山药(Dioscorea opposita)块茎不同发育时期相关差异基因的cDNA-AFLP分析

cDNA-AFLP Analysis of Differential Genes Related to Different Development Stages of Dioscorea opposita Tubers

为分析不同品种不同生长发育时期山药块茎的相关差异基因,本研究选取毕克齐山药(细长型)与大和长芋山药(短粗型)为试验材料,采用c DNA-AFLP技术分离山药块茎生长发育差异片段。结果表明,从256对引物组合中筛选出多态性较好的25对引物组合,扩增出689条多态性条带,其中获得的上调差异片段共304条。将这些片段进行二次扩增、反向Southern blot验证,筛选获得15条阳性差异片段。经克隆和测序,最终获得12条核苷酸序列。经BLAST比对和功能分析,其中9条差异表达的核苷酸均与植物生长发育相关,主要涉及信号转导、转录调控、蛋白质代谢、能量代谢等相关功能蛋白。这些蛋白基因可进一步用于功能验证和转基因研究。为揭示两种山药块茎形态差异的可能原因提供基础资料。

In order to analyze the differentially expressed genes in yam tubers of different varieties at different growth and development stages,this study selected Bikeqi Dioscoreae rhizoma(slender type)and Dahechangyu Dioscoreae rhizoma(stubby type)as experimental materials.The differentially expressed gene fragments related to tuber growth and development of Chinese yam were analyzed by cDNA-AFLP.The results showed that 25 pairs of primer combinations with better polymorphism were screened out from 256 pairs of primer combinations,and a total of 689 polymorphic bands were amplified,of which 304 were up-regulated differential fragments.15 positive TDFs were obtained after the secondary amplification and reverse Southern blot verification.After cloning and sequencing,12 nucleotide sequences were finally obtained.By BLAST comparison and functional analysis,9 differentially expressed nucleotides were tightly related to growth and development of plants,which were mainly involved in signal transduction,transcriptional regulation,protein metabolism,energy metabolism and other related functional proteins.These protein genes might be further used in functional verification and transgenic experi ments.This study might provide basic data for revealing the possible causes of tuber morphological difference between two kinds of yam tubers.