关键词: 龙眼胚性愈伤组织, ACC氧化酶, 基因克隆, 序列分析, 实时荧光定量PCR 法

Abstract:

【Objective】 In this study, the 1-aminocyclopropane-1-carboxylate oxidase gene (ACO) was cloned from embryogenic calli of Longan (Dimocarpus longan Lour.). And the expression of ACO was determined during Longan somatic embryogenesis. 【Method】 The RT-PCR(reverse transcription polymerase chain reaction) with RACE (rapid amplification of cDNA ends) method were used to clone the complete cDNA sequence and DNA sequence of ACO from embryogenic calli of Longan. And bioinformatics method was used to analyze sequences obtained and putative amino acid sequence. Then qRT-PCR (real-time reverse transcription PCR) method was used to determine the cDNA transcription level of this gene. 【Result】 The full length ACC oxidase cDNA from Dimocarpus longan embryogenic callus, about 1 315 bp[including a 13 bp poly(A) tail], consisted of an open reading frame of 948 bp , and 5' and 3' utranslated regions of 86 bp and 281 bp, respectively. The sequence had been submitted to the DDBJ / EMBL/ GenBank database, the accession number was AY521566. The putative protein has 315 amino acids, and the identity with the other polypeptides varied between 86%-47%. Its DNA sequence (the accession number is GU123929) was 1660 bp, and the splice sites of three introns contained were obeyed to the “GT-AG” rule. ACO from embryogenic calli of Longan expressed in the different stages during somatic embryogenesis. And it showed approximately a “M” curve. 【Conclusion】 It is inferred that the full length cDNA and DNA sequence from embryogenic calli of Longan are obtained. The peak cDNA transcription level of Longan. ACO gene occurred at the incomplete compact pro-embryogenic culture stage and the heart embryo stage.

Key words: Dimocarpus longan embryogenic callus, ACC oxidase, gene cloning, sequence analysis, real-time reverse transcription PCR (q-PCR)