大豆7S球蛋白α'亚基缺失新种质中黄608的分子鉴定
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摘要
大豆7S球蛋白α'亚基含量与大豆的营养品质和加工特性关系密切。本研究利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和免疫印迹法(Western blot)从中品661的EMS突变库中筛选出α'亚基缺失突变体中黄608。利用中黄608与登科1号创建了一个由210个个体组成的F2分离群体。遗传分析表明,α'亚基缺失由1对隐性单基因控制。利用连锁分析方法将该基因定位于第10染色体标记SSR10-1489与SSR10-1612之间,其中,包括控制α'亚基合成基因Cgy-1(Glyma.10G246300),序列分析发现,中黄608在Cgy-1第1外显子第84个碱基发生单碱基突变(G84→A84),导致氨基酸翻译提前终止。根据新发现的变异位点开发了共显性分子标记,并检测F2个体基因型,结果表明, Cgy-1基因型与α'亚基表型共分离。本研究不仅为大豆优质育种提供了新材料,同时也为分子育种提供了技术支持。
The content of alpha' subunit of 7 S globulin has an important effect on the nutritional quality and processing characteristics in soybean. In this study, using polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting, Zhonghuang 608(ZH608), an α' subunit deletion mutant, was identified from the Zhongpin 661(ZP661) EMS mutant library. Zhonghuang 608 was crossed to the soybean variety Dengke 1, and self-pollinated to establish an F2 segregating population composed of 210 individuals. Genetic analysis showed that the alpha' subunit deletion trait in ZH608 was controlled by a single recessive nuclear gene. By linkage analysis we mapped the locus between SSR10-1489 and SSR10-1612 on chromosome 10. Coincidentally, in the mapping region Cgy-1(Glyma.10 G246300) was annotated as an alpha' subunit synthesis-related gene according to the Williams 82 reference genome. Further sequence analysis showed that the mutant had a single base change(G84 to A84) in the first exon of Cgy-1, which resulted in the premature termination of the amino acid sequence. Based on the newly discovered SNP mutation, a co-dominant molecular marker was developed, and used to detect the genotypes of those F2 individuals. The results indicated that the Cgy-1 genotype cosegregated with the alpha' subunit phenotype in the F2 segregating population of Zh608 and Dengke 1. This study not only provides new materials for quality improvement, but also offers technical support for molecular breeding in soybean.
The content of alpha' subunit of 7 S globulin has an important effect on the nutritional quality and processing characteristics in soybean. In this study, using polyacrylamide gel electrophoresis(SDS-PAGE) and Western blotting, Zhonghuang 608(ZH608), an α' subunit deletion mutant, was identified from the Zhongpin 661(ZP661) EMS mutant library. Zhonghuang 608 was crossed to the soybean variety Dengke 1, and self-pollinated to establish an F2 segregating population composed of 210 individuals. Genetic analysis showed that the alpha' subunit deletion trait in ZH608 was controlled by a single recessive nuclear gene. By linkage analysis we mapped the locus between SSR10-1489 and SSR10-1612 on chromosome 10. Coincidentally, in the mapping region Cgy-1(Glyma.10 G246300) was annotated as an alpha' subunit synthesis-related gene according to the Williams 82 reference genome. Further sequence analysis showed that the mutant had a single base change(G84 to A84) in the first exon of Cgy-1, which resulted in the premature termination of the amino acid sequence. Based on the newly discovered SNP mutation, a co-dominant molecular marker was developed, and used to detect the genotypes of those F2 individuals. The results indicated that the Cgy-1 genotype cosegregated with the alpha' subunit phenotype in the F2 segregating population of Zh608 and Dengke 1. This study not only provides new materials for quality improvement, but also offers technical support for molecular breeding in soybean.
引文
[1]Shu X O,Jin F,Dai Q,Wen W,Potter J D,Kushi L H,Ruan Z,Gao Y T,Zheng W.Soyfood intake during adolescence and subsequent risk of breast cancer among Chinese women.Cancer Epidemiol Biomarkers Prev,2001,10:483-488.
[2]Fukui K,Tachibana N,Wanezaki S,Tsuzaki S,Takamatsu K,Yamamoto T,Hashimoto Y and Shimoda T.Isoflavone-free soy protein prepared by column chromatography reduces plasma cholesterol in rats.J Agric Food Chem,2002,50:5717-5721.
[3]Huang W,Wood C,L’AbbéM R,Gilani G S,Cockell K A,Xiao C W.Soy protein isolate increases hepatic thyroid hormone receptor content and inhibits its binding to target genes in rats.JNutr,2005,135:1631-1635.
[4]Lo W M Y,Li-Chan E C Y.Angotensin I converting enzyme inhibitory peptides from in vitro pepsin-pencreatin digestion of soy protein.J Agric Food Chem,2005,53:3369-3376.
[5]Tsuruki T,Takahata K,Yoshikawa M.Anti-alopecia mechanisms of soymetide-4,an immunostimulating peptide derived from soyβ-conglycinin.Peptides,2005,26:707-711.
[6]Cordle C T.Soy protein allergy:Incidence and relative severity.JNutr,2004,134:1213S-1219S.
[7]Fukushima D.Recent progress of soybean protein foods:chemistry,technology and nutrition.Food Rev Int,1991,7:323-351.
[8]刘春,王显生,麻浩.大豆种子贮藏蛋白亚基特异种质的蛋白功能性评价.中国油脂,2008,33(8):31-36.Liu C,Wang X S,Ma H.Evaluations on functional properties of soybean protein isolates prepared from cultivars with seed storage protein subunits variation.China Oils Fats,2008,33(8):31-36(in Chinese with English abstract).
[9]Wolf W J,Briggs D R.Ultracentrifugal investigation of the effect of neutral salts on the extraction of soybean proteins.Archives Biochem Biophys,1956,63:40-49.
[10]Krishnan H B,Kim W S,Jang S,Kerley M S.All three subunits of soybean beta-conglycinin are potential food allergens.J Agric Food Chem,2011,57:938-943.
[11]周瑞宝,周兵.大豆7S和11S球蛋白的结构和功能性质.中国粮油学报,1998,(6):39-42.Zhou R B,Zhou B,The structure and functional properties of soybean 7S and 11S globulin proteins.J Chin Cereal Oil Ass,1998,(6):39-42(in Chinese with English abstract).
[12]Mohamad Ramlan B M,Maruyama N,Takahashi K,Yagasaki K,Higasa T,Matsumura Y,Utsumi S.Gelling properties of soybean beta-conglycinin having different subunit compositions.Biosci Biotechnol Biochem,2004,68:1091-1096.
[13]Fukushima D.Structures of plant storage proteins and their functions.Food Reviews Int,1991,7:353-381.
[14]Fukushima D.Recent progress of soybean protein foods:chemistry,technology,and nutrition.Food Reviews Int,1991,7:323-351.
[15]Kitamura K,Kaizuma N.Mutant strains with low level of subunits of 7S globulin in soybean(Glycine max Merr.)seed.Jpn JBreed,2008,31:353-359.
[16]Takahashi K,Banba H,Kikuchi A,Ito M,Nakamura S.An induced mutant line lacking the alpha-subunit of beta-conglycininin soybean(Glycine max(L.)Merrill).Jpn J Breed,1994,44:65-66.
[17]Kitamura K,Davies C S,Nielsen N C.Inheritance of alleles for Cgy 1 and Gy 4 storage protein genes in soybean.Theor Appl Genet,1984,68:253-257.
[18]Teraishi M,Takahashi M,Hajika M,Matsunaga R,Uematsu Y,Ishimoto M.Suppression of soybeanβ-conglycinin genes by a dominant gene,Scg-1.Theor Appl Genet,2001,103:1266-1272.
[19]宋波,蓝岚,田福东,拓云,白月,姜自芹,申丽威,李文滨,刘珊珊.大豆7S球蛋白α'亚基缺失及(α'+α)亚基双缺失品系的回交转育.作物学报,2012,38:2297-2305.Song B,Lan L,Tian F D,Tuo Y,Bai Y,Jiang Z Q,Shen L W,Li W B,Liu S S.Development of soybean lines withα'-subunit or(α'+α)-subunits deficiency in 7S globulin by backcrossing,Acta Agron Sin,2012,38:2297-2305(in Chinese with English abstract).
[20]刘珊珊,王志坤,葛玉君,刁桂珠,田福东,高丽辉,李文滨.大豆7S球蛋白亚基相对含量与品质性状间的相关分析.中国油料作物学报,2008,30:284-289.Liu S S,Wang Z K,Ge Y J,Diao G Z,Tian F D,Gao L H,Li WB.Correlations between relative content of individual subunit of7S globulin and quality characteristics in soybean germplasm.JChin Cereals Oils Assoc,2008,30:284-289(in Chinese with English abstract).
[21]Song Q J,Jia G F,Zhu Y L,Grant D,Nelson R T,Hwang E Y.Abundance of SSR motifs and development of candidate polymorphic SSR markers(barcsoyssr_1.0)in soybean.Crop Sci,2010,50:1950-1960.
[22]Li Z F,Jiang L X,Ma Y S,Wei Z Y,Hong H L,Liu Z X,Lei J H,Liu Y,Guan R X,Guo Y,Jin L G,Zhang L J,Li Y H,Ren Y L,He W,Liu M,Nan M P,Liu L,Guo B F,Song J,Tan B,Liu G F,Li M Q,Zhang X L,Liu B,Shi X H,Han S N,Hua S N,Zhou FL,Yu L L,Li Y F,Wang S,Wang J,Chang R Z,Qiu L J.Development and utilization of a new chemically induced soybean library with a high mutation density.J Integr Plant Biol,2017,59:60-74.
[23]Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA.Nucl Acids Res,1980,8:4321-4325.
[24]Yagasaki K,Takagi T,Sakai M,Kitamura K.Biochemical characterization of soybean protein consisting of different subunits of glycinin.J Agric Food Chem,1997,45:656-660.
[25]Harada J J,Barker S J,Goldberg R B.Soybean beta-conglycinin genes are clustered in several DNA regions and are regulated by transcriptional and posttranscriptional processes.Plant Cell,1989,1:415-425.
[26]Poysa V,Woodrow L,Yu K.Effect of soy protein subunit composition on tofu quality.Food Res Int,2006,39:309-317.
[27]刘珊珊,滕卫丽,姜自芹,张彬彬,葛玉君,刁桂珠,郑天慧,曾蕊,李文滨.大豆7S球蛋白α-亚基缺失型种质创新.作物学报,2010,36:1409-1413.Liu S S,Teng W L,Jiang Z Q,Zhang B B,Ge Y J,Diao G Z,Zheng T H,Jiang Z Q,Zeng R,Wu S,Li W B.Development of soybean germplasm lacking of 7S globulinα-subunit.Acta Agron Sin,2010,36:1409-1413(in Chinese with English abstract).
[28]宋波,蓝岚,田福东,拓云,白月,姜自芹,申丽威,李文滨,刘珊珊.大豆7S球蛋白α'亚基缺失及(α'+α)亚基双缺失品系的回交转育.作物学报,2012,38:2297-2305.Song B,Lan L,Tian F D,Tuo Y,Bai Y,Jiang Z Q,Shen L W,Li WB,Liu S S.Development of soybean lines withα'-subunit or(α'+α)-subunits deficiency in 7S globulin by backcrossing.Acta Agron Sin,2012,38:2297-2305(in Chinese with English abstract).
[29]张国敏,张亚琴,舒英杰,麻浩.三种大豆种子贮藏蛋白亚基缺失种质的筛选与鉴定.大豆科学,2015,34:1-8.Zhang G M,Zhang Y Q,Shu Y J,Ma H.Screening and identification of three type of soybean lines lacking different seed storage protein subunits.Soybean Sci,2015,34:1-8(in Chinese with English abstract).
[30]姜振峰,赫卫,汪洋,李文滨.大豆种子7S、11S球蛋白及7S球蛋白亚基的研究.中国油料作物学报,2007,29:138-141.Jiang Z F,He W,Wang Y,Li W B.Study on 7S,11S globulin of soybean seed.Chin J Oil Crop Sci,2007,29:138-141(in Chinese with English abstract).
[31]Liu S,Ohta K,Dong C,Thanh VC,Ishimoto M,Qin Z,Hirata Y.Genetic diversity of soybean(Glycine max(L.)Merrill)7Sglobulin protein subunits.Genet Resour Crop Evol,2006,53:1209-1219.
[32]Kim S,Kim M Y,Van K,Lee Y H,Kim H S,Cai C M,Park B S,Seo H S,Lee S H.The development of a co-dominant marker for theβ-conglycininα′subunit in soybeans.Euphytica,2011,177:355-363.
[33]张国敏.种子贮藏蛋白亚基缺失大豆种质的鉴定及其蛋白质功能性评价与α'亚基缺失的分子机制.南京农业大学硕士学位论文,江苏南京,2014.Zhang G M.Identification of Soybean Lines Lacking Different Seed Storage Protein Subunits,Their Functional Properties of Proteins and the Molecular Mechanism of Lackingα'Subunit.MS Thesis of Nanjing Agriculture University,Nanjing,Jiangsu,China,2014(in Chinese with English abstract).
[34]Kitamura K,Kaizuma N.Mutant Strains with low level of subunits of 7S globulin in soybean(Glycine max Merr.)seed.Jpn JBreed,2008,31:353-359.
[35]Krishnan,Hari B.Engineering soybean for enhanced sulfur amino acid content.Crop Sci,2005,45:454-461.
[2]Fukui K,Tachibana N,Wanezaki S,Tsuzaki S,Takamatsu K,Yamamoto T,Hashimoto Y and Shimoda T.Isoflavone-free soy protein prepared by column chromatography reduces plasma cholesterol in rats.J Agric Food Chem,2002,50:5717-5721.
[3]Huang W,Wood C,L’AbbéM R,Gilani G S,Cockell K A,Xiao C W.Soy protein isolate increases hepatic thyroid hormone receptor content and inhibits its binding to target genes in rats.JNutr,2005,135:1631-1635.
[4]Lo W M Y,Li-Chan E C Y.Angotensin I converting enzyme inhibitory peptides from in vitro pepsin-pencreatin digestion of soy protein.J Agric Food Chem,2005,53:3369-3376.
[5]Tsuruki T,Takahata K,Yoshikawa M.Anti-alopecia mechanisms of soymetide-4,an immunostimulating peptide derived from soyβ-conglycinin.Peptides,2005,26:707-711.
[6]Cordle C T.Soy protein allergy:Incidence and relative severity.JNutr,2004,134:1213S-1219S.
[7]Fukushima D.Recent progress of soybean protein foods:chemistry,technology and nutrition.Food Rev Int,1991,7:323-351.
[8]刘春,王显生,麻浩.大豆种子贮藏蛋白亚基特异种质的蛋白功能性评价.中国油脂,2008,33(8):31-36.Liu C,Wang X S,Ma H.Evaluations on functional properties of soybean protein isolates prepared from cultivars with seed storage protein subunits variation.China Oils Fats,2008,33(8):31-36(in Chinese with English abstract).
[9]Wolf W J,Briggs D R.Ultracentrifugal investigation of the effect of neutral salts on the extraction of soybean proteins.Archives Biochem Biophys,1956,63:40-49.
[10]Krishnan H B,Kim W S,Jang S,Kerley M S.All three subunits of soybean beta-conglycinin are potential food allergens.J Agric Food Chem,2011,57:938-943.
[11]周瑞宝,周兵.大豆7S和11S球蛋白的结构和功能性质.中国粮油学报,1998,(6):39-42.Zhou R B,Zhou B,The structure and functional properties of soybean 7S and 11S globulin proteins.J Chin Cereal Oil Ass,1998,(6):39-42(in Chinese with English abstract).
[12]Mohamad Ramlan B M,Maruyama N,Takahashi K,Yagasaki K,Higasa T,Matsumura Y,Utsumi S.Gelling properties of soybean beta-conglycinin having different subunit compositions.Biosci Biotechnol Biochem,2004,68:1091-1096.
[13]Fukushima D.Structures of plant storage proteins and their functions.Food Reviews Int,1991,7:353-381.
[14]Fukushima D.Recent progress of soybean protein foods:chemistry,technology,and nutrition.Food Reviews Int,1991,7:323-351.
[15]Kitamura K,Kaizuma N.Mutant strains with low level of subunits of 7S globulin in soybean(Glycine max Merr.)seed.Jpn JBreed,2008,31:353-359.
[16]Takahashi K,Banba H,Kikuchi A,Ito M,Nakamura S.An induced mutant line lacking the alpha-subunit of beta-conglycininin soybean(Glycine max(L.)Merrill).Jpn J Breed,1994,44:65-66.
[17]Kitamura K,Davies C S,Nielsen N C.Inheritance of alleles for Cgy 1 and Gy 4 storage protein genes in soybean.Theor Appl Genet,1984,68:253-257.
[18]Teraishi M,Takahashi M,Hajika M,Matsunaga R,Uematsu Y,Ishimoto M.Suppression of soybeanβ-conglycinin genes by a dominant gene,Scg-1.Theor Appl Genet,2001,103:1266-1272.
[19]宋波,蓝岚,田福东,拓云,白月,姜自芹,申丽威,李文滨,刘珊珊.大豆7S球蛋白α'亚基缺失及(α'+α)亚基双缺失品系的回交转育.作物学报,2012,38:2297-2305.Song B,Lan L,Tian F D,Tuo Y,Bai Y,Jiang Z Q,Shen L W,Li W B,Liu S S.Development of soybean lines withα'-subunit or(α'+α)-subunits deficiency in 7S globulin by backcrossing,Acta Agron Sin,2012,38:2297-2305(in Chinese with English abstract).
[20]刘珊珊,王志坤,葛玉君,刁桂珠,田福东,高丽辉,李文滨.大豆7S球蛋白亚基相对含量与品质性状间的相关分析.中国油料作物学报,2008,30:284-289.Liu S S,Wang Z K,Ge Y J,Diao G Z,Tian F D,Gao L H,Li WB.Correlations between relative content of individual subunit of7S globulin and quality characteristics in soybean germplasm.JChin Cereals Oils Assoc,2008,30:284-289(in Chinese with English abstract).
[21]Song Q J,Jia G F,Zhu Y L,Grant D,Nelson R T,Hwang E Y.Abundance of SSR motifs and development of candidate polymorphic SSR markers(barcsoyssr_1.0)in soybean.Crop Sci,2010,50:1950-1960.
[22]Li Z F,Jiang L X,Ma Y S,Wei Z Y,Hong H L,Liu Z X,Lei J H,Liu Y,Guan R X,Guo Y,Jin L G,Zhang L J,Li Y H,Ren Y L,He W,Liu M,Nan M P,Liu L,Guo B F,Song J,Tan B,Liu G F,Li M Q,Zhang X L,Liu B,Shi X H,Han S N,Hua S N,Zhou FL,Yu L L,Li Y F,Wang S,Wang J,Chang R Z,Qiu L J.Development and utilization of a new chemically induced soybean library with a high mutation density.J Integr Plant Biol,2017,59:60-74.
[23]Murray M G,Thompson W F.Rapid isolation of high molecular weight plant DNA.Nucl Acids Res,1980,8:4321-4325.
[24]Yagasaki K,Takagi T,Sakai M,Kitamura K.Biochemical characterization of soybean protein consisting of different subunits of glycinin.J Agric Food Chem,1997,45:656-660.
[25]Harada J J,Barker S J,Goldberg R B.Soybean beta-conglycinin genes are clustered in several DNA regions and are regulated by transcriptional and posttranscriptional processes.Plant Cell,1989,1:415-425.
[26]Poysa V,Woodrow L,Yu K.Effect of soy protein subunit composition on tofu quality.Food Res Int,2006,39:309-317.
[27]刘珊珊,滕卫丽,姜自芹,张彬彬,葛玉君,刁桂珠,郑天慧,曾蕊,李文滨.大豆7S球蛋白α-亚基缺失型种质创新.作物学报,2010,36:1409-1413.Liu S S,Teng W L,Jiang Z Q,Zhang B B,Ge Y J,Diao G Z,Zheng T H,Jiang Z Q,Zeng R,Wu S,Li W B.Development of soybean germplasm lacking of 7S globulinα-subunit.Acta Agron Sin,2010,36:1409-1413(in Chinese with English abstract).
[28]宋波,蓝岚,田福东,拓云,白月,姜自芹,申丽威,李文滨,刘珊珊.大豆7S球蛋白α'亚基缺失及(α'+α)亚基双缺失品系的回交转育.作物学报,2012,38:2297-2305.Song B,Lan L,Tian F D,Tuo Y,Bai Y,Jiang Z Q,Shen L W,Li WB,Liu S S.Development of soybean lines withα'-subunit or(α'+α)-subunits deficiency in 7S globulin by backcrossing.Acta Agron Sin,2012,38:2297-2305(in Chinese with English abstract).
[29]张国敏,张亚琴,舒英杰,麻浩.三种大豆种子贮藏蛋白亚基缺失种质的筛选与鉴定.大豆科学,2015,34:1-8.Zhang G M,Zhang Y Q,Shu Y J,Ma H.Screening and identification of three type of soybean lines lacking different seed storage protein subunits.Soybean Sci,2015,34:1-8(in Chinese with English abstract).
[30]姜振峰,赫卫,汪洋,李文滨.大豆种子7S、11S球蛋白及7S球蛋白亚基的研究.中国油料作物学报,2007,29:138-141.Jiang Z F,He W,Wang Y,Li W B.Study on 7S,11S globulin of soybean seed.Chin J Oil Crop Sci,2007,29:138-141(in Chinese with English abstract).
[31]Liu S,Ohta K,Dong C,Thanh VC,Ishimoto M,Qin Z,Hirata Y.Genetic diversity of soybean(Glycine max(L.)Merrill)7Sglobulin protein subunits.Genet Resour Crop Evol,2006,53:1209-1219.
[32]Kim S,Kim M Y,Van K,Lee Y H,Kim H S,Cai C M,Park B S,Seo H S,Lee S H.The development of a co-dominant marker for theβ-conglycininα′subunit in soybeans.Euphytica,2011,177:355-363.
[33]张国敏.种子贮藏蛋白亚基缺失大豆种质的鉴定及其蛋白质功能性评价与α'亚基缺失的分子机制.南京农业大学硕士学位论文,江苏南京,2014.Zhang G M.Identification of Soybean Lines Lacking Different Seed Storage Protein Subunits,Their Functional Properties of Proteins and the Molecular Mechanism of Lackingα'Subunit.MS Thesis of Nanjing Agriculture University,Nanjing,Jiangsu,China,2014(in Chinese with English abstract).
[34]Kitamura K,Kaizuma N.Mutant Strains with low level of subunits of 7S globulin in soybean(Glycine max Merr.)seed.Jpn JBreed,2008,31:353-359.
[35]Krishnan,Hari B.Engineering soybean for enhanced sulfur amino acid content.Crop Sci,2005,45:454-461.