Basic HTML Version
分子植物育种
(
网络版
), 2013
年
,
第
11
卷
,
第
1106
-
1113
页
Fenzi Zhiwu Yuzhong (Online), 2013, Vol.11, 1106
-
1113
http://mpb.5th.sophiapublisher.com
Copyright © 2013 5
th
Publisher 1106
技术主题
Technology Feature
Bt
水稻
T1C-19
中外源基因的三引物多重
PCR
检测方法
查中萍
,
万丙良
,
杜雪树
湖北省农业科学院粮食作物研究所
,
湖北省粮食作物种质资源创新与遗传育种重点实验室
,
武汉
, 430064
通讯作者：
ricewanbl@126.com
；
作者
分子植物育种
, 2013
年
,
第
11
卷
,
第
16
篇
doi: 10.5376/mpb.cn.2013.11.0016
这是一篇采用
Creative Commons Attribution License
进行授权的开放取阅论文。只要对本原作有恰当的引用
,
版权所有人允许并同意第三方无条
件的使用与传播。
引用格式
(
中文
)
：
查中萍等
, 2013, Bt
水稻
T1C-19
中外源基因的三引物多重
PCR
检测方法
,
分子植物育种
(online), 11(16): 1106-1113 (doi:
10.5376/mpb.cn.2013.11.0
0
16)
引用格式
(
英文
)
：
Zha et al., 2013, A Tri-primer Multiplex PCR Method for the Exogenous Genes in Bt Rice T1C-19, Fenzi Zhiwu Yuzhong (online) (Molecular Plant
Breeding), 11(16): 1106-1113 (doi: 10.5376/mpb.cn.2013.11.0016)
摘
要
转
cry1C
基因水稻
T1C-19
是一个优良的抗螟虫水稻育种资源。本研究目的在于研发一种用于检测
T1C-19
及其衍
生材料中外源基因的三引物多重
PCR (polymerase chain reaction, PCR)
检测方法。根据
T1C-19
中插入的外源基因序列和插入
位点左右旁侧水稻基因组序列设计了
3
个
PCR
引物
C1
、
C2
和
C3
，并对多重
PCR
体系进行了优化。结果表明，
0.5 μL 10 μmol/L
C1+0.4 μL 10 μmol/L C2+0.2 μL 10 μmol/L C3
的多重
PCR
体系可以准确区分
cry1C
基因纯合、杂合和阴性三种基因型，纯合
体和阴性材料中均只扩增出一条
DNA
片段，纯合体中的片段大小为
512 bp
，而阴性材料中的片段大小为
386 bp
，杂合体中
则同时扩增出这两条片段。该体系的建立为利用
T1C-19
进行抗螟虫水稻育种提供了一个简便有效的基因鉴定技术。
关键词
转基因水稻
;
多重
PCR;
共显性标记
;
cry1C
; T1C-19
ATri-primer Multiplex PCR Method for the Exogenous Genes in Bt Rice T1C-19
Zha Zhongping , Wan Bingliang , Du Xueshu
Food Crop Research Institute, Hubei Academy of Agricultural Science, Hubei Key Laboratory of Crop Germplasm and Genetic Improvement, Wuhan, 430064,
P.R. China
Corresponding author, ricewanbl@126.com;
Authors
Abstract
T1C-19 is a transgenic rice possessing
Bacillus thuringiensis
(Bt) insecticide crystal protein encoding gene cry1C,
which has a good resistance to leaf folder and stem borer, and can be used as an elite germplasm in the breeding of insect-resistant
rice. The genotype identification of
cry1C
could greatly improve the breeding efficiency. This research aimed at developing a
tri-primer multiplex PCR method to identify the genotype of
cry1C
in T1C-19 and its relative lines. Three primers of C1, C2 and C3
were designed according to the genomic sequence T1C-19. C2 was located on the T-DNA sequence where
cry1C
was located, and C1
and C3 were located on two flanking rice genomes of the T-DNA, respectively. The quantity of primers in multiplex PCR system was
optimized. The result indicated that the tri-primer multiplex PCR system with 0.5 μL 10 μmol/L C1+0.4 μL 10 μmol/LC2+0.2 μL 10
μmol/L C3 could correctly identify three genotypes of
cry1C
. Only one DNA band was amplified in the homozygote and the negative,
the DNA band was 512bp in the homozygote, but 386bp in the negative. The heterozygote had both two DNA bands above. A few
varieties and two breeding populations from T1C-19 were used to verify the accuracy of the tri-primer multiplex PCR system, the
result confirmed that the tri-primer multiplex PCR system could provide a convenient and effective method for the identification of
cry1C
gene in the breeding of insect-resistant rice with T1C-19 as a parent.
Keywords
Transgenic rice; Multiplex PCR; Co-dominant marker;
cry1C
; T1C-19
研究背景
稻纵卷叶螟、二化螟和三化螟是水稻的主要害
虫，其危害会造成叶片枯白、枯心苗、白穗，瘪谷
大量增加，严重影响水稻产量。转
Bt
毒蛋白基因
水稻对稻纵卷叶螟、二化螟和三化螟具有良好的抗
收稿日期：
2013
年
03
月
20
日
接受日期：
2013
年
04
月
27
日
发表日期：
2013
年
06
月
08
日
基金项目：本研究由国家转基因生物新品种培育重大专项
(2011ZX08001-001)
及湖北省农业科技创新中心资助项目
(2007-620-001-03)
共同资助