利用改良型cDNA-AFLP技术筛选木薯干旱胁迫相关基因

cDNA-AFLP是一种经济且高通量的筛选和识别非生物胁迫下基因差异表达的实验技术.本研究对传统cDNA-AFLP技术进行改良,并利用改良型技术对木薯KU50和SC124不同程度干旱胁迫RNA样品进行基因差异表达分析.共获得68条差异表达的转录获得片段(transcript derived fragment,TDF),其中带有引入酶切位点的TDF为64条,预示cDNA-AFLP技术的改良初步获得成功.通过对TDF序列进行功能注释,其中31条TDF可找到已知功能的同源基因;20条为功能不明确的预测基因或蛋白;17条无同源序列,可能为新基因.按功能将31条已知功能的TDFs分类,分别为:能量代谢、糖类代谢、脂类代谢、细胞生长与凋亡、信号转导、转移运输、转录调节、遗传信息处理、多肽类合成与代谢.选择8个差异表达的TDF进行荧光定量PCR验证,发现这些TDF的确在干旱胁迫条件下显著上调或下调表达,证明改良型cDNA-AFLP技术体系为筛选差异表达基因的有效方法.

Modified cDNA-AFLP Technique Analysis of Drought-resistance Related Genes Screened in Cassava(Manihot esculenta Crantz)

cDNA-AFLP is a high-throughput and economical experimental technology for screening and identifying differentially expressed genes under abiotic stress. The study aimed to improve the traditional cDNA-AFLP technique and use the modified method to isolate the drought-resistance genes by using the samples of KU50 and SC124 with varied degree of drought treatment. Sixty-eight differentially expressed transcript-derived fragments (DE-TDFs)were obtained and 64 of them had the introduced restriction enzyme cutting site, suggesting that the modified cDNA-AFLP technique was useful to meet the initial target. The DE-TDFs could be classified to 31 DE-TDFs with functional annotation, 20 DE-TDFs predicted genes or proteins with unknown function, the rests with non-homologous sequences, maybe novel genes based on functional annotation. The functions of the 31 annotated DE-TDFs involved in: energy metabolism, carbohydrate metabolism, lipid metabolism, cell growth and apoptosis, signal transduction, transfer and transport, DNA combined, genetic information processing, transcription regulation, peptide synthesis and metabolism. The significantly up-regulated or down-regulated expressions of eight randomly selected DE-TDFs were validated by real-time PCR analyzing. The results indicated that the modified cDNA-AFLP technique was a feasible and efficient tool to screen differentially expressed gene under abiotic stress.