大豆籽粒不同发育时期基因表达谱的研究
摘要
大豆是世界上最重要的油料作物和最主要的经济作物之一。大豆中含有丰富的蛋白质、脂肪和多种对人体有益的生理活性物质,既是人类蛋白质、油脂及保健活性物质的重要来源,又是食品、饲料等多种加工工业的优质原料。
大豆的品质是其加工利用的主要依据,而油脂和蛋白质成分则是衡量大豆品质的两个最重要的指标。大豆籽粒中含有18%-27%的油脂和40%左右的蛋白质,油脂中90%以上的成分是脂肪酸,蛋白质中最主要的成分是多种氨基酸和贮藏蛋白。
随着基因工程技术和分子生物学技术的飞速发展,改良这两种营养物质的含量和成分一直受到科学家们的关注,但是,油脂和蛋白质合成的生理过程十分复杂,尤其是蛋白质中含有的多种氨基酸,多数氨基酸各有其合成通路,且涉及众多基因和酶的参与,其作用位点及启动功能的时期及如何使细胞内各组分发生变化尚无定论,因此,对大豆种子品质改良的基因工程研究应选用高效、高通量的方法对其进行整体监测,从而挖掘出起调控作用的关键基因。
创新点：基因的时空表达成为生物学家研究的热点,基因的表达受时间和空间以及内外因子的影响,与基因组的静态相比,基因表达状况往往呈现出复杂的动态过程,基因表达谱的分析正是通过对基因表达在时间和空间上的分析,来探索生物体内复杂的生理过程。Solexa (Illumina)是第二代新型大规模、高通量的测序平台,它要比基因芯片技术具有检测数据范围更大、检测精度更高的优点,本研究应用Solexa数字基因表达谱测序对大豆籽粒发育的4个不同时期的基因表达情况进行了深入研究,将15DAF作为对照,对后续3个时期基因表达水平与之相比较,从mRNA水平对大豆籽粒发育过程中差异表达基因进行了探测,首次全面、精确地检测出了影响脂肪和蛋白质积累的关键基因及其作用位点。
主要结果如下：
1.通过Solexa测序,比对出的所有差异表达的基因数分别为29632、29511和29572个。符合条件FDR≤0.001和(?)log2Ratio|≥1的差异表达基因数目分别为11592、16594和16255个,其中共有的差异表达基因为9905个,这些共有的差异表达基因即可认定为在大豆籽粒发育过程中持续表达且起关键作用的基因。
2.通过Gene Ontology分析,共有11963个基因至少含有一种功能注释,大部分基因功能集中在基因的分子功能本体的binding和catalytic活性上,细胞组件的cel、cell parts和organelles上,并主要参与cellular和metabolic等生物学过程,在三个比较组中其GO功能分类模式类似。
3.在脂肪酸和油脂合成的功能显著性富集分析中,共有112个三组共有的差异表达基因被筛选出来,在对蛋氨酸和其他多种氨基酸合成相关的功能分析中总共有159个三组共有的差异表达基因被筛选出来。
4.在本研究中通过Solexa测序一共鉴定出124条pathway,共注释到11228个差异表达基因,筛选出5条与油脂和氨基酸合成相关的pathway。
5.在三个比较组中的pathway分析中,筛选出25个脂肪酸合成途径中的共有差异表达基因,86个氨基酸合成代谢相关途径的共有差异表达基因。
6.为验证测序结果的准确性,本研究随机选择55DAF的大豆籽粒作为检测样本,12个候选基因进行荧光定量PCR检测,检测结果基本与测序结果相符,证明Solexa RNA测序结果准确可靠。
这些结果为大豆籽粒发育提供了一个分子生物学背景,尤其对大豆籽粒发育过程中油脂和蛋白质的合成过程提供全面而详细的信息,所有在本研究中检测到的与油脂和蛋白质合成相关的关键基因可作为今后大豆优质种质改良的重要资源。
Soybean (Glycine max (L.)Merrll.) is one of the most important oil crops and also one of the most important economic crops in the world. Soybean contains rich proteins, lipid and various materials which are beneficial to human. It is the main sources of proteins, greases and raw materials of food and feed.
The quality of soybean is the basic principle for its processing and utilization, while oil and protein composition of soybean are the two main factors of its quality. There are about18%-27%of lipid and40%protein Soybean seeds contain. More than90%of the lipid composition is fatty acid, and the majority of protein components is a variety of amino acids and storage protein.
As the rapid development of genetic engineering and molecular biology techniques, the improvement of these two nutrients content and composition has always being the focus of scientists. However, the physiological processes of lipid and protein biosynthesis are very complex, especially the protein contains lots of amino acids. Majority of these amino acids has their own different pathways which many genes and enzymes are involves in. For these genes and enzymes, it is uncertain their action sites and the period of their action and how to make the various components of the cell changes. So, genetic engineering improvement of soybean seeds quality should choose an efficient and high-throughput method for the overall monitoring so as to discovering the key regular genes.
Innovation:Gene space-time expression has become the hot spot, and the expression of genes is reflected by space-time and some other factors. Compared with the static genome, gene expression always presents a complex dynamic process. Gene expression profiling analysis is just the method that analysis gene space-time expression to detect the complex physiological processes. Solexa (Illumina) is the second geneRation platform as the new large-scale, high throughput sequencing. It has a larger wider range and higher resolution than microarray. So in this study we selected Solexa digital gene expression profiling sequencing to detect the soybean seeds at four different stages.15DAF was taken as control, and compared with the subsequent three stages of the gene expression from mRNA levels. This is the first comprehensive and accurate detection for the key genes that regulated lipid and protein biosynthesis.
The main results are as follows:
1. By Solexa sequencing, a total of29632,29511, and29572differentially expressed unigenes were detected. Of them,11592,16594and16255differentially expressed unigenes matched the conditions of the FDR<0.001and|log2Ratio|≥1, respectively.9905differentially co-expressed unigenes were in common, and these unigenes can be identified as the key genes during the soybean seeds development.
2. By Gene Ontology analysis, a total of11963genes contained at least one functional annotation. The majority of these genes'function were concentrated in the binding and catalytic activity of the molecular function ontology, of cell parts, the cell and organelles of cellular components, and they mainly involved in cellular and metabolic biological processes. The GO function classification models are similar in these three compare groups.
3. During the GO analysis of fatty acid and lipid synthesis, a total of112differentially co-expressed genes were filtered out in these three groups. While in the analysis of methionine and other amino acids biosynthesis, a total of159differentially co-expressed genes were filtered out in these three groups.
4. In this study, a total of124pathways were identified by Solexa sequencing, in which11228differentially expressed genes are annotated, and five lipid and amino acid biosynthesis pathways were filter out.
5.25differencially co-expressed genes were identified in fatty acid synthesis pathway in the three groups, and a total of,86co-expressed genes in amino acid anabolic metabolism pathways.
6. Seeds of55DAF were randomly chosen as the sample for RNA-extraction and12differentially expressed genes were selected to analyze their expressions using qRT-PCR. The results were consistent well with those obtained from Solexa sequencing.
These results provided a molecular biology background for soybean seeds development especially provided a comprehensive and detailed information for lipid and protein biosynthesis during development of soybean seeds. All of these genes identified in our research can be the important resources for high-quality soybeans breeding.
大豆的品质是其加工利用的主要依据,而油脂和蛋白质成分则是衡量大豆品质的两个最重要的指标。大豆籽粒中含有18%-27%的油脂和40%左右的蛋白质,油脂中90%以上的成分是脂肪酸,蛋白质中最主要的成分是多种氨基酸和贮藏蛋白。
随着基因工程技术和分子生物学技术的飞速发展,改良这两种营养物质的含量和成分一直受到科学家们的关注,但是,油脂和蛋白质合成的生理过程十分复杂,尤其是蛋白质中含有的多种氨基酸,多数氨基酸各有其合成通路,且涉及众多基因和酶的参与,其作用位点及启动功能的时期及如何使细胞内各组分发生变化尚无定论,因此,对大豆种子品质改良的基因工程研究应选用高效、高通量的方法对其进行整体监测,从而挖掘出起调控作用的关键基因。
创新点：基因的时空表达成为生物学家研究的热点,基因的表达受时间和空间以及内外因子的影响,与基因组的静态相比,基因表达状况往往呈现出复杂的动态过程,基因表达谱的分析正是通过对基因表达在时间和空间上的分析,来探索生物体内复杂的生理过程。Solexa (Illumina)是第二代新型大规模、高通量的测序平台,它要比基因芯片技术具有检测数据范围更大、检测精度更高的优点,本研究应用Solexa数字基因表达谱测序对大豆籽粒发育的4个不同时期的基因表达情况进行了深入研究,将15DAF作为对照,对后续3个时期基因表达水平与之相比较,从mRNA水平对大豆籽粒发育过程中差异表达基因进行了探测,首次全面、精确地检测出了影响脂肪和蛋白质积累的关键基因及其作用位点。
主要结果如下：
1.通过Solexa测序,比对出的所有差异表达的基因数分别为29632、29511和29572个。符合条件FDR≤0.001和(?)log2Ratio|≥1的差异表达基因数目分别为11592、16594和16255个,其中共有的差异表达基因为9905个,这些共有的差异表达基因即可认定为在大豆籽粒发育过程中持续表达且起关键作用的基因。
2.通过Gene Ontology分析,共有11963个基因至少含有一种功能注释,大部分基因功能集中在基因的分子功能本体的binding和catalytic活性上,细胞组件的cel、cell parts和organelles上,并主要参与cellular和metabolic等生物学过程,在三个比较组中其GO功能分类模式类似。
3.在脂肪酸和油脂合成的功能显著性富集分析中,共有112个三组共有的差异表达基因被筛选出来,在对蛋氨酸和其他多种氨基酸合成相关的功能分析中总共有159个三组共有的差异表达基因被筛选出来。
4.在本研究中通过Solexa测序一共鉴定出124条pathway,共注释到11228个差异表达基因,筛选出5条与油脂和氨基酸合成相关的pathway。
5.在三个比较组中的pathway分析中,筛选出25个脂肪酸合成途径中的共有差异表达基因,86个氨基酸合成代谢相关途径的共有差异表达基因。
6.为验证测序结果的准确性,本研究随机选择55DAF的大豆籽粒作为检测样本,12个候选基因进行荧光定量PCR检测,检测结果基本与测序结果相符,证明Solexa RNA测序结果准确可靠。
这些结果为大豆籽粒发育提供了一个分子生物学背景,尤其对大豆籽粒发育过程中油脂和蛋白质的合成过程提供全面而详细的信息,所有在本研究中检测到的与油脂和蛋白质合成相关的关键基因可作为今后大豆优质种质改良的重要资源。
Soybean (Glycine max (L.)Merrll.) is one of the most important oil crops and also one of the most important economic crops in the world. Soybean contains rich proteins, lipid and various materials which are beneficial to human. It is the main sources of proteins, greases and raw materials of food and feed.
The quality of soybean is the basic principle for its processing and utilization, while oil and protein composition of soybean are the two main factors of its quality. There are about18%-27%of lipid and40%protein Soybean seeds contain. More than90%of the lipid composition is fatty acid, and the majority of protein components is a variety of amino acids and storage protein.
As the rapid development of genetic engineering and molecular biology techniques, the improvement of these two nutrients content and composition has always being the focus of scientists. However, the physiological processes of lipid and protein biosynthesis are very complex, especially the protein contains lots of amino acids. Majority of these amino acids has their own different pathways which many genes and enzymes are involves in. For these genes and enzymes, it is uncertain their action sites and the period of their action and how to make the various components of the cell changes. So, genetic engineering improvement of soybean seeds quality should choose an efficient and high-throughput method for the overall monitoring so as to discovering the key regular genes.
Innovation:Gene space-time expression has become the hot spot, and the expression of genes is reflected by space-time and some other factors. Compared with the static genome, gene expression always presents a complex dynamic process. Gene expression profiling analysis is just the method that analysis gene space-time expression to detect the complex physiological processes. Solexa (Illumina) is the second geneRation platform as the new large-scale, high throughput sequencing. It has a larger wider range and higher resolution than microarray. So in this study we selected Solexa digital gene expression profiling sequencing to detect the soybean seeds at four different stages.15DAF was taken as control, and compared with the subsequent three stages of the gene expression from mRNA levels. This is the first comprehensive and accurate detection for the key genes that regulated lipid and protein biosynthesis.
The main results are as follows:
1. By Solexa sequencing, a total of29632,29511, and29572differentially expressed unigenes were detected. Of them,11592,16594and16255differentially expressed unigenes matched the conditions of the FDR<0.001and|log2Ratio|≥1, respectively.9905differentially co-expressed unigenes were in common, and these unigenes can be identified as the key genes during the soybean seeds development.
2. By Gene Ontology analysis, a total of11963genes contained at least one functional annotation. The majority of these genes'function were concentrated in the binding and catalytic activity of the molecular function ontology, of cell parts, the cell and organelles of cellular components, and they mainly involved in cellular and metabolic biological processes. The GO function classification models are similar in these three compare groups.
3. During the GO analysis of fatty acid and lipid synthesis, a total of112differentially co-expressed genes were filtered out in these three groups. While in the analysis of methionine and other amino acids biosynthesis, a total of159differentially co-expressed genes were filtered out in these three groups.
4. In this study, a total of124pathways were identified by Solexa sequencing, in which11228differentially expressed genes are annotated, and five lipid and amino acid biosynthesis pathways were filter out.
5.25differencially co-expressed genes were identified in fatty acid synthesis pathway in the three groups, and a total of,86co-expressed genes in amino acid anabolic metabolism pathways.
6. Seeds of55DAF were randomly chosen as the sample for RNA-extraction and12differentially expressed genes were selected to analyze their expressions using qRT-PCR. The results were consistent well with those obtained from Solexa sequencing.
These results provided a molecular biology background for soybean seeds development especially provided a comprehensive and detailed information for lipid and protein biosynthesis during development of soybean seeds. All of these genes identified in our research can be the important resources for high-quality soybeans breeding.
引文
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