甜瓜化感自毒作用响应基因的cDNA-AFLP分析
Gene expression profiling in response to allelopathic autotoxicityin melon by cDNA-AFLP
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摘要: 本研究以甜瓜种质"新银辉"为试验材料, 利用浓度为0.03 g·mL-1的新鲜甜瓜植株水浸提液处理甜瓜幼苗, 通过cDNA-AFLP技术分析甜瓜化感自毒作用相关基因及其表达情况。试验中利用82对引物进行扩增, 共获得3 600多条差异表达转录衍生片段(TDFs), 平均每个引物组合可扩增出30~50条条带, 片段大小集中在50~800 bp之间, 根据条带强弱和有无的变化, 其表达模式可以分为持续表达、瞬时表达、上调表达和下调表达4种情况。选取其中103条TDFs进行回收、测序, 共获得75条有效序列, 其中上调表达39条, 下调表达21条, 瞬时表达15条。BLAST结果分析显示其中55条与已知功能基因具有较高的同源性, 选取其中10条TDFs, 以甜瓜Actin基因为内参照, 对其在不同胁迫处理时间的表达情况进行半定量RT-PCR验证, 分析结果与cDNA-AFLP结果基本吻合, 表明cDNA-AFLP技术是研究甜瓜化感自毒作用相关基因差异表达的有效方法。55条TDFs的BLAST结果显示, 甜瓜自毒作用涉及到的基因表达情况较为复杂, 与信号转导、能量代谢、蛋白合成、离子运输、逆境响应和转录调控等过程均有关系, 这一结果为探讨甜瓜化感自毒作用的分子机理和相关基因的克隆提供了理论依据。Abstract: As an important horticultural crop worldwide, melon cultivation has been significantly affected by increasingly serious cultivations problems in recent years due to changes in cultivation systems and environment. Critical for continuous melon cultivation was allelopathy. However, little has been done in terms of the molecular biology of melon allelopathy. In this research, allelopathic autotoxicity genes of melon germplasm 'New Silverlit' were analyzed using the cDNA-AFLP technique. Seedlings were treated with 0.03 g·mL 1 concentrations of aqueous extracts from melon roots and stems for cDNA-AFLP analysis. A total of 3 600 transcript-derived fragments (TDFs) were obtained via cDNA-AFLP with 82 primer pairs. Each primer pair was amplified to 30~50 bands with fragment size of 50~800 bp. The TDF expression patterns were divided into four ― up-regulation, down-regulation, transient-expression and continuous-expression. From 103 selected TDFs, 75 had reliable sequences. Also 55 (73%) of the reliable TDF sequences functions were determined through BLAST search in GenBank database. Most of the 55 TDF genes involved in energy and metabolism (17.31%), signal transduction and regulation (15.38%), protein synthesis and ion transport (34.62%) and disease defense (32.69%) were predictable. Of the 75 reliable TDFs, 10 homologous genes were critical for disease defense conditions and signal transductions. Uncharacterized genes were validated in cDNA-AFLP expression patterns using semi-quantitative reverse-transcription polymerase chain reaction (PCR) analyses. This analysis used melon actin as the internal reference gene. The cDNA-AFLP technique confirmed altered expression patterns of 9 (90%) genes. The results show that cDNA-AFLP was a reliable technique for analyzing expression patterns of genes involved in melon allelopathy. Genes involved in melon allelopathy were identified and their expression patterns determined. This study was helpful in shedding more light on molecular mechanisms of melon allelopathy. It was also useful in identifying genes that could alleviate allelopathic autotoxicity in melon.