基于cDNA-AFLP的N^+离子注入甘草当代根部基因表达分析

The Analysis of Differential Rxpression of Glycyrrhiza uralensis Root Gene by Nitrogen Ions Implantationbased on cDNA-AFLP Method

为研究离子束注入对甘草基因表达的影响,本文用低能N+离子束注入甘草种子,以未经处理的同批甘草种子作为对照,利用c DNA-AFLP技术分析甘草幼根基因的表达差异;以甘草幼根总RNA为模板,通过反转录合成c DNA双链,使用Pst I和Mse I酶切双链c DNA,后经接头连接,预扩增,选择性扩增,变性聚丙烯酰胺凝胶电泳得到c DNA-AFLP图谱;9对选择性扩增引物总共出现条带1576条,差异条带286条,约占总条带的20%,对其中105个表达差异显著的片段进行测序,最终得到92个差异片段的核苷酸序列。通过Blast分析,其中53条测序序列检索到与之同源性高的DNA序列或已知功能的蛋白mRNA序列;成功构建离子注入甘草的当代根部基因表达c DNA-AFLP体系,证实了离子束注入甘草种子对当代甘草根部基因表达的影响,为建立甘草品质育种提供参考。

In order to study the differential expression of gene in licorice root induced by N＋implantation,c DNA-AFLP approach was employed. Total RNA was isolated from root of licorice. synthesizing the c DNA double chain by reverse transcription,using the Pst I and Mse I enzymes to digest the double standed c DNA,connecting the joint,pre-amplification,selective amplification. 286 differential expression c DNA fragments were obtained with 81 selective amplification primer pairs combinations. Which accounted for 20% of the total 1576 bands. The differential c DNA fragments were eluted,cloned and sequenced. 92 fragments of c DNA were obtained,53 pieces of which retrieved high-homology DNA or protein mRNA sequences with known function by BLAST analysis. Functional alaysis indicated that some c DNA fragments might participate in metabolism pathways,signal transduction,stress responses etc. 30 fragments were showed homolory to unknown and hypothetical function genes.