Cloning And Identification Of Three Blossom Correlation Genes From Alfalfa (Medicago Sativa L.)

Alfalfa is an important leguminous forage plant and study its mechanism of flowering has great significance.In this study, the cDNA sequences of alfalfa LEAFY (LFY) gene, Flowering Locus T (FT) gene and NMH7 gene was obtained by using homology cloning and RACE technique. The main results of preliminary identification are as follows:The genomic DNA sequence of MsLFY was 2138bp and has two introns.The ORF(Open reading Frame) was 1176bp,encoded a protein of 392 amino acids and the protein was located in nucleus.The semi-quantitative RT-PCR analysis of MsLFY expression patterns showed that the expression of MsLFY gene is with a low level in roots,stems,leaves and pods, the expression level in floral buds and flowers are higher than other tissues; The expression of MsLFY is induced by GA and long photoperiod.Plant expression vector was constructed and transformed into tobacco by the Agrobacterium-mediated methods. PCR amplification with the transgenic tobacco genome DNA indicated that MsLFY gene had integrated in tobacco genome;RT-PCR analysis showed that MsLFY gene can be transcribed into mRNA in transgenic tobacco.The genomic DNA sequence of MsFT was 1705bp and has three introns.The ORF was 528bp,It encoded a protein of 176 amino acids and the protein was located in nucleus.The semi-quantitative RT-PCR analysis of MsFT expression patterns showed that the expression of MsFT gene is with a low level in roots,stems and pods, the expression level in leaves and flowers are higher than other tissues; The expression of MsFT is induced by SA and long photoperiod.The 1235bp sequence before the gene transcription site was obtained by TAIL-PCR technique, sequence analysis showed that it contains a number of light-induced component,and also contains salicylic acid response element, heat responsive element and the MYB binding element,.The ORF of NMH7 gene was 687bp and encoded an protein of 229 amino acids,the protein was located in nucleus.To identify the subcellular location of Alfalfa NMH7 gene,a recombinant plasmid with a green fluorescent protein gene(GFP-NMH7) has been constructed and was transformed into onion epidermis cells by gene gun method.Transient expression analysis indicated that GFP-NMH7 fusion protein was localized in the nucleus of onion epidermal cells.Semi-quantitative RT-PCR analysis was used to determine the tissue expression patterns of NMH7 gene in alfalfa,the result revealed that it expressed itself in roots,floral buds,flowers,pods and there was the highest expression level in flowers.The results of Semi-quantitative RT-PCR analysis showed that the expression of NMH7 gene in alfalfa was not relate to gibberellin and reduced by abscisic acid and Jasmonic acid methyl ester.