| Acyl carrier protein (ACP) is a small acidic protein with a conservative serine residue. In the de novo synthesis of fatty acid in plastid, ACP carrying acyl-chains with different lengths takes part in the cycles of condensation, reduction, and dehydration steps. ACP is also a cofactor for desaturation and acyl-transfer reaction of fatty acid with different chains catalyzed by stearoyl-ACP desarurase and acyl transferase .It has much to do with the species and differential expression of the isomer of ACP,the content and the composition of unsaturated fatty acid in the storage fatty acid in the plants and the proportion of unsaturated fatty acid in the total fatty acid .It has been shown that transgenic Arabidopsis plants, transformed with ACP-1 and its upstream 400 bp region of the transcription start site which is driven by the cauliflower mosaic virus 35S promoter, expressed high levels of ACP-1, and expression of this isoform increased 3 to 8 fold in leaf tissue, but no significant changes in seed. It also revealed that overexpression of ACP-1 in leaf tissue alters fatty acid composition. Levels of 16:3 significantly decreased while 18:3 increased, however, the levels of total fatty acid had no changes.Based on the cloning of peanut AhACP1-1 and AhACP1-1 gene, the expression patterns of these two genes in peanut root, shoot, leaf, flower and seed were analyzed. and, to identify the functions of these genes, transformation of Arabidopsis, tobacco and peanut with construct pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 was carried out. The main results were shown as follows.1. The expression patterns of AhACP1-1 and AhACP1-2 in root, shoot, leaf, flower and seed were analyzed by RT-PCR. It was found that the expression level of AhACP1-1 was highest in seed while nearly no expression in shoot, and similar in root, leaf and flower, and that AhACP1-2 expressed in every parts of transgenic plants, but, the highest expression level was in seed and leaf, and the second in root and flower, and the least in shoot.2. Used pROKⅡas the initial vector, the code region, upstream 54bp and downstream 20bp of AhACP1-1 and AhACP1-2 was forward, or reversely, inserted in the same site of vector, and 4 plant expression vectors were constructed. 3. the genetic transformation of Arabidopsis, tobacco and peanut(1) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into Arabidopsis by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 10 lines of transformants for each construct were obtained. The subsequent generations of transgenic plants were still selected on kanamycin-containing medium.(2) The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into tobacco by Agrobacterium-mediated transformation. After selection on kanamycin-containing medium and PCR analysis, 25 transformants were obtained. Among the transformants obtained, for AhACP1-1, 6 transformants were overexpression type and 6 were antisense expression type, and for AhACP1-2, 7 transformants were overexpression type and 6 were antisense expression type. In comparison with that of the control tobacco, the expression level of the foreign gene in most transgenic tobacco plants was increased.(3) To improve the efficiency of transformation, the efficient system of genetic transformation as the explants of leaflet of LuHua14 was established; and the constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were introduced into peanut by Agrobacterium-mediated transformation. After selection on kanamycin containing medium and PCR analysis, 15 transformants were obtained. Among of them, 5 transformants were overexpression type and 10 were antisense expression type.4. Functional Identification of the AhACP1-1and AhACP1-2 in the transgenic tobaccoThe content of fatty acid in leaf of some transgenic tobacco was detected by gas chromatograph. The results indicated that the content of unsaturated fatty acid was increased in comparison with the control. Then, the concentration of malondialdehyde, free proline and soluble sugar of the transgenic plants and the control plant, which were treated at 8℃in 0h,0.5h,1h,2h,4h,6h and 8h, was detected by the method of Spectrophotometer detection The results indicated that the concentration of free proline and soluble sugar of the transgenic plants were increased in comparison with the control, and that the concentration of malondialdehyde was reduced.To sum up, this study modified the peanut regeneration and transformation system. The constructs of pROKⅡ:AhACP1-1 and pROKⅡ:AhACP1-2 were transformed into Arabidopsis, tobacco and peanut by Agrobacterium-mediated transformation; the functional analysis of the transgenic plants indicated that the constitutive expression of AhACP1-1and AhACP1-2 in tobacco could change the composition of fatty acid in leaf of the transgenic plants, and the increasing of content of unsaturated fatty acid may improve the cold resistance of the transgenic plants. Next,we would analyze the function of AhACP1-1and AhACP1-1 in transgenic Arabidopsis and peanut, and overexpress them in seed of peanut to change the content and the composition of fatty acid in seed of peanut by gene engineering.
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