Partial cloning and sequencing of some floral organ genes in thermopsis turcica, an endemic legume species with an unusual structure of multi-carpellated flower

The molecular mechanisms controlling unusual floral morphology that is producing 3-4 free carpels from a single flower in the wild legume Thermopsis turcica were aimed to be evaluated. The partial cloning and characterization of A-[APETALA1 (AP1), APETALA2 (AP2)], B-[ APETALA3 (AP3), PISTILLATA (PI)], C-[AGAMOUS (AG)], D-[SEEDSTICK (STK), SHATTERPROOF (SHP)] and E-[SEPALLATA (SEP)] function genes that controlling the initiation and development of floral organs such as sepals, petals, stamens, carpels and seed pods in T. turcica was aimed in this study to reach our major objective. The total RNA extracted from the floral buds of T. turcica was used to produce first strands cDNAs. The degenerate primers were designed from the nucleotide and protein sequences of target genes especially known for the legume species. PCR reactions were set up with cDNA template, gene specific degenerate primer pairs and PhusionTM Polymerase. After separating PCR products on agarose gel, target DNA fragments were cut, removed and then purified from the agarose. After ligating the target DNA fragments into the pJET1.2TM cloning vectors, ligation products were transformed in to competent DH5 E. coli strains. The colony PCR technique was used to identify whether transformants have the right PCR fragment or not. The recombinant plasmid DNA holding the PCR product was isolated from randomly selected transformants and was sequenced. The results of nucleotide and deduced protein sequence clearly indicated that all floral meristem identity genes of our target were partially obtained from T. turcica. 667 bp for AP1, 447 bp for AP2, 480 bp for AP3, 249 bp for PI, 402 bp for AG, 296 bp for SHP, 350 bp for STK and 441 bp for SEP were identified with at least three independent sequencing. In addition to these sequencings, 451 bp for 18S rRNA and 276 bp for -ACTIN were also sequenced to be used as internal control genes in further functional analysis, such as qPCR. BLASTn, BLASTp and CLUSTALw analyses obviously indicated that nucleotide/deduced protein sequences of T. turcica floral meristem identity genes obtained in this study were very similar (100%-70%) to their homologues determined for the other legume species. Consequently, eight different floral meristem identity genes for T turcica were partially cloned and sequentially characterized in the present study. These sequence information would be used to determine the full length cDNA sequences and their functional analyses of the target genes.