Song K, Osborn T C
Department of Agronomy, University of Wisconsin, Madison 53706.
Plant Mol Biol. 1994 Nov;26(4):1065-71. doi: 10.1007/BF00040689.
One of the essential issues regarding evolution of polyploid species is how duplicate genes are expressed. Most studies on gene expression in polyploids have been based on isozyme analyses; RNA analysis has not been widely used partially due to difficulties in distinguishing homologous transcripts which usually have the same length and similar or almost identical sequences. In this study, a method combining RT-PCR with RFLP was used to analyze transcripts of homologous genes in natural and synthetic Brassica amphidiploids. Sequences coding for several known genes were selected and used to synthesize gene-specific primers. Total RNAs were used as templates for RT-PCR to amplify homologous transcripts in three diploid parental species, three cultivated amphidiploid species and six synthetic amphidiploids. For each gene, initial PCR products amplified in all species had identical length; however, homologous transcripts in the diploid and amphidiploid species could be distinguished after digesting the PCR products with restriction enzymes. Preliminary results based on three genes indicated that both transcripts from the diploid parents were expressed in the synthetic and natural amphidiploids. This study represents the first application of RT-PCR and RFLP analysis to investigate expression of homologous genes in higher plants. The technique is a sensitive, simple and efficient method for distinguishing homologous transcripts in a mixed RNA population and can be applied to many types of studies on expression of homologous genes.
多倍体物种进化的一个关键问题是重复基因如何表达。大多数关于多倍体基因表达的研究都基于同工酶分析；RNA分析尚未得到广泛应用，部分原因是难以区分通常长度相同且序列相似或几乎相同的同源转录本。在本研究中，采用RT-PCR与RFLP相结合的方法分析天然和人工合成的芸苔属双二倍体中同源基因的转录本。选择了几个已知基因的编码序列并用于合成基因特异性引物。以总RNA为模板进行RT-PCR，以扩增三个二倍体亲本物种、三个栽培双二倍体物种和六个合成双二倍体中的同源转录本。对于每个基因，在所有物种中扩增的初始PCR产物长度相同；然而，在用限制性酶消化PCR产物后，可以区分二倍体和双二倍体物种中的同源转录本。基于三个基因的初步结果表明，来自二倍体亲本的转录本在合成和天然双二倍体中均有表达。本研究首次应用RT-PCR和RFLP分析来研究高等植物中同源基因的表达。该技术是一种在混合RNA群体中区分同源转录本的灵敏、简单且高效的方法，可应用于许多类型的同源基因表达研究。
