水稻胚乳特异性表达基因的鉴别及应用性探索
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摘要
本论文研究对象为水稻胚乳文库及其生物信息数据库和从嘉兴市农科院及四川省农科院生物技术核技术研究所采集的966个不同水稻品种胚乳总RNA。
为探索水稻胚乳中特异表达基因，我们将水稻胚乳EST经BLASTN比对分析，获576个有功能注释的基因。统计结果显示，贮藏蛋白以及相应的核糖体蛋白，起始因子，延伸因子以及转录因子等基因高丰度表达。
为进一步确认这些胚乳特异表达基因，我们将来自水稻胚乳文库的2013个TUT制作cDNA阵列，以水稻花期和花后10-15天的胚乳，胚，穗，叶片，茎节间等总RNA为探针与其杂交。利用贝叶氏T检验，对这些基因的表达谱进行分析，检测胚乳与其他水稻组织的差异表达基因，以期揭示灌浆期胚乳特异表达基因。设定Bayesian p-value为0.05对所有探针杂交结果进行差异分析，检测到230个TUT具有显著差异，其中129个基因在胚乳中明显下调表达。101个TUT在胚乳中高表达，包括75个具有功能注释。这些基因大部分编码或者参与编码贮藏蛋白和淀粉。29个TUT编码贮藏蛋白，4个TUT参与淀粉合成。而贮藏蛋白主要包括谷蛋白，醇溶谷蛋白和过敏性蛋白等；淀粉合成包括直链淀粉和支链淀粉的合成。另外还有α-淀粉酶，光诱导蛋白，光受体，转录因子以及一些信号传递物质等基因表达量也很高。在显著表达的101个基因中有26个是未知基因，占有较大的比例。随后的Northern印迹结果验证了cDNA微阵列的准确性。同时我们证实，谷蛋白、醇溶谷蛋白、脂类转运蛋白、可溶性淀粉合成酶Ⅱ-3、18 kDa油体蛋白、α-淀粉酶等基因为胚乳特异表达基因。另外发现Rbe1基因有一较大转录本在水稻胚乳中特异表达。
为检测不同水稻样本中Wx基因表达差异，我们将966个水稻品种总RNA制作微阵列，以Wx基因为探针，进行杂交实验。结果表明该基因表达丰度在总体上与水稻胚乳AC呈正相关。对两个转录本RNA微阵列的杂交结果经贝叶氏T检验分析表达差异显著性，发现fold与AC之间呈正相关，该值的转折点处，的水稻品种米质较好。另外发现非成熟转录本也存在于AC较高的水稻品种(AC大于20％)中。
其他的淀粉分支酶RBE1、ADP葡萄糖焦磷酸化酶和可溶性淀粉合成酶Ⅱ-3等淀粉合成关键基因以及其他19个水稻胚乳特异表达的基因，未发现与直链淀粉含量之间有直接的关系。
利用RNA微阵列对水稻胚乳贮藏蛋白，淀粉合成相关的基因，脂类基因等19个基因进行表达谱分析，发现H105D02是GluB组基因或者GluA-1和GluA-2基因，H063F03是GluA组中与GluA-3表达一致的基因。
对未知基因H098F12和H051G06与其他10个已知基因的聚类，发现H098F12与过敏性蛋白(克隆ID：H097G05)具有相似的表达调控机制。而H051G06与10 kDa油体蛋白(克隆ID：H129F01)表达模式最相近，其次为可溶性淀粉合成酶Ⅱ-3(克隆ID：H144A06)和13kDa醇溶谷蚩白(克隆ID：H110F01)，该未知基因与胚乳贮藏物质基因共调控表达，而且
Northem印迹实验证实其在胚乳中特异表达，推测H05lG06可能参与胚乳贮藏物质的积累。
制作RNA微阵列的9“个水稻品种多为育种材料，如在嘉兴早稻杂交一代的ZR卫6样
本中，醇溶谷蛋白，谷蛋白以及参与淀粉合成的基因等都具有表达量远远高于其他的杂交一
代的特点。这可为以后FZ代的选育提供参考。本研究所检测基因的表达谱可以应用于选育
优良品种，优化选择。
In the study, rice endosperm library, its bioinformatics database and 966 different rice endosperm total RNA from Jiaxing Academy of Agricultural Sciences and Institute of Biotechnology and Nuclear Technology, Sichuan Academy of Agricultural Sciences, were used.
576 TUTs were annotated after ESTs searching against oryza sativa database of NCBI. Of them, most highly-expressed genes encoded storage proteins, starch, ribosome proteins, initiation factors, elongation factors and transcription factors.
In order to analyze genes expression profiles of different rice tissues and to find the highly-expressed genes in endosperm, 2013 TUTs were printed on cDNA array. And different tissues included endosperm, embryo, ear, leaf and stem internode during anthesis and 10-15 days after anthesis. Differential expression analysis, especially comparing endosperm with other tissues, was performed by Cyber-T Bayesian statistical framework. While Bayesian p-value was set to be 0.05, expression of 203 TUTs changed remarkablly. Except for 129 down-regulated genes, a total of 101 TUTs were up-regulated prominently in endosperm, and 75 TUTs were annotated. The up-regulated genes were mostly concerned with storage protein and starch synthesis. There were 29 TUTs encoding storage protein, such as glutelin and prolamin. 4 TUTs participated in amylose and amylopectin synthesis. Other genes mostly encoded ct-amylase, light-inducible protein, transduction factor, signal recognition particle and so on. Unkown genes occupied about a
quarter of all up-regulated genes.
And the veracity of cDNA array was validated by following Northern blot hybridization. The result had confirmed the credibility of cDNA-array. And glutelin, prolamin, lipid transfer protein, soluble starch synthase, 18 kDa oleosin and ot-amylase were found only expressed in endosperm, the same as the larger Rbel transcription.
The hybridization with RNA array of 966 RNA indicated that Wx gene had linear relationship with rice AC on the whole. But some rice samples were not coincident with the rule. Wx gene mature mRNA and an un-mature mRNA were tested by Cyber-T. And the result indicated that fold had positive relationship with AC. AC was 17%-20% while fold between -1 and +1.
19 genes expression profiles were analyzed by RNA array. These genes coded storage protein, starch and lipid. And we found the expression of homologous genes may be different.
According to the expression profile of four glutelin genes, the expression of H105D02 is similar to GluB, and the expression of H063F03 simialr to GluA.
Two unkown genes (H098F12, H051G06) and other 10 known genes were clustered. And the result indicated that H098F12 had closer expressional pattern with allergenic protein (H097G05).
H051G06 was similar to 10 kDa oleosin (H129F01), soluble starch synthase (H144A06), and 13kDa prolamin (H110F01). We deduced they were respectively co-regulated genes. And northern had confirmed H051G06 as endosperm-specific gene; we deduced it was concerned with storage substance genes.
Our results could provide reference for rice breeding, because a large number of samples were generations during breeding. For example, ZRF6 was one of F1 of early rice from Jiaxing agricultural academy of sciences. The expression of prolamin, glutelin and the genes concerned with starch synthesis in ZRF6 was higher than other samples of Fl of early rice. This could direct to select high-quality F2. And this was also adapt to new rice combination of 2003 and fertility restoring line select of 2003 from Sichuan agricultural academy of sciences.
为探索水稻胚乳中特异表达基因，我们将水稻胚乳EST经BLASTN比对分析，获576个有功能注释的基因。统计结果显示，贮藏蛋白以及相应的核糖体蛋白，起始因子，延伸因子以及转录因子等基因高丰度表达。
为进一步确认这些胚乳特异表达基因，我们将来自水稻胚乳文库的2013个TUT制作cDNA阵列，以水稻花期和花后10-15天的胚乳，胚，穗，叶片，茎节间等总RNA为探针与其杂交。利用贝叶氏T检验，对这些基因的表达谱进行分析，检测胚乳与其他水稻组织的差异表达基因，以期揭示灌浆期胚乳特异表达基因。设定Bayesian p-value为0.05对所有探针杂交结果进行差异分析，检测到230个TUT具有显著差异，其中129个基因在胚乳中明显下调表达。101个TUT在胚乳中高表达，包括75个具有功能注释。这些基因大部分编码或者参与编码贮藏蛋白和淀粉。29个TUT编码贮藏蛋白，4个TUT参与淀粉合成。而贮藏蛋白主要包括谷蛋白，醇溶谷蛋白和过敏性蛋白等；淀粉合成包括直链淀粉和支链淀粉的合成。另外还有α-淀粉酶，光诱导蛋白，光受体，转录因子以及一些信号传递物质等基因表达量也很高。在显著表达的101个基因中有26个是未知基因，占有较大的比例。随后的Northern印迹结果验证了cDNA微阵列的准确性。同时我们证实，谷蛋白、醇溶谷蛋白、脂类转运蛋白、可溶性淀粉合成酶Ⅱ-3、18 kDa油体蛋白、α-淀粉酶等基因为胚乳特异表达基因。另外发现Rbe1基因有一较大转录本在水稻胚乳中特异表达。
为检测不同水稻样本中Wx基因表达差异，我们将966个水稻品种总RNA制作微阵列，以Wx基因为探针，进行杂交实验。结果表明该基因表达丰度在总体上与水稻胚乳AC呈正相关。对两个转录本RNA微阵列的杂交结果经贝叶氏T检验分析表达差异显著性，发现fold与AC之间呈正相关，该值的转折点处，的水稻品种米质较好。另外发现非成熟转录本也存在于AC较高的水稻品种(AC大于20％)中。
其他的淀粉分支酶RBE1、ADP葡萄糖焦磷酸化酶和可溶性淀粉合成酶Ⅱ-3等淀粉合成关键基因以及其他19个水稻胚乳特异表达的基因，未发现与直链淀粉含量之间有直接的关系。
利用RNA微阵列对水稻胚乳贮藏蛋白，淀粉合成相关的基因，脂类基因等19个基因进行表达谱分析，发现H105D02是GluB组基因或者GluA-1和GluA-2基因，H063F03是GluA组中与GluA-3表达一致的基因。
对未知基因H098F12和H051G06与其他10个已知基因的聚类，发现H098F12与过敏性蛋白(克隆ID：H097G05)具有相似的表达调控机制。而H051G06与10 kDa油体蛋白(克隆ID：H129F01)表达模式最相近，其次为可溶性淀粉合成酶Ⅱ-3(克隆ID：H144A06)和13kDa醇溶谷蚩白(克隆ID：H110F01)，该未知基因与胚乳贮藏物质基因共调控表达，而且
Northem印迹实验证实其在胚乳中特异表达，推测H05lG06可能参与胚乳贮藏物质的积累。
制作RNA微阵列的9“个水稻品种多为育种材料，如在嘉兴早稻杂交一代的ZR卫6样
本中，醇溶谷蛋白，谷蛋白以及参与淀粉合成的基因等都具有表达量远远高于其他的杂交一
代的特点。这可为以后FZ代的选育提供参考。本研究所检测基因的表达谱可以应用于选育
优良品种，优化选择。
In the study, rice endosperm library, its bioinformatics database and 966 different rice endosperm total RNA from Jiaxing Academy of Agricultural Sciences and Institute of Biotechnology and Nuclear Technology, Sichuan Academy of Agricultural Sciences, were used.
576 TUTs were annotated after ESTs searching against oryza sativa database of NCBI. Of them, most highly-expressed genes encoded storage proteins, starch, ribosome proteins, initiation factors, elongation factors and transcription factors.
In order to analyze genes expression profiles of different rice tissues and to find the highly-expressed genes in endosperm, 2013 TUTs were printed on cDNA array. And different tissues included endosperm, embryo, ear, leaf and stem internode during anthesis and 10-15 days after anthesis. Differential expression analysis, especially comparing endosperm with other tissues, was performed by Cyber-T Bayesian statistical framework. While Bayesian p-value was set to be 0.05, expression of 203 TUTs changed remarkablly. Except for 129 down-regulated genes, a total of 101 TUTs were up-regulated prominently in endosperm, and 75 TUTs were annotated. The up-regulated genes were mostly concerned with storage protein and starch synthesis. There were 29 TUTs encoding storage protein, such as glutelin and prolamin. 4 TUTs participated in amylose and amylopectin synthesis. Other genes mostly encoded ct-amylase, light-inducible protein, transduction factor, signal recognition particle and so on. Unkown genes occupied about a
quarter of all up-regulated genes.
And the veracity of cDNA array was validated by following Northern blot hybridization. The result had confirmed the credibility of cDNA-array. And glutelin, prolamin, lipid transfer protein, soluble starch synthase, 18 kDa oleosin and ot-amylase were found only expressed in endosperm, the same as the larger Rbel transcription.
The hybridization with RNA array of 966 RNA indicated that Wx gene had linear relationship with rice AC on the whole. But some rice samples were not coincident with the rule. Wx gene mature mRNA and an un-mature mRNA were tested by Cyber-T. And the result indicated that fold had positive relationship with AC. AC was 17%-20% while fold between -1 and +1.
19 genes expression profiles were analyzed by RNA array. These genes coded storage protein, starch and lipid. And we found the expression of homologous genes may be different.
According to the expression profile of four glutelin genes, the expression of H105D02 is similar to GluB, and the expression of H063F03 simialr to GluA.
Two unkown genes (H098F12, H051G06) and other 10 known genes were clustered. And the result indicated that H098F12 had closer expressional pattern with allergenic protein (H097G05).
H051G06 was similar to 10 kDa oleosin (H129F01), soluble starch synthase (H144A06), and 13kDa prolamin (H110F01). We deduced they were respectively co-regulated genes. And northern had confirmed H051G06 as endosperm-specific gene; we deduced it was concerned with storage substance genes.
Our results could provide reference for rice breeding, because a large number of samples were generations during breeding. For example, ZRF6 was one of F1 of early rice from Jiaxing agricultural academy of sciences. The expression of prolamin, glutelin and the genes concerned with starch synthesis in ZRF6 was higher than other samples of Fl of early rice. This could direct to select high-quality F2. And this was also adapt to new rice combination of 2003 and fertility restoring line select of 2003 from Sichuan agricultural academy of sciences.
引文
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