초록 ▼

본연구는 ① 유전자형 판별기술 확립, ② 판별기술 향상, ③ 판별기술의 정확도 검증을 거친 후, ④ 유전양상 확인과 열성 유전자형이 성장에 미치는 효과 파악, ⑤ 국내 집단 내에서의 열성 대립형질의 빈도 파악과 유전자형에 따른 경제형질 측정. ⑥ 그리고 형질에 대한 유전자형 효과를 분석하였으며, 결론적으로 PSS 유전자와 관련된 종돈 육종기법에 대한 방향성을 제시하고자 하였다. 우선, 능력검정중인 자돈의 혈액, 정액, 조직 등을 이용하여 적절한 DNA분리 방법을 확립하였으며, 효율적인 DNA 증폭조건을 설정하기 위해 여러 가지의 P

본연구는 ① 유전자형 판별기술 확립, ② 판별기술 향상, ③ 판별기술의 정확도 검증을 거친 후, ④ 유전양상 확인과 열성 유전자형이 성장에 미치는 효과 파악, ⑤ 국내 집단 내에서의 열성 대립형질의 빈도 파악과 유전자형에 따른 경제형질 측정. ⑥ 그리고 형질에 대한 유전자형 효과를 분석하였으며, 결론적으로 PSS 유전자와 관련된 종돈 육종기법에 대한 방향성을 제시하고자 하였다. 우선, 능력검정중인 자돈의 혈액, 정액, 조직 등을 이용하여 적절한 DNA분리 방법을 확립하였으며, 효율적인 DNA 증폭조건을 설정하기 위해 여러 가지의 PCR primer를 합성하였다. 선별된 PCR primer를 이용하여 증폭된 산물을 제한효소로 단편화 한 후 열성 돌연변이의 존재 여부를 확인하였는데, 1차 처리한 효소에 의한 단편화 여부와 상반되는 결과를 보이는 2차 제한효소 처리를 통해 유전자형 판별에 오차를 제거하였다. 유전자형 판별기법이 확립된 이후에 판별기법의 효율과 용이성을 높이기 위해, 제한효소 처리과정을 생략하여 유전자형을 판별할 수 있는 기법인 유전자형 특히 증폭방법(AS-PCR, allele specific PCR)을 도입하였고, DNA 분리과정을 생략할 수 있는 혈액 직접 증폭법(blood direct PCR)을 확립하였을 뿐만 아니라, 채혈이나 조직시료 채취과정을 간편화하기 위해 모근을 이용한 유전자형 판별을 시도하였다. 이러한 과정을 통해 유전자형 판별기술을 향상하고 난 다음으로, 열성 유전자변이에 의한 스트레스 감수성 여부를 확인하기 위해 halothane 가스를 이용한 발작 여부를 검사하여 유전자형과의 관계를 관찰하였다. 또한, 열성유전자를 가지는 개체들을 교배하여 얻어지는 서로 다른 유전자형의 자손들 간의 성장시의 특성을 비교하였다. 이러한 확립된 기술을 이용하여 국내 능력검정집단에 대해 PSS 유전자형을 판별하였으며, 능력검정에서 측정된 자료를 바탕으로 유전자형의 효과를 분석하였다. 얻어진 결론을 이용하여 PSS 유전자를 종돈육종에 활용할 수 있는 방법에 대하여 고찰하였다.

Abstract ▼

To increase the productivity of swine and the income from pig farming in Korea, it is important to remove the PSS (Porcine Stress Syndrome) gene from Korean pig population. Therefore, this research was conducted to develop the screening methods for PSS gene and the breeding system which will adopt t

To increase the productivity of swine and the income from pig farming in Korea, it is important to remove the PSS (Porcine Stress Syndrome) gene from Korean pig population. Therefore, this research was conducted to develop the screening methods for PSS gene and the breeding system which will adopt the PSS screening method. Experimental animals were secured from the pig farms located in many parts of Korea. Genomic DNA was extracted from blood, semen and tissues of pig. The primers used for PSS screening were synthesized using DNA sequence deposited in GenBank. Two sets of them were optimized to screen the PSS gene and were used for the population screening. The optimal condition for restriction digestion and electrophoresis was also established. The optimal condition of primer for amplification was as follows ; The optimum omount of primer was 10pmole and genomic DNA for PCR amplification was from 100ng to 200ng. The gel electrophoresis in agarose gel or 15％ polyacrylamide gel were suitable for the genotyping of PSS. PCR for 479bp and 83bp fragments including point-mutated 1843 nucleotide(C$\rightarrow$T) in ryanodine receptor 1 gene was performed and then, PCR products were digested by restriction enzyme, Hha I($G^\downarrow$CGC) and Asp H I. As a result of PCR-RFLP, normal homozygous type(N/N) showed two fragments, normal heterozygous type(N/n) had two digested and one undigested fragments, and PSS homozygous type(n/n) had only one undigested fragment. After the establishment of screening techniques, the effort was continued to enhance the methods for technical relief. First, blood direct PCR methods was performed using diluted several white blood cells, resulting successful amplification. Next, direct PCR amplification using the hair-root cell from 100 pulled out hairs or so. Hair root cell was broken by microwave oven for 3-5 minutes. Also successful, stable amplification was obtained using treated hair-root cells. Finally, allele specific PCR was carried out for labour saving in genetic screening. Using one set of outer sequences and two oligonucleotides for each specific allele, genotype differentiating amplification pattern was obtained. Based on the above screening condition, the genotype of 1,463 individuals were determined. The frequency of PSS gene based on the test was 0.098 in L. Yorkshires, 0.224 in Landraces and 0.015 in Durocs. The frequency of PSS gene estimated in this study was similar to the estimates of other countries in L. Yorkshires and Durocs, but was higher in Landraces. The frequency of PSS gene estimated was higher in Kyungsang-do than in 3 other provinces. Variation among the breeding farms in the frequency of PSS gene was also observed in this study. Of 23 farms studied, the frequency of PSS gene was zero only in 5 farms. In view of the overall frequency, 0.105 of PSS gene estimated in this study, it appears that the PSS genes were not eliminated systematically in Korea. A total of 27 progenies were produced from the carrier(Nn) × carrier(Nn) matings with the cross between Pietrain and Large Yorkshire at the Experimental Farm of Seoul National University. The segregation ratio in the progenies from the mating was 8 normal(NN):14 heterozygote(Nn):5 PSS(nn), which was not significantly different from the segregation ratio expected on the basis of the autosomal inheritance of the PSS gene. The viability to 2 weeks of age estimated in the progenies produced at the SNU Experimental Farm was 100％ for normal. 78.6％ for the heterozygote and 40％ for the PSS pigs. The normal and heterozygote pigs genotyped by PCR-RFLP showed the negative reaction to the halothane test and the PSS pigs genotyped by PCR-RFLP showed the positive reaction to the halothane test which indicates the complete agreement between the results of PCR-RFLP and halothane test. The effect of the PSS genotype on the economic traits was estimated on the basis of the data obtained from the performance test of 1,463 pigs that were genotyped with respect to PSS. The nn pigs(PSS) reached at 30kg at significantly older age than the NN pig(Normal) by 4.2 days. The age at 90kg was significantly older in the nn pigs than in the NN pigs by 9.3 days. Average daily gain was significantly lower in the nn pigs than in the NN pigs by 78.9g. The difference between the two homozygotes(NN-nn) in backfat thickness estimated was 0.055cm in males and -0.068cm in females, indicating the significant interaction between PSS genotype and sex for backfat thickness. The difference between NN and nn pigs estimated was -0.03％ for lean meat percent and 7.6 for selection index. The difference between the heterozygote and the mean of the two homozygotes, Nn-(NN+nn)/2, estimated was -0.7 day for the age at 30kg, -2.75 days for the age at 90kg, 34.45g for average daily gain, -0.035 for feed efficiency, 0.0165cm for backfat thickness in males, -0.057cm in backfat thickness in females, 0.05％ for the lean meat content and 4.2 for selection index. In view of the results obtained in this study, it was recommended to establish the breeding system which can effectively eliminate the PSS gene from the nucleus hards or from other breeding stocks in Korea by means of the genotyping of the breeding stock with respect to PSS using the PCR-RFLP technique developed in this study and by selecting against the PSS pigs and the carriers of the PSS gene.

목차 Contents

• 표지...1
• 제출문...2
• 요약서...3
• SUMMARY...13
• 목차...19
• 제 1 장 서론...21
• 제 1 절 연구개발의 목적과 범위...21
• 제 2 절 연구개발사업 목표...24
• 1. 최종연구 사업개발 목표...24
• 2. 연구범위...25
• 제 2 장 실험내용 및 방법...30
• 제 1 절 시험축 선정, 구입 및 시료 채취...30
• 제 2 절 유전자형 판별 기술 확립...30
• 1. DNA 추출...30
• 2. PCR primer의 작성 및 PCR 증폭조건 확립...33
• 제 3 절 유전자형 판별 기술의 효율 향상...34
• 1. 혈액 직접 PCR기법 확립...34
• 2. 모근을 이용한 PCR 기법 확립...35
• 3. AS - PCR 기법을 이용한 유전자형 판별 기술 확립...35
• 제 4 절 유전자형 파악의 정확도 검증...36
• 1. PCR을 이용한 유전자형 파악...36
• 2. Halothane 검사...37
• 제 5 절 유전자형 파악, 능력검정 및 PS S 유전자의 응용...37
• 1. 유전자형 파악...37
• 2. 유전자형확인 종돈의 능력 검정...37
• 3. 조사자료의 통계분석...38
• 4. 종돈개량에 있어 PS S 유전자의 응용...38
• 제 3 장 연구개발 결과...39
• 제 1 절 시험축군의 확보...39
• 제 2 절 유전자형 판별기술 확립...39
• 1. Genomic DNA추출...39
• 2. Primer 준비 및 PCR 증폭조건 확립...43
• 3. 제한효소 처리 및 전기영동조건 확립...45
• 제 3 절 유전자형 판별기술의 효율 향상...49
• 1. 혈액 직접 PCR기법 확립...49
• 2. 모근을 이용한 PCR기법 확립...50
• 3. AS - PCR 기법을 이용한 유전자형 판별 기술 확립...52
• 제 4 절 유전자형 파악의 정확도 검증과 유전자형의 유전양상 확인...56
• 1. 유전자형 파악 및 정확도 검증...56
• 2. PSS 유전자의 유전양상 파악...57
• 제 5 절 PSS 유전자형의 빈도...61
• 제 6 절 PSS 유전자형이 경제형질에 미치는 효과...65
• 1. 능력검정과 PSS 유전자형의 효과...65
• 2. 통계분석...69
• 제 7 절 종돈육종에 있어 PSS 유전자의 응용...78
• 1. 종돈 개량 측면에서의 PSS 유전자...78
• 2. PSS 유전자에 의한 피해를 예방하기 위한 방법...80
• 제 8 절 결론...81
• 제 9 절 참고문헌...82
• 제 4 장 기대되는 성과 및 활용성...85
• 제 1 절 기대되는 성과...85
• 제 2 절 활용 및 건의...86