Cloning And Expression Analysis Of GPAT Genes Of Ammopiptanthus Nanus

In this study, AnGPAT gene which is a cold-related enzyme gene in the highly cold hardiness plant Ammopiptanthus nanus was analyzed about partial sequence and molecular evolution. The prokaryotic expression and the P. pastoris Eukaryotic expression of AnGPAT gene were done in vitro, then extracted AnGPAT enzyme from it. The transgenic expression of AnGPAT was done and introduced into agrobacterium to Function Identification of gene. That provided an important reference on discovering the new cold resistance genes and application of genetic engineering in forest tree breeding.1.GPAT gene of Ammopiptanthus nanus was isolated by PCR, named as AnGPAT and analysis proves that AnGPAT gene contained a maximum open reading frame of 1380 bp, encoding a protein of 460 amino acids. According to the estimation of AnGPAT, molecular weight of the putative protein is 40.9 kD and isoelectric point is 7.38. Conservative functional area search found that amino acid residues from 95 to 171 was GPAT_N domain; Amino acid residues from 201 to 436 was LPLATs domain, which is one of conserved domain of GPAT enzyme. Phylogenetic tree analysis showed that AnGPAT had homology with GPAT protein of Glycine soja, the result of its homology was 83%.2.Inserted open reading frames of the AnGPAT gene into prokaryotic expression vector pET30a(+), obtained the pET30a-AnGPAT expression vector. Transformed into E.coli BL21, induced expression AnGPAT gene. Then purified approximately 40kDa protein, the size same with the prediction; Inserted open reading frames of the AnGPAT gene into P. pastoris Eukaryotic expression vector pPIC9, obtained the pPIC9-AnGPAT expression vector. Transformed into P. pastoris GS115, induced expression AnGPAT gene. Then purified approximately 45KDa protein, the size same with the prediction.3. The fatty acid content of transgenic E.coli BL21 was detected by liquid chromatography, the results showed that the content of unsaturated fatty acid of transgenic E.coli BL21 was higher than that of control. It is proved that prokaryotic expression of AnGPAT has biological activity.4.Inserted open reading frames of the AnGPAT gene into Eukaryotic expression vector pBI121, obtained the pBI121-AnGPAT expression vector. Transformed into Agrobacterium LBA4404. Transformed into tobacco, successfully constructed the transgenic tobacco. The results of the PCR analysis among transgenic tabacum indicated that AnGPAT gene was integrated into the genomes of transgenic tabacum.