| Tobacco is an important economic crop in China, early flowering has a great influence on the yield and quality. CO gene and FT gene are two important regulatory genes in the photoperiod pathway in plants blossom. CO gene induces the expression of the downstream gene FT in the light condition, thus promotes the flowering of plants. The three tobacco cultivars (Yunyan 85, Yunyan 97 and Xingyan No.1) were closely related but had different flowering time. Studying the molecular differences of homologous gene CO and FT between three cultivars can help to reveal the mechanism of the different flowering time between them, and provide the molecular basis for the further utilization of genetically engineered cultivars. In this article, we chose three tobacco cultivars:Yunyan 85, Yunyan 97 and Xingyan No.1 as experimental materials. We designed primers COF and COR for NtCO gene (GenBank accession number JQ686941) and FTF and FTR for N. tomentosiformis FT1 gene (GenBank ID: KC485968). Using those primers, we cloned CO, FT gene of three tobacco cultivars from their genome DNA. Then, we sequenced the obtained genes. The phylogenesis was analyzed by sequence alignment of DNA and predicted amino acid sequences according to the conserved domain. The main results are as follows:(1) Phylogenetic relationships between 3 tobacco cultivars (Yunyan 97, Yunyan 85 and Xingyan No.1) were identified by SSR-PCR by using 14 pairs of SSR core primers.(2) We obtained partial DNA sequences of CO and FT gene from three tobacco cultivars using PCR amplification. We named those CO genes Yunyan85 CO, Yuanyan97 COa, Yunyan97 COb, Yuanyan97 COc, Xingyan No.1 COa and Xingyan No.1COb, which are 1534bp expected in Yunyan85 CO(1531bp); and named those FT genes Yunyan85 FTa, Yunyan85 FTb, Yuanyan97 FTa, Yunyan97 FTb, Xingyan No.l FTa, Xingyan No.l FTb and Xingyan No.1 FTc, which are 1409bp expect in Yuanyan97 FTa (1407bp).(3) Their introns and exons were identified and the corresponding amino acid sequence was deduced by analyzing of three cultivars of CO homology gene and FT homology gene which were cloned by PCR and according to the coding sequence of the corresponding gene of NCBI database:NtCO gene (JQ686941) and N. tomentosiformis FT1 gene (KC485968). Homology comparison corresponding amino acid of Yunyan85 CO gene to corresponding amino acid of the other two cultivars of CO homologous gene shows there are 18 amino acid mutations. Homology comparison corresponding amino acid of Yuanyan97 COa gene and Yunyan97 COb gene to corresponding amino acid of the other two cultivars of CO homologous gene shows there are 3 amino acid mutations. Homology comparison corresponding amino acid of XingyanNo.1 COa gene to corresponding amino acid of the other two cultivars of CO homologous gene shows there are 3 amino acid mutations. Homology comparison corresponding amino acid of Yuanyan97 COc gene to corresponding amino acid of the other two cultivars of CO homologous gene shows there are 1 amino acid mutations. Homology comparison corresponding amino acid of Xingyan No.l FTa gene to corresponding amino acid of the other two cultivars of FT homologous gene shows there are 2 amino acid mutations.(4) Analysis of predicted amino acid sequences of CO and FT gene from three tobacco cultivars using CD-search (Conserved Domains search) shows that the predicted amino from three tobacco cultivars acid sequences of CO gene have two BBOX domains in amino terminal and one CCT domains in carboxyl terminal; The predicted amino acid sequences of FT gene from three tobacco cultivars share a PEPB homology structure of FT gene family.(5) We compare the amino acid sequence deduced from the cloned gene and the amino acid sequence of the reported amino acid sequence using MEGA6 software with the method of Neighbor-Joining algorithm. The result shows that Yunyan85 CO has relatively far relationship compared with other cloned CO gene; Xingyan No.1 FT1 also has a relatively far relationship compared with other cloned FT gene. |
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