铜胁迫下小白菜叶片基因表达谱的cDNA-AFLP分析

Gene expression profiling in response to copper stress in Brassica rapa L. Chinensis by cDNA-AFLP

    摘要
  • 摘要: 本研究以小白菜品种'上海青'为试验材料, 利用Cu2+质量浓度为10 mg·L-1的营养液水培小白菜幼苗, 通过cDNA-AFLP技术分析小白菜响应铜胁迫相关基因及其表达情况。试验中利用多态性较好的135对引物进行扩增, 共获得5 800多条转录衍生片段(TDFs), 平均每个引物组合扩增出30~60条, 片段大小主要集中在100~1 000 bp, 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。通过筛选获得180条差异表达TDFs, 其中上调表达77条(42.8%), 下调表达61条(33.9%), 瞬时表达42条(23.3%)。选取其中152条差异表达TDFs进行回收测序, 最终得到151个有效序列, 其中上调表达68条(45%), 下调表达50条(33.1%), 瞬时表达33条(21.9%)。Blastx比对结果显示, 112条TDFs序列与已知基因具有较高的相似度, 涉及能量代谢、物质合成与运输、逆境响应、信号转导等多种途径, 16条TDFs序列与功能未知或假定基因的同源性较高, 其余23条TDFs序列与已知功能基因的同源性较低或零匹配, 可能是一些新的铜胁迫应答基因。从151条TDFs选取4条TDFs, 以小白菜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荧光定量RT-PCR验证, 分析结果与cDNA-AFLP结果基本吻合, 表明cDNA-AFLP技术是研究小白菜铜胁迫相关基因差异表达的有效方法。小白菜铜胁迫相关基因表达情况较为复杂, 涉及多种代谢途径。本研究结果可为探讨小白菜铜胁迫的分子机理和相关基因的克隆提供理论依据。

     

    Abstract: With the discharge of industrial wastewater, and application of copper-containing pesticides and fertilizers, copper content in soils has increased steadily. This has had severe effect on yield and quality of Brassica rapa L. Chinensis. However, little has been done in terms of analyzing the molecular biology of copper stress in B. rapa. In this research, copper stress responding genes of B. rapa variety 'Shanghaiqing' were analyzed using the cDNA-AFLP technique. Seedlings were cultivated in solution with 10 mg·L-1 Cu2+ concentration for the cDNA-AFLP analysis. A total of 5 800 transcript-derived fragments (TDFs) were obtained via cDNA-AFLP, with 135 primer pairs. Each primer pair was amplified to 30 60 bands with fragment size of 100 1 000 bp. The TDF expression patterns were divided into four - up-regulation, down-regulation, transient-expression and continuous-expression. A total of 180 differentially-expressed TDFs were identified from a total of 5 800 TDFs, which included 77 up-regulated (42.8%), 61 down-regulated (33.9%) and 42 transient-expressed (23.3%) genes. About 152 TDFs were recovered and sequenced, and 151 reliable sequences obtained. The sequences included 68 up-regulated (45.0%), 50 down-regulated (33.1%) and 33 transient-expressed (21.9%) genes. Also the functions of 151 of the reliable TDF sequences were determined through BLAST search in GenBank database. Out of the 151 TDFs, 112 were homolog genes with known functions, 16 were similar genes with unknown functions and 23 had no sequence homology in GenBank entries. The functions of homologous genes were involved in energy and metabolism, protein synthesis and ion transport, stress response, signal transduction and regulation. Of the 151 reliable TDFs, 4 homologous genes were selected for fluorogenic quantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses. The analysis used actin of B. rapa as internal reference gene. The results showed that cDNA-AFLP was a reliable technique for analyzing expression patterns of genes involved in copper stress response. Genes involved in copper stress response were identified and their expression patterns successfully determined. This study laid the theoretical basis for the analysis of molecular mechanism and cloning of genes related with copper stress in B. rapa.

     

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