以龙眼胚性愈伤组织为材料,采用RT-PCR结合RACE法克隆生长素应答因子基因(auxinresponsefac-tor,即DL-ARFl),运用生物信息学方法对序列进行分析,并通过实时荧光定量PCR法研究其在龙眼体细胞胚胎发生过程中的表达。结果表明:DL-ARF1基因的mRNA全长序列为2695bp(GenBank登录号GQ923778),包含2046bp开放阅读框,163bp5’非编码区(5’UTR),486bp3’非编码区(3’UTR);推定的氨基酸序列含681个氨基酸,与其它植物ARF基因相似性达40%~81%。DL-ARF1基因可能属于转录抑制因子,在龙眼体胚的各阶段均有表达,整个变化趋势呈双峰形,在胚性愈伤Ⅱ(Stage2)中的表达量最高。
The auxin response factor gene (DL-ARF1) was cloned from embryogenic calli of longan (Dimo- carpus longan Lour. ) by the RT-PCR with RACE method. The obtained sequence and putative amino acid sequence were analyzed by bioinformatics methods. Then the mRNA transcription level of the gene in the process of somatic embryogenesis was determined by qRT-PCR (real-time reverse transcription PCR) method. The results were as follows: The full length mRNA sequence of DL-ARF1 gene(GenBank, GQ923778) was about 2 695 bp,consisting of an open reading frame(ORF) of 2 046 bp,5' and 3' untrans- lated regions(UTR) of 163 bp and 486 bp,respectively. The putative protein had 681 amino acids,and the i- dentity with other polypeptides varied between 40% ~81%. DL-ARF1 gene was probably belonged to the transcription inhibitor and expressed in 8 different stages during somatic embryogenesis,which showed ap- proximately a bimodal curve. The peak mRNA transcription level of DL- ARF1 gene occurred at the em- bryogenic callus ]1 (Stage 2).