龙眼胚性愈伤组织ACC氧化酶基因的克隆及其在龙眼体胚发生过程中的表达分析

Cloning of ACO Gene from Embryogenic Calli of Longan(Dimocarpus longan Lour.) and Its Expression During Longan Somatic Embryogenesis

【目的】分离克隆龙眼(Dimocarpus longan Lour.)胚性愈伤组织乙烯合成关键酶ACO(1-aminocyclopropane-1-carboxylate oxidase)基因,并分析该基因在龙眼体细胞胚胎(以下简称龙眼体胚)发生过程中的表达情况。【方法】采用RT-PCR结合RACE法,获得龙眼胚性愈伤组织ACO基因的cDNA全长序列和DNA序列,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(q-PCR)法研究该基因在龙眼体胚发生过程中的表达【。结果】克隆得到龙眼胚性愈伤组织ACO基因1315bp的cDNA全长序列(GenBank登录号为FJ534854),该cDNA的开放阅读框推定的氨基酸序列(含315个氨基酸)与其它植物ACO具有86%-47%同源性,包含了5'非编码区为86bp,3'非编码区为281bp,3'poly(A)尾长13bp;该基因的DNA序列(GenBank登录号为GU123929)长为1660bp,包含3个内含子,内含子的剪切位点均符合真核生物"GT-AG"规则;该基因在龙眼体胚各阶段均有表达,整个变化趋势呈字母"M"状。【结论】确定所获得的序列是龙眼胚性愈伤组织ACO基因的cDNA序列和DNA全长序列;该基因在不完全胚性紧实结构和心形胚的表达量为两个峰值。

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.