记技术已成为辅助育种及鉴别特异种质资源的重要手段,利用分子标记辅助选择可以在育种早期如苗期进行鉴定;选择过程中不受环境等因素的影响。作物育种常用的分子标记有RFLP、RAPD、SSR以及SCAP标记等。该方法采用特异分子标记技术获得芥菜蔬菜抗芜菁花叶病毒的分子标记,并用于芥菜类蔬菜抗芜菁花叶病毒(TuMV)特异种质资源的筛选鉴定。
芜菁花叶病毒(TuMV)属于马铃薯Y病毒科(Potyviridae)马铃薯Y病毒属(Potyvirus Y),是一个危害范围广、危害程度深的世界性病原,自1992年Schultz发现TuMV以来,TuMV至少可以侵染43科318种植物,其中以重要的十字花科经济作物危害最为严重。十字花科芥菜类蔬菜是我国重要的经济作物,包括叶芥菜、茎芥菜、根芥菜、苔芥菜等,浙江、四川、江苏、湖南、湖北、上海、重庆等省市是芥菜类蔬菜的主要产地,其加工产品深受消费者喜爱。而芜菁花叶病毒(Turnip mosaic virus,TuMV)是危害芥菜类蔬菜,特别是茎用芥菜(榨菜)的主要病害,有些年份甚至绝产,已严重影响其稳产高产和芥菜加工业的发展。中国是芥菜类蔬菜的起源中心,演化至今,类型十分丰富,有几千份芥菜类蔬菜种质资源。丰富的芥菜种质为我们寻找抗TuMV基因提供了丰富的基因资源。作为异源四倍体(AABB),理论上比白菜(AA)、黑芥(BB)、甘蓝(CC)有更加丰富的遗传背景,我们推定在芥菜类蔬菜中可能存在高抗/免疫TuMV的种质资源及基因资源。迄今为止,在十字花科作物中,关于TuMV抗性的遗传特性已经有所报道,如白菜抗TuMV为隐性基因控制,甘蓝型油菜抗TuMV为超显性基因控制等(Yoon er al.,1993;Suh et al.,1995;Hughes et al.,2002;Walsh et al.,1999,2002;Shattuck and Stobbs 1987;Jenner et al.,2002),并且已经将抗TuMV的QTL或基因(TuRB01、TuRB01b及TuRB03)定位在相应的染色体上(Walsh et al.,1999;Rusholme 2000;),为抗TuMV基因的克隆奠定了基础,十字花科蔬菜中未见抗TuMV基因被克隆到。到目前为止,尚无芥菜类蔬菜抗TuMV相关的分子标记报道。
发明内容
本发明的目的是提供鉴别芥菜类蔬菜抗芜菁花叶病毒的分子标记方法。
本发明的鉴别芥菜类蔬菜抗TuMV的分子标记方法,步骤如下:
提取芥菜类蔬菜不同种质的基因组DNA,以其为模板,根据已克隆的芥菜类蔬菜抗芜菁花叶病毒基因BjRT设计权利要求1叙述的引物,进行链式聚合酶反应,扩增产物在琼脂糖凝胶电泳,在芥菜类蔬菜检测抗芜菁花叶病毒特征带。
在高抗TuMV的种质中检测到抗TuMV标记基因的特征带,而在不抗TuMV的种质中未能检测到该特征带。
上述分子标记方法中所述的引物,即人工合成的核苷酸分子是:
正向引物:5’-GCTGACCCAATAGTCTTGAT-3’
反向引物:5’-TAGACGGAAATCCAAAGAG-3’
上述链式聚合酶反应(PCR)的具体步骤如下:
(1)首先将下列试剂混和在一起
10×Taq DNA聚合酶缓冲液 5微升(μl)
模板DNA 1μl
正向引物(1.25μg/l) 0.4μl
反向引物(1.25μg/l) 0.4μl
脱氧核苷酸混合物(dNTP) 1μl
Taq DNA聚合酶 0.4μl
灭菌水 41.8μl
总体积 50μl
(2)将上述混和液94℃变性5分钟,然后进入下来循环:94℃变性1分钟,50℃退火45秒,72℃延伸1分钟,35个循环后72℃延伸10分钟,结果扩增到663的一条带。
(3)将扩增产物采用1%琼脂糖凝胶电泳检测
本发明首次提出采用人工合成的核苷酸分子标记在芥菜类蔬菜种质中鉴别抗TuMV种质,为鉴别芥菜类蔬菜中抗TuMV种质资源奠定基础。
附图说明
图1芥菜类蔬菜抗TuMV分子标记在不同芥菜类蔬菜种质中的特征标记。其中3、5、6及7为抗TuMV种质,1、2、4、8及9为不抗TuMV种质。
具体实施方式
实施例
芥菜类蔬菜抗TuMV的分子标记方法
芥菜类蔬菜(Brassica juncea var.)由本实验室提供,材料在浙江大学农业与生物技术学院园艺系蔬菜研究所种植。
(1)引物设计:根据Genebank数据库中Brassica juncea抗TuMV相关基因BjRT,(BjRT基因Genebank登录号:DQ789595,可以在http://www.ncbi.nlm.nih.gov/中查找到)设计引物,人工合成的核苷酸分子:
正向引物:5’-GCTGACCCAATAGTCTTGAT-3’
反向引物:5’-TAGACGGAAATCCAAAGAG-3’
(2)CTAB法提取植物总DNA
(a)取0.5g嫩叶于研钵中,液氮状态下研磨,加入600ul 65℃预热的2×CTAB,(30μl巯基乙醇混匀),转移到离心管中,65℃保温1-2小时。
(b)加入等体积氯仿:异戊醇(24/1)轻轻摇匀,4℃,13000rpm离心,10分钟。
(c)取上清,重复(2)步骤一次。
(d)取上清于另一eppendorf管中,加入等体积异丙醇,颠倒混匀,静置5分钟,13000rpm,离心10分钟。
(e)倒去上清,加入700ul Wash Buffer漂洗DNA沉淀,离心,重复一次。
(f)加15微升TE-Rnase,37℃保温1小时。
(g)加入2mol/L NaCl 100ul+100%乙醇250ul混匀,-20℃静置30min,13000rpm离心10分钟。
(h)倒去上清,加入500ul 70%乙醇,漂洗,倒去乙醇,重复一次,常温晾干。
(i)加入30-50ul ddH2O或TE,回溶,-20℃保存备用。
注:2×CTAB配方,200ul:
CTAB: 4g
Tris(pH8.0): 20ml
EDTA: 1.488g
NaCl: 16.352g
TE-RNase:15ul TE加入1/100体积的RNase
Wash Buffer:10mmol/L乙酸氨
76%乙醇
(3)链式聚合酶反(PCR)的试剂
首先将下列试剂混和在一起
10×Taq DNA聚合酶缓冲液 5微升(μl)
模板DNA 1μl
正向引物(1.25μg/l) 0.4μl
反向引物(1.25μg/l) 0.4μl
脱氧核苷酸混合物(dNTP) 1μl
Taq DNA聚合酶 0.4μl
灭菌水 41.8μl
总体积 50μl
(4)PCR反应条件:将上述混和液94℃变性5分钟,然后进入下来循环:94℃变性1分钟,50℃退火45秒,72℃延伸1分钟,35个循环后72℃延伸10分钟。
(5)分子标记检测:扩增产物在1%琼脂糖凝胶电泳,溴化乙锭(ethidiumbromide)中染色,紫外灯下观察,凝胶成像系统(美国Bio-Rad公司产品)拍照记录,结果在高抗TuMV的种质中检测到抗TuMV标记基因的特征带,而在不抗TuMV的种质中未能检测到该特征带。如图1箭头所示标记。
Claims (3)
1.一种人工合成的核苷酸分子,其特征在于具有下列碱基组成的特定序列的寡聚体:
正向引物:5’-GCTGACCCAATAGTCTTGAT-3’
反向引物:5’-TAGACGGAAATCCAAAGAG-3’
2.一种鉴别芥菜类蔬菜抗芜菁花叶病毒的分子标记方法,其特征是步骤如下:提取芥菜类蔬菜不同种质的基因组DNA,以其为模板,根据已克隆的芥菜类蔬菜抗芜菁花叶病毒基因BjRT设计权利要求1叙述的引物,进行链式聚合酶反应,扩增产物在琼脂糖凝胶电泳,在芥菜类蔬菜检测抗芜菁花叶病毒特征带。
3.根据权利要求2所述的方法,其特征在于链式聚合酶反应的具体步骤如下:
(1)首先将下列试剂混和在一起
10×Taq DNA聚合酶缓冲液 5微升(μl)
模板DNA 1μl
正向引物(1.25μg/l) 0.4μl
反向引物(1.25μg/l) 0.4μl
脱氧核苷酸混合物(dNTP) 1μl
Taq DNA聚合酶 0.4μl
灭菌水 41.8μl
总体积 50μl
(2)将上述混和液94℃变性5分钟,然后进入下来循环:94℃变性1分钟,50℃退火45秒,72℃延伸1分钟,35个循环后72℃延伸10分钟;
(3)将扩增产物采用1%琼脂糖凝胶电泳检测。
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN102586478A (zh) * | 2012-02-17 | 2012-07-18 | 北京农业生物技术研究中心 | 检测芜菁花叶病毒的一步多重rt-pcr法及其专用引物 |
CN105755011A (zh) * | 2016-04-12 | 2016-07-13 | 浙江大学 | 用于芥菜芜菁花叶病毒病抗性鉴定的分子标记及其用途 |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102586478A (zh) * | 2012-02-17 | 2012-07-18 | 北京农业生物技术研究中心 | 检测芜菁花叶病毒的一步多重rt-pcr法及其专用引物 |
CN105755011A (zh) * | 2016-04-12 | 2016-07-13 | 浙江大学 | 用于芥菜芜菁花叶病毒病抗性鉴定的分子标记及其用途 |
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