摘要
烟草的N基因是一个较为有效的抗烟草花叶病毒(tobacco mosaic virus,TMV)基因,在抗病育种中发挥着重要的作用。为建立其标记基因PCR检测体系,评估已知目的基因cDNA序列作为分子标记的目的基因辅助育种的可行性,本文依据N基因cDNA序列设计4对扩增范围在150～950bp的不同片段长度引物,并对含有N基因的coker176基因组DNA进行PCR扩增。扩增结果表明,有3对可扩增出特异产物。测序结果表明,有2对引物的扩增产物含有内含子。根据所获得的DNA序列设计出2对用于分子标记辅助育种的引物,并分别对C151、NC89、coker176等11个品种的基因组DNA进行扩增,结果表明在coker176、龙江912、吉烟9号和龙江911品种中有扩增产物,除龙江911外,这一结果与已知的N基因在11个品种的分布相一致,成功建立了N基因PCR检测体系,并证明以cDNA序列设计引物扩增DNA序列的目的基因辅助育种技术完全可行。并对实验中出现的问题进行了研究。
N gene of tobacco, an efficient gene conferring resistance to tobacco mosaic virus （TMV）, plays an important role in disease resistance breeding. In this paper, we designed four pairs of primers with the amplification fragment ranged from 150 bp to 950 bp based on the eDNA sequence of N gene, and carried out PCR amplification of genomic DNA extracted from coker176 harboring N gene in order to develop a PCR detection system for marker gene and assess the availability of molecular marker assistant breeding by employing known eDNA sequence of target gene as molecular marker. The PCR amplification showed that three pairs of primers generated specific products. Sequencing results demonstrated that amplification products by two primer pairs contained introns Moreover, we designed two pairs of primers used in molecular marker assisted breeding based on the obtained DNA sequence, and conducted PCR amplification in genomic DNA from 11 tobacco varieties, such as C151, NC89, coker176 and so on. The amplification results indicated that target products was found in coker176, Longjiang912, JiyanNo.9 and Longiiang911, which is consistent with the distribution of known N gene in these 11 varieties but longjiang911. We established PCR detection system ofN gene successfully and proved that it is feasible for the target gene-assisted breeding technology by designing primers on the basis of cDNA sequences.
出处
《分子植物育种》
CAS
CSCD
2010年第1期167-171,共5页
Molecular Plant Breeding
基金
黑龙江省烟草专卖局资助项目(HN200805)资助
关键词
N基因
分子标记辅助育种
PCR检测
N gene, Molecular marker assistant breeding, PCR detection
