Construction Of StGID1b Gene Expession Vector And Its Genetic Transformation In Potato

GID1(Gibberellin insensitive dwarf1,GID1)is a receptor protein in the gibberellin(GA)signaling pathway and one of the key factors of this pathway.Three classes of genes encoding this protein are founded in Arabidopsis thaliana: GID1 a,GID1b,and GID1 c.The GID1 gene has been cloned in a variety of angiosperms but less reported in potato.In order to study the role of GA in potato dormancy,11 potato GID1 s family genes(St GID1-1~10 and StGID1b)were cloned.Amino acid sequence analysis showed that there existed family conserved domains in potato GID1 proteins StGID1-1~10 and StGID1 b.qRT-PCR(Real time quantitative PCR,real-time fluorescence quantitative polymerase chain reaction)analysis showed that the expression levels of StGID1-1~10 and StGID1 b genes in potato tubers varied during different storage periods;Transgenic potato was obtained by Agrobacterium-mediated method.The results obtained in this study are as follows:1.Using the nucleotide sequences of 11 GID1 s family genes in the potato database as probes,homologous sequences were obtained by BLAST comparison in NCBI.Through phylogenetic analysis,the homology of the family genes to tomato and pepper was high.Bioinformatics analysis of the family genes identified eleven GID1 s genes on chromosomes 1,6,9,11 and 12.2.qRT-PCR analysis showed that the expression levels of StGID1-1~10 and StGID1 b genes in potato tubers were different at different time periods(0,20,50,61 and 78 d),among which StGID1-b and StGID1-4.StGID1-6 was up-regulated in potato tubers,StGID1-8 and StGID1-10 were down-regulated in potato tubers,while StGID1-b gene was up-regulated.3.Overexpression vector pCPB-StGID1 b was constructed by selecting StGID1-b gene with the most obvious up-regulation,and the interference expression vector pHellsgate8-StGID1 b was constructed using Gateway cloning technology.The potato cultivar was transformed by Agrobacterium mediated transformation.The transgenic plants were obtained 4 and 5 transgenic plants were obtained by PCR.4.By analyzing the 3000 bp upstream of the promoter region of StGID1-b gene,part of the functional cis-acting elements in the promoter region of the gene was obtained;as the bait vector pHis2.1-4TATC-box was constructed by tandem repeat of the TATC-box cis-acting element.Yeast one-hybrid technique was used to predict the interaction between TATC-Box and StGID1 b gene.The results showed that TATC-Box interacts with StGID1 b.