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作物学报 ›› 2019, Vol. 45 ›› Issue (1): 18-25.doi: 10.3724/SP.J.1006.2019.84078

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

大豆7S球蛋白α'亚基缺失新种质中黄608的分子鉴定

李俊英1,2(),孙如建2,3,李忠峰2,魏中艳2,任玉龙2,王俊1,*(),邱丽娟2,*()   

  1. 1 长江大学农学院, 湖北荆州434025
    2 中国农业科学院作物科学研究所 / 国家农作物基因资源与遗传改良重大科学工程 / 农业部种质资源利用重点实验室, 北京100081
    3 呼伦贝尔市农业科学研究所, 内蒙古扎兰屯162650
  • 收稿日期:2018-06-02 接受日期:2018-10-08 出版日期:2018-11-06 网络出版日期:2018-11-06
  • 通讯作者: 王俊,邱丽娟
  • 基金资助:
    本研究由“十三五”国家重点研发计划(2016YFD0100201);国家大豆种质资源平台项目资助(2016-004)

Molecular identification of a new soybean germplasm Zhonghuang 608 lacking of 7S globulin alpha' subunit

Jun-Ying LI1,2(),Ru-Jian SUN2,3,Zhong-Feng LI2,Zhong-Yan WEI2,Yu-Long REN2,Jun WANG1,*(),Li-Juan QIU2,*()   

  1. 1 College of Agriculture, Yangtze University, Jingzhou 434025, Hubei, China
    2 Key Laboratory of Crop Germplasm Utilization, Ministry of Agriculture, Institute of Crop Sciences, Chinese Academy of Agricultural Science, Beijing 100081, China / National Key Facility for Gene Resources and Genetic Improvement, Beijing 100081, China
    3 Hulun Buir Institution of Agricultural Sciences, Zhalantun 162650, Inner Mongolia, China
  • Received:2018-06-02 Accepted:2018-10-08 Published:2018-11-06 Published online:2018-11-06
  • Contact: Jun WANG,Li-Juan QIU
  • Supported by:
    The study was supported by the National Key R&D Program for Crop Breeding(2016YFD0100201);the National Soybean Germplasm Resources Platform.(2016-004)

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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(G 84→A 84), 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。

关键词: 大豆, α'亚基, 突变体, 7S球蛋白, dCAPs标记

Abstract:

The content of alpha' subunit of 7S 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.10G246300) 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 (G 84 to A 84) 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.

Key words: soybean, alpha' sunbunit, mutant, 7S globulin, dCAPS marker

表1

基因Cgy-1 PCR扩增引物"

引物名称
Name		 引物序列
Sequence of primer (5'-3')		 PCR产物长度
PCR product size (bp)		 退火温度
Annealing temperature (℃)
Cgy1-A Forward: AGCCCAAAACATTCACCAAC 1598 58
Reverse: AGGACTGTTGAGCTTGAGTGC
Cgy1-B Forward: CGCCATACCCGTTAACAAAC 1702 58
Reverse: TTGTGGCAGGACATTGCTAC

表2

dCAPS标记的引物序列和内切酶"

标记
Marker		 突变位点
SNP		 酶
Enzyme		 引物序列
Sequence of primer (5'-3')		 退火温度
Tm (℃)		 PCR片段长度
PCR product
Size (bp)		 酶切后片段长度
Product size after digestion by Taq I
GM7S-1 G/A Taq I F: TCTCATTTGGCATTGCGTATCG
R: TTGACCTTCTTCGCATTCT		 55 158 158 bp/(138 bp+20 bp)

图1

α'亚基缺失突变体的表型鉴定 a: α'亚基缺失材料中黄608与野生型中品661的SDS-PAGE电泳结果。b: 中黄608子代株行的α'亚基表型鉴定结果, 1~8分别为8个子代。"

表3

α'亚基缺失突变体F2群体遗传分析"

表型
Phenotype		 观测值
Observed value		 期望值
Excepted value		 χ2(3:1) P值
P-value
突变体(α'亚基缺失) Mutant (α' subunit deletion) 64 53 1.43 0.08
野生型 Wild type 146 157
共计 Total 210 210

图2

α'亚基缺失突变体的基因定位 a: 用210个F2单株将候选基因Cgy-1定位到标记SSR10-0982与SSR10-1638之间。b: 通过多态性标记筛选和加密, 将候选基因定位区间缩小至SSR10-1489与SSR10-1619之间。c: 突变体中黄608在Cgy-1基因第1外显子的第84位碱基发生突变(核苷酸G变为A), 造成编码氨基酸序列的提前终止。蓝色方框代表外显子, 白色方框代表5'和3'UTR, 黑线代表基因的内含子。"

图3

野生型和突变体的SDS-PAGE (a)和Western blot (b)实验结果 a: SDS-PAGE电泳结果显示突变体中黄608 (ZH608)缺少α'亚基条带。b: Western blot结果: 抗体杂交结果表明野生型(ZH608)有2条带; 突变体(ZP661)缺失了α'亚基条带。"

图4

dCAPS分子标记开发 a: 引物GM7S-1分别扩增野生型及突变体, PCR产物为150 bp的单一条带。b: ZH608的扩增产物被Taq I酶酶切为2条更小的片段。"

图5

F2代dCAPs标记基因型检测结果 P1, P2为亲本; G为野生型基因型(父本), A为突变基因型(母本), H为杂合基因型。"

图6

F2群体α'亚基缺失和α'亚基正常材料11S/7S比值分布"

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