Identification Of Core Promoter Of Pig CuZnSOD Gene And The Study Of CuZnSOD Gene Transgenic Rabbit By Sperm-mediated Gene Transfer

With the development of modern large scale farming, oxidative stress has appeared in theproduction of commercial pigs. Pig copper zinc superoxide dismutase (CuZnSOD) is animportant antioxidant enzyme，and plays an important role in maintaining the balance ofoxygen free radicals. CuZnSOD is widely distributed in the cytoplasmic matrix, accountingfor about90%of total SOD. With the development of biotechnology, the CuZnSOD geneomesequences of many species have been cloned. Now CuZnSOD gene research more focused onits function, and regulation mechanism is not clear. In order to further investigate the structureand function of CuZnSOD gene, promoter of CuZnSOD gene was amplified by PCR.CuZnSOD gene transgenic rabbit model was producted to verify the function of theCuZnSOD gene for improving muscle antioxidant capacity. The main results are as follows:1.853bp DNA sequence of5′-flanking promoter was amplified by PCR. A series ofCuZnSOD promoter fragments with gradually truncated5′-end were produced by nested PCRand inserted into the pGL3-Basic vector. The promoter activities were measured by thedual-luciferase assay system after transient transfection. The results demonstrated that theregion from-383bp to+67bp in CuZnSOD gene promoter showed higher activity than otherregions, and further deletion analysis demonstrated that the region from-75bp to-32bpincludes an essential promoter sequence for pig CuZnSOD gene transcription. In addition,several potential transcriptional factor binding sites were predicted with bioinformaticsmethod，suggesting that these transcription factors binding sites may be involved in thetranscriptional regμlation of CuZnSOD gene.2. By restriction enzyme identification, PCR and sequence analysis, we successfμllyconstructed the eukaryotic expression vector. The green fluorescent protein-expressing cellswere observed after transfection.The expression of target gene was detected by RT-PCR.3. Six healthy adult New Zealand male rabbits were selected, and they were treated bybilateral testicular injection. The injection composed of the pIRES2-AcGFP1-CuZnSOD,transfection reagents and appropriate DMEM (The former two have been incubated15 minutes before mingled with the latter). Another two rabbits were selected as the controlgroup, and they were also treated by bilateral testicular injection except the injectioncontained same amount of saline only. The injection continued three weeks with one time foreach week. One month after the last injection, all of the eight male rabbits mated with normalfemales at a ratio of1:6to get the F1generation. Then the F1hybridized to get the F2generation.4. In our experiment, the test group gave birth to169F1offsprings (54transgenicpositive ones and115negative ones) with a transgenic positive rate of31.95%. The controlgroup of F1generation gave birth to48offsprings. The F1generation gave birth to58F2offsprings (23transgenic positive ones and35negative ones) with a transgenic positive rateof39.66%. The control group of F2generation gave birth to29offsprings. The result ofcarcass traits showed daily gain in the F1transgenic positive group was significantly lowerthan those of the negative and blank groups（P<0.05）. The eviscerated weight of the testgroup differed significantly with the control group (P<0.01). The result of meat qualityshowed drip loss in the F1transgenic positive group was significantly less than that of thenegative and blank groups（P<0.01）. The T-SOD activity was elevated significantly in the F1transgenic positive group compared to the negative ones and the control group (P<0.05). Theprotein expression of the CuZnSOD was higher in the F1transgenic positive group than theother two groups(P<0.05). The fluorescence of the transgenic positive rabbits was moreintense than the control group, and it fluoresced in the limbs and tail mainly. We measured themeat quality of the F2generation, too. Daily gain of the F2transgenic positive ones weresignificantly lower compared to the negative and blank groups(P<0.05). Drip loss in the F2transgenic positive group was significantly less than that of the negative and blankgroups(P<0.05). The T-SOD activity was elevated significantly in the F2transgenic positivegroup compared to the negative ones and the control group (P<0.05).