보고서 정보
주관연구기관 |
경북대학교 KyungPook National University |
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2016-02 |
과제시작연도 |
2015 |
주관부처 |
농촌진흥청 Rural Development Administration(RDA) |
등록번호 |
TRKO201600003256 |
과제고유번호 |
1395039918 |
사업명 |
국책기술개발 |
DB 구축일자 |
2016-06-25
|
DOI |
https://doi.org/10.23000/TRKO201600003256 |
초록
▼
Ⅳ. 연구개발결과
○ 콩 지방산 개량에 사용될 유전자원 확보 및 DNA 마커 개발
- PE1690(low linolenic acid, 4-5%)의 FAD3A 유전자 돌연변이를 밝히고 돌연변이를 검출할 수 있는 DNA 마커를 개발하였다.
- PE980(high stearic acid, 17%)의 SPACD_C 유전자의 돌연변이를 밝히고 돌연변이가 high stearic acid 함량과 관련이 있다는 것을 밝혔다.
- PE451(high oleic acid, 32%), PE2166(high linolenic aci
Ⅳ. 연구개발결과
○ 콩 지방산 개량에 사용될 유전자원 확보 및 DNA 마커 개발
- PE1690(low linolenic acid, 4-5%)의 FAD3A 유전자 돌연변이를 밝히고 돌연변이를 검출할 수 있는 DNA 마커를 개발하였다.
- PE980(high stearic acid, 17%)의 SPACD_C 유전자의 돌연변이를 밝히고 돌연변이가 high stearic acid 함량과 관련이 있다는 것을 밝혔다.
- PE451(high oleic acid, 32%), PE2166(high linolenic acid, 17%), PE2742(high oleic acid, 34%), PE3058(high palmitic acid, 15%)에 대하여 각각 계통별로 지방산 생합성 관련 유전자의 염기서열을 분석하였으나 특이한 이상을 발견하지 못하였다.
- PE451(high oleic acid), PE2166(high linolenic acid), PE2742(high oleic acid), PE3058(hihg palmitic acid)과 풍산 또는 우람콩과 인공교배에서 만들어진 F2 집단에 대하여 지방산 분석을 실시하여 분리 정도를 확인하였다.
○ Group A 사포닌 결핍 콩 육성을 위한 유전자원 확보 및 DNA 마커개발
- LC/MSMS 분석을 통해 PE1515의 사포닌 표현형을 확정 지었다.
- PE1515의 Sg-1 유전자 염기서열을 분석하여 돌연변이를 발견하고 그것이 사포닌 group A 생성과 어떤 영향을 미치는 것에 대한 결과를 얻었다.
- PE1515의 돌연변이를 검출할 수 있는 DNA 마커를 만들었고, 그것을 바탕으로 유전 분석을 하여 한 개의 열성 유전자에 의해 지배된다는 것을 밝혔다.
Abstract
▼
Mutation breeding, the method of treating seeds with chemical mutagen like ethyl methanesulfonate (EMS) to generate mutants with desirable traits, has been utilized in developing new soybean cultivars. The use of EMS as a chemical mutagen and screening for induced changes in a gene target of interes
Mutation breeding, the method of treating seeds with chemical mutagen like ethyl methanesulfonate (EMS) to generate mutants with desirable traits, has been utilized in developing new soybean cultivars. The use of EMS as a chemical mutagen and screening for induced changes in a gene target of interest, provides an allelic series of mutants that can be used functional genomics and crop improvement. Hence, the objective of this project was to determine the genetic basis of selected mutant lines for altered fatty acid concentrations and group-A acetylsaponin-deficient EMS-induced mutant.
In one of the previous studies, a mutant population was developed by treatment of seeds of soybean cultivar Pungsannamul with 0.3% EMS (Chae et al., 2013) and advancing to the further generations in the field. By analyzing the total fatty acid content of the M4 mutant population (n=892), several mutants with altered fatty acid compositions, for instance, PE1690 (low linolenic acid, 5%) PE980 (higher stearic acid, 17%), PE451 (higher oleic acid, 32%), PE2166 (higher linolenic acid, 18%), PE2742 (higher oleic acid, 34%), and PE3058 (higher palmitic acid) were identified. Besides, we also selected a group-A acetylsaponin-deficient mutant (PE1515). These above-mentioned lines were used in this project to determine the genetic basis of the respective traits.
1. Characterization of mutants with altered fatty acid compositions
DNA isolated from the mutant PE1690 was used to amplify the genic sequence of a candidate gene GmFAD3A. Upon sequencing of the PCR products, we detected a point mutation generating a substitution of adenine with guanine in the 2nd intron splice-site of the GmFAD3A gene. Mutation of PE1690 at the first base of the 2nd intron caused missplicing which led to the inclusion of 334 bp of the 2nd intron into the coding sequence. The amino acid sequence of the mutant was analyzed based on cDNA sequence of GmFAD3A in PE1690. The amino acid positioned at 128, which codes for tryptophan (TGG) in PE1690 was changed to a premature stop codon (TGA). This showed that a single base mutation in GmFAD3A gene in the mutant PE1690 renders the desaturase enzyme nonfunctional. Based on a genetic marker that we developed, the association of the PE1690 derived GmFAD3A allele with linolenic acid level was tested in 89 F2 progenies of a cross between Pungsannamul (wild type) x PE1690. The results showed that the mutation of GmFAD3A in PE1690 was associated with reduced linolenic acid content in the soybean seed oil.
The mutant PE980 was identified as a high stearic acid (SA) mutant from the M4 mutant population. The candidate gene Δ 9-stearoyl-acyl carrier protein-desaturase (SACPD) genes are responsible for the conversion of SA content and oleic acid content. Mutation in one of these genes may block the conversion of SA to oleic acid, resulting in the increasing the SA content in the seed. A total of three SACPD genes have been reported to be present at three different locations in soybean chromosomes. To identify the candidate for high SA in PE980, genomic sequence of SACPD gene was downloaded from phytozome and primers were synthesized. The genomic DNA of mutant and wild type were used to amplify and sequence the SACPD exonic regions and sequenced. We detected a mutation in 1st exon at 703 position in SACPD_C gene. The single base mutation caused valine to methionine change in the amino acid sequence. No mutations were detected in other two genes SACPD_A and SACPD_B in PE980. To identify whether the mutation was associated with SA, Bulk Segregant Analysis (BSA) was performed by constituting the high (DNA from 10 F2 plants with high SA) and low (DNA from 10 F2 plants with low SA) bulks and sequencing the bulks along with parental samples using 180K SNP chip. The BSA showed high association of the mutation with the increased SA levels in the PE980 mutant. The CAPS-based marker designed for the respective SNP, further confirmed the results.
To find out the mutation for genes related fatty acid biosynthesis, we have sequenced KASII genes of PE3058 for palmitic acid, FAD2 genes of PE451 and PE2742 for oleic acid, and FAD3 genes of PE2166. The sequences of these candidate genes were compared with those of wild type Pungsannamul and we did not find any non-synonymous mutations. These results indicated that mutations in other genomic regions probably might have altered the fatty acid content in the mutant lines.
2. Characterization of a mutant with a group-A acetylsaponin deficient soybean mutant
Saponins are major components of secondary metabolites in soybean seed that have been broadly studied for their beneficial effects on human health. In soybean, group A acetylsaponins can be classified into two types, Aa- and Ab-series, both of which cause bitterness and astringent after taste. Consequently, group A acetylsaponins are considered as unwanted components that have negative effects on soybean use in food industries.
Therefore, group A acetylsaponin-deficient cultivars solve the purpose for high use of soybeans in food industry. The objective of this study was to investigate a group A acetylsaponin deficient soybean mutant (PE1515) generated from the cultivar 'Pungsannamul' (containing Ab-series saponins) by EMS treatment. The phenotypic and genotypic variation was investigated for the mutant PE1515. Our analyses showed that in the mutant PE1515, the Ab-series saponins were absent and A0-αg saponin accumulated as the major saponin. DNA of the mutant and wild type was utilized to amplify and sequence the candidate gene Sg-1. PE1515 had a single base substitution from G to A at 1004 bp of the Sg-1 gene which introduced a premature stop codon in the Sg-1 gene. A DNA marker to detect the single nucleotide substitution was developed for breeding purpose. Using this marker, association of sg-1 allele in PE1515 was verified using 120 F2 progenies of a cross Pungsannamul x PE1515. The results showed that the mutation of the Sg-1 gene in PE1515 contributed to the lack of Ab-series saponins in the seed hypocotyl.
In conclusion, the mutants PE1690 (low linolenic acid), PE980 (high stearic acid), and PE1515 (group A acetylsaponin deficiency) characterized in the present study, may find useful as an alternate genetic resources to improve the quality of soybean oil. In addition, these mutants can be good materials to understand the genetic and molecular basis of fatty acid biosynthesis in soybean.
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