【摘要】 分离克隆龙眼(Dimocarpus longan Lour.)胚性愈伤组织14-3-3基因,并分析该基因在龙眼体胚发生过程中的表达情况。采用RT-PCR结合RACE法,获得龙眼胚性愈伤组织14-3-3基因的cDNA全长序列和DNA序列,采用TAIL-PCR法,获得龙眼胚性愈伤组织14-3-3基因的启动子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(qRT-PCR)研究该基因在龙眼体胚发生过程中的表达。结果表明:经克隆得到龙眼胚性愈伤组织14-3-3基因786 bp的cDNA全长序列(GenBank检索号为GU573765),该cDNA开放阅读框推定的氨基酸序列(含261个氨基酸)与其它植物14-3-3具有较高同源性;该基因的DNA序列(GenBank检索号为GU573766)长为1 859 bp,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;该基因的启动子DNA序列(GenBank检索号为GU573766)长为910 bp;该基因在龙眼体胚各阶段均有表达,其中在松散型胚性愈伤组织阶段和心形胚及鱼雷形胚阶段呈现高表达,整个变化趋势呈"倒S"状。更多还原

【Abstract】 In this study,14-3-3 gene and its promotor were cloned from embryogenic calli of longan(Dimocarpus longan Lour.).And the expression of 14-3-3 was determined during longan somatic embryogenesis.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 14-3-3 from embryogenic calli of longan.And bioinformatics methods were used to analyze sequences obtained and putative amino acid sequence.Then qRT-PCR(real-time reverse transcription PCR)method was used to determine the mRNA transcription level of this gene.The full length 14-3-3 cDNAwas about 786 bp.The sequence was submitted to the DDBJ/EMBL/GenBank database,and the accession number was GU573765.The putative protein had 261 amino acids,and the identity to the other polypeptides varied between 96%～73%.Its DNA sequence(the accession number was GU573766)was 1 859 bp,and the splice sites of three introns contained were obeyed to the "GT-AG rule".The length of 14-3-3 promotor was 910 bp(GenBank: GU573766).14-3-3 from embryogenic calli of longan expressed in different stages during somatic embryogenesis.And it showed approximately a "inverted S" curve.It was inferred that the full length cDNA and DNA sequence from embryogenic calli of longan were obtained.The peak mRNA transcription level of longan 14-3-3 gene occurred at the incomplete compact pro-embryogenic culture stage and the heart embryo stage and the Topedo embryo stage.更多还原