Cloning and Analysis of 14-3-3 Gene and Its Promotor and Its Expression during Longan(Dimocarpus longan Lour.) Somatic Embryogenesis
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.