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

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.