水稻谷蛋白的质谱和Western Blot鉴定与贮藏蛋白分析
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摘要
水稻的营养品质主要取决于两个方面,蛋白质含量和氨基酸含量。其中特别重要的是可消化的蛋白组成含量,以及必需氨基酸的含量。分析水稻种子贮藏蛋白营养组成为水稻育种选取高营养组分品种提供了理论基础。本研究围绕稻米的蛋白营养品质主要进行了两方面的工作：水稻谷蛋白分子鉴定分析和谷蛋白基因启动子初步分析；水稻种子贮藏蛋白组成以及氨基酸含量分析。
主要研究结果如下：
1.以水稻品种93-11(Oryza sativa L. ssp. Indica cv.93-11)和日本晴(Oryza sativa L. ssp. Japonica cv. Nipponbare)为材料,鉴定和分析粳稻和籼稻的谷蛋白亚基组成。通过2D-PAGE分离谷蛋白亚基并通过软件比较各亚基组成,运用LC-MS/MS和特异性亚基抗体两种方法鉴定谷蛋白亚基。鉴定结果表明,粳稻(cv. Nipponbare)和籼稻(cV.93-11)有80%的谷蛋白亚基被准确定性到Swiss-Prot数据库里已知亚基,10%的亚基与GluB亚家族序列高同源性；除此之外,发现存在新的谷蛋白亚基和GluA(或者GluB)亚家族序列同源性比较低。粳稻和籼稻的谷蛋白多样性体现在谷蛋白亚基等电点的漂移,具有差异的谷蛋白亚基为GluA-1和GluA-3。从谷蛋白组成来看,籼稻的GluA-3和GluB-1百分含量是粳稻的两倍。从赖氨酸含量来看,籼稻的谷蛋白营养组成要高于粳稻。运用质谱和免疫两种方法鉴定谷蛋白亚基是本研究的创新,总结了籼稻和粳稻的谷蛋白组成特点。
2.以35个优质栽培水稻品种作为材料,分析不同品种的种子贮藏蛋白营养组成。通过SDS-PAGE分析种子贮藏蛋白组成,运用凯氏定氮法和Bradford方法测定蛋白含量,并测定氨基酸含量。分析结果表明,水稻贮藏蛋白通过SDS-PAGE分成清蛋白、球蛋白、醇溶蛋白和谷蛋白4个组分,其中谷蛋白占50-80%绝对优势。在营养水平上,谷蛋白易消化,其赖氨酸含量是其他组分的9倍。稻米中的蛋白含量和氨基酸含量有一定的规律：谷蛋白组分和醇溶蛋白组分呈极其显著负相关,Lys含量和总蛋白含量呈极其显著正相关(P<0.01)。运用凯氏定氮法、Bradford法、氨基酸分析以及SDS-PAGE等方法分析稻米的营养品质,总结了不同水稻品种之间营养水平的相关性,为水稻育种选取高营养品质资源提供了理论依据。
3.选取蛋白含量存在显著差异的5个优质栽培水稻品种作为亲本,得到4个水稻亲本子代的组合,其中包括3个籼稻组合和1个粳稻组合。通过不同品种的谷蛋白启动子序列比对和对应谷蛋白2D-PAGE图谱比较,初步分析粳稻和籼稻的谷蛋白组成和水稻组合之间谷蛋白组成差异。启动子分析结果表明,水稻谷蛋白不同亚基的启动子序列中重要转录元件排列有一定特异性；粳稻和籼稻的启动子序列具有几个特异性核苷酸突变。水稻组合谷蛋白2D-PAGE图谱结果表明：粳稻和籼稻各自的杂交组合的谷蛋白含量都具有叠加性。
The protein contents and components in rice (Oryza sativa L.) play a significant role in the quality of rice seeds, especially the digestible protein content and its essential amino acids content. Therefore, it is essential to understand the protein contents and components of rice seed storage protein (SSPs), and to improve the nutritive composition in rice breeding.
The main results are as follows:
1. The glutelin subunits of cv.93-11and cv.Nipponbare selected from O. sativa L. ssp. japonica and ssp. indica were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combining with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blot with highly subunit-specific antibodies. The results showed that～80%of glutelin content from the total glutelin extract consisted of seven major subunits in the SwissProt database, and～10%of glutelin consisted of a member of the glutelin type-B subfamily. In addition, based on the sequence heterogeneity at the amino acid level, the glutelin components of unknown identity seem to be new members of a new glutelin subfamily. The diversity of rice glutelin subunits between japonica and indica were identified as GluA-1and GluA-3, and the diversity of the glutelin composition between japonica and indica was reflected by the difference in glutelin subunit composition between glutelin type-A and type-B and the special subunits identified as GluA-1, and the percentage composition of GluA-3and GluB-1was twice more in the indica cultivars than that in the japonica cultivars. The result also revealed that the indica cultivars were superior to the japonica cultivars in terms of the nutritional value at the lysine level. The innovation was the combination of two methods, LC-MS/MS and western blot.
2. The SSPs components of thirty-five prevailing rice cultivars were analyzed. First, they were separated by SDS-PAGE, and then their protein contents were determined by the Kjeldahl and Bradford method, and finally their amino acid contents were identified using the amino acid analyzer. The results showed that four components were separated, including albumin, globulin, prolamine, and glutelin, and the glutelin was found to be the main components in a range of50-80%. Moreover, as one of the digestible proteins for human, the Lys content of the glutelin was nine times higher than the other three. In further analysis, certain relationship was found between the protein contents and the amino acid components:the glutelin content was in significantly negative relation to the prolamine content; while the Lys content showed positive relation to the total SSPs content (P<0.01). In the study, the relativity of the nutritive value will provide the theoretical evidence for rice genetics and breeding.
3. DNA sequences of Glu promoters in five cultivars with difference protein contents were selected for sequence slignment analysis, which were as hybrid rice parents new rice groups were analyzed the glutelin subunits by2D-PAGE These five cultivars as hybrid rice parents breed and analysis of glutelin composition. The results showed that the transcription factors were known to play central roles in regulating gene transcription from promoters. The diversity of Glu promoters between japonica and indica were4-5mutation nucleotides. The certain protein content of the Hybrid group had the assumption at certain protein content level.
主要研究结果如下：
1.以水稻品种93-11(Oryza sativa L. ssp. Indica cv.93-11)和日本晴(Oryza sativa L. ssp. Japonica cv. Nipponbare)为材料,鉴定和分析粳稻和籼稻的谷蛋白亚基组成。通过2D-PAGE分离谷蛋白亚基并通过软件比较各亚基组成,运用LC-MS/MS和特异性亚基抗体两种方法鉴定谷蛋白亚基。鉴定结果表明,粳稻(cv. Nipponbare)和籼稻(cV.93-11)有80%的谷蛋白亚基被准确定性到Swiss-Prot数据库里已知亚基,10%的亚基与GluB亚家族序列高同源性；除此之外,发现存在新的谷蛋白亚基和GluA(或者GluB)亚家族序列同源性比较低。粳稻和籼稻的谷蛋白多样性体现在谷蛋白亚基等电点的漂移,具有差异的谷蛋白亚基为GluA-1和GluA-3。从谷蛋白组成来看,籼稻的GluA-3和GluB-1百分含量是粳稻的两倍。从赖氨酸含量来看,籼稻的谷蛋白营养组成要高于粳稻。运用质谱和免疫两种方法鉴定谷蛋白亚基是本研究的创新,总结了籼稻和粳稻的谷蛋白组成特点。
2.以35个优质栽培水稻品种作为材料,分析不同品种的种子贮藏蛋白营养组成。通过SDS-PAGE分析种子贮藏蛋白组成,运用凯氏定氮法和Bradford方法测定蛋白含量,并测定氨基酸含量。分析结果表明,水稻贮藏蛋白通过SDS-PAGE分成清蛋白、球蛋白、醇溶蛋白和谷蛋白4个组分,其中谷蛋白占50-80%绝对优势。在营养水平上,谷蛋白易消化,其赖氨酸含量是其他组分的9倍。稻米中的蛋白含量和氨基酸含量有一定的规律：谷蛋白组分和醇溶蛋白组分呈极其显著负相关,Lys含量和总蛋白含量呈极其显著正相关(P<0.01)。运用凯氏定氮法、Bradford法、氨基酸分析以及SDS-PAGE等方法分析稻米的营养品质,总结了不同水稻品种之间营养水平的相关性,为水稻育种选取高营养品质资源提供了理论依据。
3.选取蛋白含量存在显著差异的5个优质栽培水稻品种作为亲本,得到4个水稻亲本子代的组合,其中包括3个籼稻组合和1个粳稻组合。通过不同品种的谷蛋白启动子序列比对和对应谷蛋白2D-PAGE图谱比较,初步分析粳稻和籼稻的谷蛋白组成和水稻组合之间谷蛋白组成差异。启动子分析结果表明,水稻谷蛋白不同亚基的启动子序列中重要转录元件排列有一定特异性；粳稻和籼稻的启动子序列具有几个特异性核苷酸突变。水稻组合谷蛋白2D-PAGE图谱结果表明：粳稻和籼稻各自的杂交组合的谷蛋白含量都具有叠加性。
The protein contents and components in rice (Oryza sativa L.) play a significant role in the quality of rice seeds, especially the digestible protein content and its essential amino acids content. Therefore, it is essential to understand the protein contents and components of rice seed storage protein (SSPs), and to improve the nutritive composition in rice breeding.
The main results are as follows:
1. The glutelin subunits of cv.93-11and cv.Nipponbare selected from O. sativa L. ssp. japonica and ssp. indica were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) combining with liquid chromatography-tandem mass spectrometry (LC-MS/MS) and western blot with highly subunit-specific antibodies. The results showed that～80%of glutelin content from the total glutelin extract consisted of seven major subunits in the SwissProt database, and～10%of glutelin consisted of a member of the glutelin type-B subfamily. In addition, based on the sequence heterogeneity at the amino acid level, the glutelin components of unknown identity seem to be new members of a new glutelin subfamily. The diversity of rice glutelin subunits between japonica and indica were identified as GluA-1and GluA-3, and the diversity of the glutelin composition between japonica and indica was reflected by the difference in glutelin subunit composition between glutelin type-A and type-B and the special subunits identified as GluA-1, and the percentage composition of GluA-3and GluB-1was twice more in the indica cultivars than that in the japonica cultivars. The result also revealed that the indica cultivars were superior to the japonica cultivars in terms of the nutritional value at the lysine level. The innovation was the combination of two methods, LC-MS/MS and western blot.
2. The SSPs components of thirty-five prevailing rice cultivars were analyzed. First, they were separated by SDS-PAGE, and then their protein contents were determined by the Kjeldahl and Bradford method, and finally their amino acid contents were identified using the amino acid analyzer. The results showed that four components were separated, including albumin, globulin, prolamine, and glutelin, and the glutelin was found to be the main components in a range of50-80%. Moreover, as one of the digestible proteins for human, the Lys content of the glutelin was nine times higher than the other three. In further analysis, certain relationship was found between the protein contents and the amino acid components:the glutelin content was in significantly negative relation to the prolamine content; while the Lys content showed positive relation to the total SSPs content (P<0.01). In the study, the relativity of the nutritive value will provide the theoretical evidence for rice genetics and breeding.
3. DNA sequences of Glu promoters in five cultivars with difference protein contents were selected for sequence slignment analysis, which were as hybrid rice parents new rice groups were analyzed the glutelin subunits by2D-PAGE These five cultivars as hybrid rice parents breed and analysis of glutelin composition. The results showed that the transcription factors were known to play central roles in regulating gene transcription from promoters. The diversity of Glu promoters between japonica and indica were4-5mutation nucleotides. The certain protein content of the Hybrid group had the assumption at certain protein content level.
引文
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