【作者】 刘顺湖；

【导师】 盖钧镒； 周瑞宝；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 大豆[Glycine max（L.）Merr.]原产于我国,是当今世界上最重要的植物蛋白与食用植物油的来源。大豆含有丰富的蛋白质和人体必需的氨基酸,蛋白质组分（主要为11S和7S及其比值）和亚基是大豆蛋白质品质的重要性状。以往蛋白质组分及其含量的测定主要采用超速离心分离或碱溶、酸沉、凝胶过滤纯化方法,这些方法适于小样本大样品的情况。对于育种和资源研究,需要一种能处理大样本小量样品、简单易行的技术。国内外不同学者曾经对蛋白质组分做过SDS-PAGE分析,电泳分析比较简易,但所分析材料多局限于局部地区的个别品种,缺乏代表性,所获蛋白质组分和亚基分子量的研究结果差异很大甚至相互矛盾,参考价值受到限制。因此,分析材料的范围有待扩大,材料代表性有待提高,以便把研究结果用于大豆蛋白质品质改良的研究中,因为育种者期望用简易方法研究资源和育种材料,提高育种成效并节省人力、物力和时间。在方法未解决时,育种者只对蛋白质含量做了分析测定,也做了一些遗传分析与QTL定位,但对11S、7S组分及其亚基相对含量的遗传分析和QTL定位研究很少。鉴于以上情况,本研究目的为:（1）在前人研究的基础上,通过640份具有代表性的栽培大豆品种的蛋白质SDS-PAGE分析,揭示蛋白质组分和亚基的电泳条带分布规律、确定鉴别大豆蛋白质11S和7S组分及其亚基的分子量标准,在此基础上建立一种相对简单的测定其相对含量的方法。（2）以原产于我国各个生态区的代表性野生豆、地方品种和育成品种为材料,在同一试验条件和同一直接测定方法下研究自然进化和人工进化对蛋白质含量、11S和7S组分相对含量的影响,分析不同生态区不同类型资源变异的特点,并从中优选特异资源以供蛋白质品质育种利用。（3）在资源研究的基础上,利用南京农业大学大豆所提供的重组自交系群体NJRIKY和重组回交自交系群体NJBIEX,探讨蛋白质含量、11S和7S组分相对含量的遗传机制并进行QTL定位,筛选相关的分子标记,为大豆蛋白质品质育种提供参考。本研究得到以下主要结果:1.640份栽培大豆品种的提取蛋白SDS-PAGE分析结果,品种间电泳条带数和条带分子量（MW）变异很大,在SDS-PAGE谱带中不同分子量的电泳条带呈现连续分布的趋势,没有间断点。参照前人结果,根据分布峰谷状况,按分子量把SDS-PAGE谱带划分成两个区域:分子量MW＜44 KDa区域和分子量MW≥44 KDa的区域。第一个区域对应为11S组分,第二个区域对应为7S组分。进一步按照电泳条带次数分布的峰谷变化将条带分组称为亚基组。第一个区域的电泳条带归为4个亚基组,即11S-1（14.4-22 KDa）、11S-2（22-26 KDa）、11S-3（26.34 KDa）和11S-4（34-44 KDa）;第二个区域的电泳条带归为6个亚基组,即7S-1（44-49KDa）、7S-2（49-55 KDa）、7S-3（55-67 KDa）、7S-4（67-73 KDa）、7S-5（73-82 KDa）和7S-6（82-91KDa）。11S-1～11S-4相对含量之和作为11S组分的相对含量,7S-1～7S-6相对含量之和作为7S组分的相对含量,从而计算11S/7S的比值。2.对全国138份野生豆、409份地方品种和148份育成品种以及83份国外引进品种（合计778份）的蛋白质组分有关性状分析结果,全国野生豆蛋白质含量、油脂含量和蛋脂总含量变幅分别为39.2～54.2%、7.5～17.5%和47.3～64.6%,地方品种为38.8～51.5%、11.5～23.4%和55.6～70.6%,国内育成品种为41.7～49.4%、12.9～24.9%和55.6～72.0%。野生豆驯化为栽培豆并经人工选育后油脂含量和蛋脂总含量有大幅增加,而蛋白质含量平均数和变异度则有减小,说明以往人工进化着重在油脂含量的改进。蛋白质含量、油脂含量和蛋脂总含量3性状各群体在各生态区内均有较大变异,区平均间差异并不大,各区都有优良变异。野生豆蛋白质含量、油脂含量和蛋脂总含量与来源地纬度并未发现相关;栽培豆地方品种和育成品种的油脂含量与地理纬度出现显著正相关;育成品种蛋白质含量与地理纬度还出现显著负相关;野生自然状态下蛋白质含量和油脂含量之间无相关,而栽培豆地方品种和育成品种依次增强了负相关,说明形成这种相关的原因在于地区间油脂含量人工进化程度的差异。全国野生豆、地方品种和育成品种11S相对含量平均分别为54.7%、64.8%和71.7%,变幅28.8～82.6%、38.8～79.4%和48.2～88.9%;7S相对含量平均分别为44.7%、34.9%和27.9%,变幅20.6～71.2%、20.6～61.1%和15.7～47.8%;11S/7S比值平均分别为1.4、2.0和2.7,变幅0.4～3.9、0.6～3.9和0.9～4.0。野生豆驯化为栽培豆并经选育后11S相对含量和11S/7S比值上升,7S相对含量下降,变幅均减小;亚基组11S-2和11S-3相对含量增加;7S的6个亚基组,尤其7S-1和7S-6,相对含量下降。11S、7S、11S/7S在各群体各生态区内均有较大变异,与来源地纬度、蛋白质和油脂含量均无显著相关。从各生态区和国外引进品种中优选出高蛋白质（≥50%）、高油脂（≥23%）和高蛋脂总含量（≥68%）种质各10份,优选到11S/7S比值大于3.7、11S相对含量为78.9-88.9%的8份种质,发现有11S的4个亚基组相对含量分别大于37%、7S的6个亚基组相对含量分别大于24%、以及11S-1和7S的6个亚基组缺失的种质。3.以蛋白质组分有关性状差异较大的科丰1号与南农1138-2衍生的RIL群体（NJRIKY,简称KY）和ZDD2315与Essex衍生的BIL群体（NJBIEX,简称EX）为材料,用主基因+多基因混合遗传模型分析大豆蛋白质组分有关性状的遗传机制,结果在KY中蛋白质含量主基因和多基因的遗传率分别为31.3%和53.7%,11S组分相对含量的为14.3%和50.7%,7S组分相对含量的为34.5%和45.1%,11S/7S比值的为74.8%和20.1%,4个11S亚基组的为45.2～77.9%和15.5～41.2%,6个7S亚基组的为38.9～67.8%和29.2～45.5%。在EX中蛋白质含量的为40.9%和37.2%,11S组分相对含量的为60.7%和17.0%,7S组分相对含量的为44.1%和21.6%,11S/7S比值的为56.6%和10.1%,4个11S亚基组的为45.4～67.6%和26.6～53.4%,6个7S亚基组的为76.2～92.6%和5.0～22.2%。在KY中蛋白质含量、11S和7S组分相对含量的多基因遗传率高于主基因遗传率,而11S和7S的亚基组则相反。EX中多数性状的主基因遗传率高于多基因遗传率。两个群体的蛋白质组分有关性状的多基因遗传率有差异,但都比较高,在主基因+多基因混合遗传中具有重要作用。4.以KY和EX为作图群体采用Win QTL Cartographer Version 2.5程序,利用CIM法进行QTL检测,结果在KY中蛋白质含量2个QTL（B1pr和Epr1）,累计贡献率为16.5%。11S组分相对含量2个QTL（A211S和D1a11S）,累计贡献率为13.3%。7S组分相对含量2个QTL（I7S1和I7S2）,累计贡献率为12.7%。11S/7S比值3个QTL（D1arat、Irat1和Irat2）,累计贡献率为19.8%。11S和7S的亚基组QTL贡献率均低于10%。在EX中蛋白质含量1个QTL（Epr2）,贡献率为10.6%。11S组分相对含量2个QTL（E11S和B211S）,累计贡献率为23.5%。7S组分相对含量3个QTL（E7S1、E7S2和D1b-27S）,累计贡献率为38.3%。11S/7S比值1个QTL（Erat）,贡献率为14.3%。4个11S亚基组QTL贡献率为8.7～21.9%,其中11S-1的QTL（M11S-11）贡献率最高（21.9%）,6个7S亚基组QTL贡献率8.2～16.3%。在KY的D1a连锁群上的分子标记GMKF008b-GMKF008a之间和在EX群体的E连锁群上的分子标记sat₃80-satt263之间各检测到3个QTL,GMKF008b-GMKF008a与11S组分的OTL D1a11S、11S/7S比值的QTL D1arat和7S-2亚基组的QTL D1a7S-2相关联,sat₃80-satt263与11S组分的QTL E11S、7S组分的QTL E7S1和11S/7S比值的QTL Erat相关联,它们是重要的分子标记。在两个群体中除了个别性状,检测到的QTL位点贡献率都很低（KY中一般低于10%,EX中约10%左右）,没有检测到贡献率高的主效QTL位点。蛋白质组分有关性状的遗传中主效QTL数量少、贡献率低,仅能解释约10%的表型变异,因此,多基因起着重要作用,这与遗传分析的结果相一致。本研究提出的亚基组划分标准和方法,经验证试验证明简单、稳定和使用方便,并在本研究的资源分析、遗传分析和QTL分析中得到应用。通过对778份资源的分析所筛选的特异种质可供蛋白质组分育种利用。遗传分析和QTL分析说明在蛋白质组分有关性状的遗传中多基因具有重要作用,在提高蛋白质含量和改善蛋白质组分时既要利用主基因又要注意多基因的积累。QTL分析发现的重要分子标记,有希望作为标记辅助选择育种的参考。更多还原

【Abstract】 Soybean[Glycine max（L.） Merr.],originated from China,is one of the most important sources of vegetable protein and edible oil world-widely.Soybean protein is rich with essential amino acids for human bodies.Soybean protein quality depends on magnitude of 11S and 7S proteins and their subunits.In the past,protein component and its content were determined by ultracentrifuge,basic dissolve,acid-precipitation and gel filtration in large sample of a few materials.But it is needed a technology that can be used to analyze amount of materials in small sample for breeding and resource studying.Several studies have been done on SDS-PAGE analysis of protein component in soybean.It was a facility method. What’s more,materials used in experiments were mostly from local regions,without representative,resulting in the limitation of reference to soybean breeding for very difference and even confliction on molecular weight of component and subunits.So,the range of materials analyzed should be extended,the material representative should also be improved,then the results studied will be used in improving protein characters of soybean. Soybean inbreeding men want a facility method to test resources and materials,to increasing effect,saving manpower,material resources and time.Before founding simple method,investigating variation and inheritance mechanism and QTL mapping of protein content and its variation range of soybeans,and some results obtained.But less concern was on relative content of 11S,7S components and their subunits.There existed few report on inheritance and QTL mapping of 11S,7S and their subunits.Herein above instance,the main objectives of this study were:（a） On the basic of former study,finding distribution law of electrophoresis bands of protein component and subunit,establishing a convenient method and molecular criterion for 11S,7S protein and their subunit in SDS-PAGE,by analyzing protein extracts of 640 soybean cultivars selected to represent different cultivar eco-regions for improving soybean protein component and their subunits,（b） With wild soybean,cultivated landraces and released domestic cultivars represent different cultivar eco-regions in China in the same experimental condition and directness test method,studying the effect of nature and artificial selection on content of protein,fat,total protein plus fat,relative content of 11S and 7S,analyzing variation characteristic of various germplasm in different eco-regions in China,screening out elite accessions for breeding purposes,（c）Revealing inheritance mechanism of protein content, 11S and 7S relative content of soybeans,mapping QTLs of them related genes with NJRIKY and NJBIEX population offering by Soybean Research Institution of Nanjing Agriculture University.The results of this study will be helpful for soybean protein quality breeding.The mostly results obtained in this study are as follows:1.Protein extracts of 640 soybean cultivars and landraces,mainly from China and a few from the US,were analyzed for their components and subunits based on distribution patterns of bands with varying molecular weights（MW） under SDS-PAGE（sodium dodecyl sulfate-polyacrylamide gel electrophoresis）.The number and molecular weight of the bands in SDS-PAGE varied among materials and showed a tendency of continuous distribution.Accordingly,referring to former results and situation of apex and vale of frequency distribution of bands,the SDS-PAGE patterns of the soybean protein extracts were divided into two regions:the region of bands with MW＜44 KDa and that with MW≥44 KDa.The first region containing mainly 11S proteins was divided into four parts, called subunit groups,i.e.11S-1（14.4-22 KDa）,11S-2（22-26 KDa）,11S-3（26-34 KDa） and 11 S-4（34-44 KDa）.The second region containing mainly 7S protein was divided into six subunit groups,i.e.7S-1（44-49 KDa）,7S-2（49-55 KDa）,7S-3（55-67 KDa）, 7S-4（67-73 KDa）,7S-5（73-82 KDa） and 7S-6（82-91 KDa）.The sum of relative contents of 11S-1～11S-4 was obtained as the relative content of 11S protein,those of 7S-1～7S-6 as that of 7S protein,and therefore,the 11S/7S ratio obtained.2.The content or relative content of traits relating to soybean protein component of 778 accessions,including 138 wild soybean（Glycine soja Sieb.et Zucc.）,409 cultivated landraces（Glycine max（L.） Merr.） and 148 released domestic cultivars sampled from various eco-regions as a representative in China,along with 83 released foreign cultivars, were tested.The results showed that the range of protein content,fat content and total of both in wild soybean were 39.2～54.2%,7.5～17.5%,47.3～64.6%,those in cultivated landraces 38.8～51.5%,11.5～23.4%,55.6～70.6%,those in released domestic cultivars 41.7～49.4%,12.9～24.9%,55.6～72.0%,respectively.The increase of fat content and protein plus fat content after domestication,especially due to scientific breeding effort,and the decrease of protein content as well as its range implied that the improvement of fat content happened during the history.There existed large amount of genetic variation of protein content,fat content and total of both contents in various eco-regions in different kinds of materials,with elite accessions in each eco-regions,but the variation was not large enough among eco-region means.No significant correlation between the three contents and the original latitude of the materials was found in wild accessions,while significant positive correlation between fat content and latitude existed in cultivated landraces and released cultivars and significant negative correlation between protein content and latitude was found only in released cultivars.There was no significant correlation between protein content and fat content in wild soybeans,but significant negative correlation between the two contents found in cultivated land races and released cultivars.The reason for this kind of change should be the differential artificial selection among the eco-regions.11S relative content mean of 54.7%,64.8%and 71.7%,11S relative content range of 28.8-82.6%,38.8-79.4%and 48.8-88.9%,7S relative content mean of 44.7%,34.9%and 27.9%,7S relative content range of 20.6-71.2%,20.6-61.1%and 15.7-47.8%,11S/7S ratio mean of 1.4,2.0 and 2.7,11S/7S ratio range of 0.4-3.9,0.6-3.9 and 0.9-4.0 in wild soybean, landraces and released cultivars,respectively.After breeding cultivars by domestication wild soybean,11S relative content and 11S/7S ratio was step up,7S relative content was step down,with their variation range decreasing.The subunit groups 11S-2 and 11S-3 relative content were increased.The relative content of 6 subunit groups of 7S protein, particularly,7S-1 and 7S-6 were decreased.There existed abundance and choiceness variation of 11S and 7S protein relative content and 11S/7S ratio of three germplasm types in each Eco-region.There was no correlation between geography latitude of origin（or protein and fat content） and three traits in three germplasm types,respectively.In addition,from the tested accessions,10 most elite accessions for each of the three traits（protein content more than 50%,or fat content more than 23%,or total of both contents more than 68%）,11S/7S ratio of 8 elite screened out from 778 accessions（about 1.03%） was more than 3.7,with their 11S relative content 78.9-88.9%.The relative content of elite accessions was more than 37%for 11S’subunit groups and 24%for 7S’ subunits groups,respectively.There existed amount of materials lacked 11S-1,or from 7S-1 to 7S-6. They will be used for high output breeding purposes.3.Soybean germplasms,the NJRIKY population（KY,from Kefeng1hao and Nannong1138-2） and NJBIEK population（EX,from Essex and ZDD2315）,with big differences in traits relating to protein component were used to reveal the genetic mechanism of them.Genetic analysis was performed under major gene+polygene mixed inheritance model.The resulted indicated that in KY population major gene heritability of protein content,11S relative content,7S relative content,11S/7S ratio,four 11S’ subunit groups and six 7S’ subunit groups were 31.3%,14.3%,34.5%,74.8%,45.2～77.9%,and 38.9～67.8%,respectively.Their polygene heritability were 53.7%,50.7%,45.1%,20.1%, 15.5～41.2%,and 29.2～45.5%,respectively.In EX population major gene heritability of protein content,11S relative content,7S relative content,11S/7S ratio,four 11S’ subunit groups and six 7S’ subunit groups were 40.9%,60.7%,44.1%,56.6%,45.4～67.6%,and 76.2～92.6%,respectively.Their polygene heritability were 37.2%,17.0%,21.6%,10.1%, 22.6～53.4%,and 5.0～22.2%,respectively.The polygene heritability of protein content, relative content of 11S and 7S were higher than major gene heritability of them in KY.It was reverse for 11S’ and 7S’ subunit groups in KY.In EX,the major gene heritability of most traits was higher than polygene heritability of them.There existed difference in polygene heritability for KY and EX,but polygene play on an important role in major gene +polygene mixed inheritance model.4.Based on the genetic research above,QTL mapping for traits relating to protein component in soybean was carried out as following.The composite interval mapping（CIM） of the software Windows QTL Cartographer Version 2.5 were used to map QTLs.In KY, there were 2 QTLs（B1pr and Epr1） for protein content with R²（R² means accumulative total account of the phenotypic variation） of 16.5%,2 QTLs（A211S and D1a11S） for 11S relative content with R² of 13.3%,2 QTLs（I7S1 and I7S2） for 7S relative content with R² of 12.7%,3 QTLs（D1arat,Irat1 and Irat2） for 11S/7S ratio with R² of 19.8%.r²（account of the phenotypic variation） of 11S’ and 7S’subunit groups was below 10%,respectively. In EX,there were 1 QTL（Epr2） for protein content with r² of 10.6%,2 QTLs（E11S and B211S） for 11S relative content with R² of 23.5%,3 QTLs（E7S1,E7S2 and D1b-27S） for 7S relative content with R² of 38.3%,1 QTL（Erat） for 11S/7S ratio with r² of 14.3%.r² （account of the phenotypie variation） of 11S’ and 7S’subunit groups was about 8.7～21.9% and 8.2～16.3%,respectively.The highest r² of 21.9%was QTL M11S-11 of 11S-1 in EX. There were 3 QTLs from marker GMKF008b to GMKF008a on linkage group D1a in KY （D1a11S,D1arat and D1a7S-2） and from marker sat₃80 to satt263 on linkage group E in EX（E11S,E7S1 and Erat）,respectively.QTL with high account of the phenotypic variation was not found.There existed a few QTL with low account of the phenotypic variation, about 10%,for the traits relating to protein component.So,polygene was important in inheritance of them.In this study,the molecular criterion and method about 11S,7S protein components and subunit groups of protein extracts were demonstrated to be simple,stable and feasible. It had been used to study protein component and subunits of soybean germplasm resources and inheritance analysis and QTL mapping in this study.The elite accessions for protein content,11S/7S ratio,11S relative content 78.9-88.9%11S’subunit groups and 7S’ subunits groups screened out from 778 accessions will be used for high output breeding purposes. The inheritance of traits relating to protein component was controlled by a few low effect major gene and lots of polygene.It was consistent with analysis results of inheritance. Based on the results,it is inferred that the markers linked to the detected QTLs should be useful for marker-assisted selection for 11S,7S and related traits in soybean.更多还原