Cloning Of SOC1 And AG Genes From Fraxinus Mandshurica And Analysis Of Genetic Transformation

SOC1 gene is a controlling gene of flowering time,AG gene is a controlling gene of higher plants flower organ development.these two genes play important roles in the flower development.In this study,we cloned the coding regions of two genes from Fraxinus mandshurica,respectively named FmSOCl and FmAG,and successfully constructed two plant expression vectors:pROK Ⅱ-35S::FmSOC1and pCAMBIA1301-35S::FmAG.By Agrobacterium-mediated genetic transformation technology,we successfully transferred them into tobaccos,obtained transgenic To plants.The main results were as follows:1.Cloning and bioinformatic analysis of FmSOCl and FmAG genesIn this study we obtained the full lengths of FmSOCl and FmA Ggenes.The length of FmSOCl gene was 654bp,the consistency with the SOC1 nucleotides sequence of Fraxinus mandshurica transcriptome was 93%,the predicted amino acid length was 218 aa,the molecular weight of encoded protein was 24920.43,the pI was 9.40,the instability index was 61.33,subcellular localization analysis showed the FmSOC1 gene is expressed in nucleus, chloroplasts and mitochondria,cluster analysis showed the FmSOCl protein has closest relationships with the SOC1 homologous proteins of Sesamum indicum,Plantago major and Antirrhinum majus.The length of FmAG gene was 726bp,the consistency with the AG nucleotides sequence of Fraxinus mandshurica transcriptome was 98%,the predicted amino acid length was 242 aa,the molecular weight of encoded protein was 27913.51,the pI was 9.25,the instability index was 67.48,subcellular localization analysis showed the FmAG gene is expressed in nucleus,cluster analysis showed the FmAG protein has a closest relationship with the AG homologous proteins of fraxinus pennsylvanica, Sesamum indicum and Antirrhinum majus.2.Construction of plant expression vectors pROK Ⅱ-35S::FmSOCl and pCAMBIA1301-35S::FmAG,optimization of genetic transformation systemWe constructed two plant expression vectors,respectively as the pROK II-35S::FmSOCl with Xba I/Sac I double enzyme digestion and the pCAMBIA1301-35S::FmAG with Nco I,then transformed into LBA4404.Meanwhile we optimized the Agrobacterium-mediated tobacco genetic transformation system.The results were as follows:pre-cultured 2 days,the OD₆₀₀ was setted as 0.8,Agrobacterium tumefaciens dipped 25 minutes,co-cultured 3 days.This system could improve the genetic transformation efficiency.The best concentrations of kanamycin and hygromycin were 50mg/L and 20mg/L.3 Genetic transformation and molecular identification of transgenic tobaccosWe detected To transgenic tobaccos through PCR and PCR-Southern blotting,initially confirmed the FmSOC1 have integrated into the tobaccos genome,the conversion rate was 31.58%.Randomly selected five PCR positive tobaccos and performed RT-PCR,we found that three of them conducted SOC1 genetic transcription,the flowering time of FmSOC1 T0 transgenic tobaccos were earlier than the wild-type by phenotype observation.