Isolation And Expression Analysis Of An AP2and A LTP Gene Involved In Flower Development From In Betula Platyphylla

The full cDNA of AP2and LTP gene of flower development was isolated from Betula platyphylla Suk. female and male inflorescence by methods of reverse transcription polymerase chain reaction (RT-PCR) and5’and3’rapid amplification of cDNA ends (RACE), respectively. The deduced protein of AP2and LTP were analyzed with bioinformatics software. Then transcription expression of the two genes was analyzed by real-time PCR in different tissues and periods in wild-type and a natural male inflorescence-abnormal mutant B. platyphylla. and the gene function was analysised preliminarily. At the same time, constructed pROK2-LTP plant expression vector, to provide help for the future of transgenic work. The main results are as follows:1. The full cDNA of AP2gene was isolated from Betula platyphylla Suk. female inflorescence by methods of reverse transcription polymerase chain reaction (RT-PCR) and5’and3’rapid amplification of cDNA ends (RACE). Results showed that AP2gene contains an open reading frame (ORF) of1554bp encoding517amino acids. Molecular weight of the deduced protein of BplAP2is56.74kDa and the theoretical isoelectric point is6.34. The AP2functional sites and characterized domains were confirmed in the sequence, so the isolated gene was named as BplAP2, and registered in GenBank with accession number JN247408. The deduced amino acid sequence shared51%～77%of identity with other twelve plant species, the maximum identity with Arabidopsis thaliana (77%) and minimum identity with with Pinus thunbergii (51%). A phylogenetic tree was constructed according to multiple sequences alignment of all the thirteen plant species.2. Transcription expression of BplAP2was analyzed by real-time PCR in different tissues and periods in B. platyphylla. Results showed that BplAP2was more highly expressed in floral organs than in vegetative organs, expression quantity more highly in young tissues than in mature tissues. It inferred that BplAP2transfactor involved in the regulation of development of floral organs and meristematic tissues in Betula. In addition, a natural male inflorescence-abnormal mutant of B. platyphylla was used for transcription analysis of BplAP2. Results showed that BplAP2gene is expressed up-regulatedly in female inflorescences, while down-regulation in male inflorescences, young leaves and young shoots, which predicted that BplAP2should be involved in regulation and expression of multiple genes, and not only be involved in the development of floral organs, but also play some roles in the development of vegetative tissues.3. The full cDNA of LTP gene was isolated from Betula platyphylla Suk. male inflorescence by methods of reverse transcription polymerase chain reaction (RT-PCR) and 5’and3’rapid amplification of cDNA ends (RACE). Results showed that LTP gene contains an open reading frame (ORF) of363bp encoding120amino acids. The protein has a conserved lipid binding motif DRQ and by eight conserved cysteine residues conserved domain of the superfamily of AAL-LTSS.Molecular weight of the deduced protein of BplLTP is12.3kDa and the theoretical isoelectric point is9.01, so the isolated gene was named as BplLTP, and registered in GenBank with accession number JQ409562. The deduced amino acid sequence shared52%～64%of identity with other fifteen plant species, the maximum identity with Gossypium hirsutum (64%) and minimum identity with with Arabidopsis thaliana (52%). A phylogenetic tree was constructed according to multiple sequences alignment of all the sixteen plant species. And constructed pROK2-LTP plant expression vector.4. Transcription expression of BplLTP was analyzed by real-time PCR in different tissues and periods in B. platyphylla. Results showed that BplLTP was more highly in young tissues than in mature tissues,expression quantity almost undetectable in mature pollen,and expression in different stages of male and female inflorescence. In addition, a natural male inflorescence-abnormal mutant of B. platyphylla was used for transcription analysis of BplLTP. Results showed that BplLTP gene is expressed up-regulatedly in female inflorescences and young leaves, while down-regulation in male inflorescences and young shoots, which predicted that BplLTP should be involved in development of a variety of organizations, but also play some important roles in plant sexual reproduction.