关键词: 消减cDNA文库, 胫骨软骨发育不良, 福美双, 抑制消减杂交(SSH), 差异表达基因

Abstract:

【Objective】An experiment was conducted to construct subtracted cDNA library for selecting time series genes differentially expressed in the TD growth plate of broiler chickens at the early stage with cDNA microarray. 【Method】 AVIAN (AV) broiler chicks at 7 days of age were randomly divided into two groups. After fasting overnight, they were fed with regular diet (control) or the same diet containing 100 mg/kg thiram for 48h to induce TD (thiram diet-fed). Forward and reverse-subtracted cDNA libraries were generated by suppression subtractive hybridization technology (SSH). Identification of the inserted cDNA fragments in subtractive library was done using PCR. One hundred clones were randomly selected for further DNA sequencing, blast homology analysis and function prediction. 【Result】 A total of 2 227 positive clones were obtained and the size of inserts was between 200 bp and 1 000 bp. There were 97 homologous gene sequences shared more than 99% identity with genes known in chicken (Gallus gallus). Non-redundancy of sequenced genes was 68.7%. Meanwhile, 3 clones were found to be novel EST as no functional clues were associated with them by bioinformatic analysis. Most of these genes are involved in matrix formation, endochondral ossification and remodelling, developmental regulation, signal transduction, electron transport in mitochondrial respiratory chain and vascularization. 【Conclusion】Successfully produced cDNA library would make a good foundation for further printing cDNA microarray, screening differential expression genes of TD growth plates at different stages, and also may provide new insights into understanding the pathogenesis of TD.

Key words: subtracted cDNA library, tibial dyschondroplasia, thiram, suppression subtractive hybridization (SSH), differential expression genes