Cloning And Expression Analysis Of Cold-Related Genes Of Ammopiptanthus Nanus

In this study, the cDNA of cold-related enzyme gene AnGPAT and partial sequence of antiretroviral factor AnICEa gene and AnICEa gene were cloned from Ammopiptanthus nanus which is highly cold hardiness plant. The genes were sequence analyzed and molecular evolution analyzed and then AnGPAT prokaryotic expression were done in vitro and transgenic expression vector were constructed. That provided an important reference on discovering the new resistance genes, as well as laid the theoretical foundation for coordination mechanisms of different cold resistance genes and breeding by genetic engineering. The main results are as follows:A full length of 1482 bp cDNA of Ammopiptanthus nanus GPAT gene was successfully isolated by homology cloning and RACE, the gene was named AnGPAT. The sequence analysis had revealed that AnGPAT contained a maximum open reading frame of 1380 bp, encoding a protein of 460 amino acids. The estimated molecular weight and isoelectric point of the putative protein were 50.9 KD and 7.38, respectively. Conservative functional area search found that amino acid residues from 201 to 436 were GPAT conserved domain: glycerol phosphate for the synthesis of lysophosphatidic acyltransferase (LPLATs). Phylogenetic tree analysis showed that AnGPAT had high homology with GPAT protein of beans(Vicia faba) and oil palm (Elaeis guineensis).Two homologous ICE genes had been isolated by homology cloning and chromosome walking techniques, named AnICEa and AnICEb, respectively. Conserved domain bHLH (basic-helix-loop-helix) had been found in the two genes. Evolution analysis on Amino acids of AnICEa and C terminal of AnICEb showed that AnICEa and soybean (Glycine max) ICE protein were clustered together, and AnICEb had high homology with ICE protein of radish (Raphanus sativus) and Arabidopsis (Arabidopsis thaliana).3. Inserted open reading frames of the AnGPAT gene into MCS district of expression vector pET-30 a (+) and then obtained the pET-30a-GPAT expression vector. Transformed into E. coli BL21, incubated at 37℃and induced by 1mM IPTG, and then approximately 51 kDa fusion proteins, the same size with the prediction, were obtained.4. By constructing intermediate expression vector pGM-35S-GPAT-NOS, AnGPAT connected with strong promoter and terminator. Then 35S-GPAT-NOS fragment was cloned into MCS district of pCAMBIA2300, successfully constructed p2300-35S-GPAT-NOS eukaryotic expression vector. That laid the foundation for subsequent research on gene function by using transgenic technology.