大豆(Glycine max (L.) Merr.)致敏蛋白Gly m Bd 30K检测方法及低致敏优异种质鉴定
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1 长江大学农学院,湖北荆州 434025; 2 农作物基因资源与遗传改良国家重大科学工程 / 农业部北京大豆生物学重点实验室 / 中国农业科学院作物科学研究所,北京 100081

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农作物种质资源保护与利用专项(2019NWB036-05);中国农科院科技创新工程项目


Detection Method of Allergen Gly m Bd 30K and Identification of Elite Accessions with Low Allergenicity in Soybean (Glycine max (L.) Merr.)
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1Yangtze University, College of Agriculture, Hubei Jingzhou 434025; 2National Key Facility for Crop Gene Resources and Genetic Improvement / Key Laboratory of Soybean Biology in Beijing( MOA) / Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081

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    摘要:

    Gly m Bd 30K 蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应。因此,发 掘低 Gly m Bd 30K 蛋白含量优异种质对于培育优质大豆品种具有重要意义。为了获得致敏蛋白 Gly m Bd 30K 低含量的优 异种质,根据 Gly m Bd 30K 蛋白的 190-379aa 多肽序列制备多克隆抗体;对来源于山西省的 29 份种质,利用 Western blot 技 术对其 Gly m Bd 30K 蛋白含量进行检测;并扩增和分析 Gly m Bd 30K 基因序列;利用荧光定量 PCR 技术对筛选出的低 Gly m Bd 30K 含量种质进行表达分析。结果表明:从参试种质中鉴定出 2 份 Gly m Bd 30K 蛋白含量低的种质, Gly m Bd 30K 蛋 白低含量种质的鉴定效率为 6.9%,分别是来自太原市的大豆种质 134( ZDD02046)和大青豆( ZDD02174),其 Gly m Bd 30K 基因序列与 Willams82 相比,在启动子上都有 TA 重复序列变异, 134( ZDD02046)有 8 次 TA 重复,大青豆( ZDD02174)有 42 次 TA 重复,表达分析结果显示, 134( ZDD02046)的转录水平显著低于大青豆( ZDD02174),推测启动子上 TA 多态性可 能影响了其转录水平。本研究建立 Gly m Bd 30K 蛋白含量测定的方法,鉴定出 2 份低蛋白含量的优异种质,为今后选育优质 蛋白组合的大豆新品种提供了技术和材料支撑。

    Abstract:

    Gly m Bd 30K protein is recognized as a major immunodominant allergen in soybean, and this protein can lead to anaphylaxis such as diarrhea and intestinal inflammation in humans and livestock. The exploration of germplasms with lower Gly m Bd 30K content is important in breeding for elite functional soybean varieties. In this study, we generated the polyclonal antibody targeting to the 190-379aa peptides of the Gly m Bd 30K protein, followed by quantifying the Gly m Bd 30K protein using Western blot in 30 soybean accessions that were derived from Shanxi province. The genomic sequence of the Gly m Bd 30K gene and its transcriptional level was further conducted. As a result, two germplasms( 134, ZDD02046, Daqingdou, ZDD02174) with lower Gly m Bd 30K protein content have been identified, the identification efficiency is 6.9%. Both accessions displayed 8 and 42 TA repeats respectively in the promoter of the Gly m Bd 30K gene if compared with that of Willams82. The transcription level in 134( ZDD02046) was found to be significantly lower than that of Daqingdou( ZDD02174) . It is speculated that TA polymorphism in the promoter may associate with the Gly m Bd 30K expression. Collectively, this study established a strategy on the quantification of Gly m Bd 30K protein content and identified two accessions with lower Gly m Bd 30K protein, which will provide technical and material supports in breeding for new soybean varieties with high-quality proteins.

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周明明,张明俊,王俊,等.大豆(Glycine max (L.) Merr.)致敏蛋白Gly m Bd 30K检测方法及低致敏优异种质鉴定[J].植物遗传资源学报,2021,22(1):149-156.

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  • 收稿日期:2020-06-18
  • 最后修改日期:2020-08-29
  • 录用日期:2020-08-31
  • 在线发布日期: 2021-01-07
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