家蚕第2白卵性状相关基因表达的研究
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摘要
家蚕(Silkworm, Bombyx mori)第2白卵(white egg2, w-2:10-16.1)突变性状适合作为家蚕转基因筛选标记,但该性状表达的生化过程和分子机制尚不明瞭。本实验以家蚕正常型黑卵品种菁松A及其第2白卵近等基因系菁松A白为生物材料,综合利用家蚕抑制消减杂交文库cDNA芯片、全基因组芯片、表达谱测序技术和实时荧光定量PCR技术,研究获取家蚕第2白卵及卵色性状相关的基因信息,为揭示家蚕第2白卵形成的分子机制提供工作基础。
1.利用抑制消减杂交和cDNA芯片检测技术研究家蚕第2白卵相关基因
构建了家蚕转色期蚕卵黑卵对第2白卵的抑制消减杂交cDNA文库,随机选取300个消减文库cDNA制作芯片进行检测,获得了转色期蚕卵黑卵和第2白卵品系差异表达基因11个。11个基因中J241文库序列在黑白卵品系之间表达量差异最大,24h和48h黑卵比白卵上调5752.60倍和7951.40倍,J241基因在黑卵品系转色期黑卵中是一种大量表达的基因,而在白卵中J241不表达,通过RACE等技术明确了差异表达的J241基因是黑卵特有序列类型。
家蚕受精卵注射J241基因的dsRNA未能有效阻止蚕卵色素合成过程,推测该基因的作用位点不在眼色素合成的主要途径上。
2.应用家蚕全基因组芯片研究家蚕第2白卵性状相关基因
利用23K家蚕全基因组芯片检测技术,对家蚕黑卵和第2白卵品系间转色期蚕卵差异表达基因进行了研究。经家蚕全基因组芯片检测,获得24h卵龄差异表达基因163个,白卵对黑卵基因表达最高上调倍数为33.00倍,最高下调倍数为32.47倍；获得48h卵龄差异表达基因186个,白卵对黑卵基因表达最高上调倍数为14.52倍,最高下调倍数为22.83倍。
GO分析表明,差异表达基因涉及到细胞组分、生物学过程、分子功能全方位的改变和调整,24h时点差异表达基因在分子功能、生物过程、细胞组分基因分别占64%、27%和9%,48h时点差异表达基因在分子功能、生物过程及细胞组份上各占56%、27%和17%。代谢通路分析表明,黑卵和白卵品系间24h时点在8个代谢通路、48h时点在23个代谢通路出现基因差异表达,24h和48h时点均呈现差异表达的基因有26个,其中明确功能的17个基因涉及8个代谢酶类基因、3个胞间和胞内运输相关蛋白、3个调控和受体蛋白等。
实时荧光定量RT-PCR验证结果显示,定量PCR和芯片检测结果高度相关,在24h或48h黑白卵样本间最大差异表达基因的差异倍数达到826倍。不同卵色品系转色卵中β-20-羟基类固醇脱氢酶、醛脱氢酶—9、thioredoxin domain containing4、microsomal glutathione transferase GSTMIC1及ATP-binding cassette transporter等色氨酸代谢、色素运输及氧化还原反应相关基因有表达差异。
3.应用表达谱测序技术研究家蚕第2白卵性状相关基因
利用Solexa高通量表达谱测序技术,对家蚕黑卵和第2白卵品系差异表达基因进行了研究。白卵样品获得有效标签(clean Tag)3493817个、有效标签种类(Distinct clean Tags)112384个；黑卵样品获得有效标签2497077个、有效标签种类95089个。饱和度分析表明本实验测序获得标签数量已能真实反映样品的基因表达情况。黑卵样本匹配(Mapping)获得13501个家蚕基因,白卵样本匹配获得14342个基因,匹配基因数已能够充分代表样本的基因表达信息。
在家蚕黑卵及第2白卵品系间转色卵检出859个表达差异的基因,其中白卵品系上调表达基因595个、下调表达基因264个。有93个匹配基因只在黑卵或白卵一方检出,认为可能包含了第2白卵性状表达相关的基因。
379个已知功能的差异表达基因涉及161个代谢通路,其中新陈代谢途径的基因89个,核糖体通路的基因19个,参与嘌呤代谢基因16个、参与氧化磷酸化的基因13个、参与嘧啶代谢的基因11个等。
与家蚕眼色素合成原料——色氨酸代谢通路有5个差异基因参与,蚕卵多巴脱羧酶(EC：4.1.1.28)基因和色氨酰-tRNA合成酶(EC：6.1.1.2)在第2白卵中的表达调控保持了卵内色氨酸代谢的动态平衡。
绝大部分基因实时荧光定量RT-PCR定量检测结果与表达谱测序结果倾向一致,黑卵和白卵之间差异表达基因的最大差异倍数达到16158倍。
上述研究获得了大量第2白卵性状相关的差异表达基因信息,通过这些信息的深入挖掘,进一步开展基因克隆和功能验证分析,将促进家蚕第2白卵和卵色性状相关分子机制的解析。
The white egg2mutant (w-2) of the silkworm, Bombyx mori, is suitable as a screening marker for screening of transgenic silkworm because of its features, such as egg and eye color, regular hereditary. However, the biochemical processe and molecular mechanism of the w-2mutant still remains unclear so far. In this study, the multidisciplinary approach combined with cDNA microarray based on SSH library, silkworm genome microarray, expression sequencing, and Real-time quantitative PCR, was employed to explore the differentially expressed genes in wild type strain Jingsong A and its near-isogenic line w-2mutant Jingsong A white, the current study provide useful data for further studying of the molecular mechanism of the white egg2mutant.
1. The differentially expressed genes identified by SSH and cDNA microarray analysis
Based on construction of suppression subtractive hybridization cDNA library from wild type strain Jingsong A and its near-isogenic line w-2mutant,300clones were randomly selected for further analysis of cDNA microarray, and finally11differentially expressed genes were identified.
J241exhibited the most different expression level between wild type Jingsong A and mutant Jingsong A white among these11genes. The expression difference between wild type and mutant reached5752.60and7951.40folds at24hour point and48hour point after normally incubated respectively. In the early development stage of egg, J241was found to be abundantly expressed in wild type, but no expression was observed in w-2mutant.. The result of RACE shows that J241is specific sequence splicing only in wild type Jingsong A.
After microinjection of double-stranded RNA (dsRNA) of J241into the wild type eggs, no inhibitory effect was observed on the ommochrome pigment formation, indicating that J241is not the core gene on the course of ommochrome biosynthesis.
2. The differentially expressed genes identified by silkworm genome microarray
At the early stage of embryonic development of wild type Jingsong A and mutant Jingsong A white,163and186differentially expressed genes were identified by23K silkworm genome microarray at24hour and48hour respectively. The maximal up-regulation in mutant was33.00folds higher, and maximal down-regulation was32.47folds lower compared to wild type at24hour point, whereas the maximal up-regulation reached14.52folds, and maximal down-regulation22.83folds at48hour point.
26genes were identified to differentially express between wild type and w-2mutant, representing8%of total differentially expressed genes, among them, there are17genes with known function including8metabolic enzymes,3intercellular or intracellular transport-related proteins, and3regulatory and receptor proteins.
Analysis of GO and metabolic pathway has demonstrated that differential expressions of genes involve in cellular components, biological processes, and molecular functions, including involvement of eight metabolic pathways at24hour point and23metabolic pathways at48hour point respectively.
The further validation using real-time quantitative RT-PCR indicated that the degree of concordance between data generated by the two methods was high, and the maximal differential expression reached826folds between wild type and w-2mutant.
3. The differentially expressed genes identified by expression profile sequencing
Solexa sequencing, a high-throughput expression profile sequencing technology, was employed to explore the differentially expressed genes during certain development stage in egg of silkworm. In this experiment, we got3493817clean tags and112384distinct clean tags from the time course mixture sample of w-2mutant, and2497077clean tags and95089distinct clean tags from wild type respectively. Saturation analysis showed that the sequence data of this experiment was a true reflection of the expressed genes in these two wild type and mutant samples.
All these distinct tags of wild type were mapped to13501silkworm genes, and the matching ratio is up to35.30%. These distinct tags of w-2mutant were mapped to14342genes, and the matching ratio is up to39.46%, suggesting that the data of sole sequence have been fully representative of gene expression profile in wild type and mutant.
In this study,859differentially expressed genes were identified by sole sequencing between wild type and white egg2mutant.595genes of them are up-regulated, and264genes are down-regulated in w-2mutant. Interestingly, there are93genes specifically expressed only in wild type or w-2mutant. It is most likely that among these differentially expressed genes, some are involved in the regulation of w-2mutant phenotype, and worth to be further investigated.
379differentially expressed genes are annotated to161metabolic pathways, including in89genes in metabolic pathway,19genes in ribosomal channels,16genes in purine metabolism,13genes in oxidative phosphorylation, and11genes in pyrimidine metabolism.
There are five differentially expressed genes involved in tryptophan pathway, which metabolites are the source of eye pigment synthesis. Among them, dopa decarboxylase (EC:4.1.1.28) gene and L-tryptophanyl-tRNA synthetase gene (EC:6.1.1.2) expressed in w-2mutant are likely required to maintain the in vivo dynamic balance of the tryptophan metabolism.
The results obtained from the expression profile sequencing analysis are perfectly consistent with data of real-time quantitative RT-PCR. The maximal differential expression level is up to16158times tested by real time quantitative RT-PCR between wild type and w-2mutant.
The present study has harvested tons of information on genes differentially expressed in wild type or w-2mutant, and will be helpful to elucidate the molecular mechanism of the white egg2mutant.
1.利用抑制消减杂交和cDNA芯片检测技术研究家蚕第2白卵相关基因
构建了家蚕转色期蚕卵黑卵对第2白卵的抑制消减杂交cDNA文库,随机选取300个消减文库cDNA制作芯片进行检测,获得了转色期蚕卵黑卵和第2白卵品系差异表达基因11个。11个基因中J241文库序列在黑白卵品系之间表达量差异最大,24h和48h黑卵比白卵上调5752.60倍和7951.40倍,J241基因在黑卵品系转色期黑卵中是一种大量表达的基因,而在白卵中J241不表达,通过RACE等技术明确了差异表达的J241基因是黑卵特有序列类型。
家蚕受精卵注射J241基因的dsRNA未能有效阻止蚕卵色素合成过程,推测该基因的作用位点不在眼色素合成的主要途径上。
2.应用家蚕全基因组芯片研究家蚕第2白卵性状相关基因
利用23K家蚕全基因组芯片检测技术,对家蚕黑卵和第2白卵品系间转色期蚕卵差异表达基因进行了研究。经家蚕全基因组芯片检测,获得24h卵龄差异表达基因163个,白卵对黑卵基因表达最高上调倍数为33.00倍,最高下调倍数为32.47倍；获得48h卵龄差异表达基因186个,白卵对黑卵基因表达最高上调倍数为14.52倍,最高下调倍数为22.83倍。
GO分析表明,差异表达基因涉及到细胞组分、生物学过程、分子功能全方位的改变和调整,24h时点差异表达基因在分子功能、生物过程、细胞组分基因分别占64%、27%和9%,48h时点差异表达基因在分子功能、生物过程及细胞组份上各占56%、27%和17%。代谢通路分析表明,黑卵和白卵品系间24h时点在8个代谢通路、48h时点在23个代谢通路出现基因差异表达,24h和48h时点均呈现差异表达的基因有26个,其中明确功能的17个基因涉及8个代谢酶类基因、3个胞间和胞内运输相关蛋白、3个调控和受体蛋白等。
实时荧光定量RT-PCR验证结果显示,定量PCR和芯片检测结果高度相关,在24h或48h黑白卵样本间最大差异表达基因的差异倍数达到826倍。不同卵色品系转色卵中β-20-羟基类固醇脱氢酶、醛脱氢酶—9、thioredoxin domain containing4、microsomal glutathione transferase GSTMIC1及ATP-binding cassette transporter等色氨酸代谢、色素运输及氧化还原反应相关基因有表达差异。
3.应用表达谱测序技术研究家蚕第2白卵性状相关基因
利用Solexa高通量表达谱测序技术,对家蚕黑卵和第2白卵品系差异表达基因进行了研究。白卵样品获得有效标签(clean Tag)3493817个、有效标签种类(Distinct clean Tags)112384个；黑卵样品获得有效标签2497077个、有效标签种类95089个。饱和度分析表明本实验测序获得标签数量已能真实反映样品的基因表达情况。黑卵样本匹配(Mapping)获得13501个家蚕基因,白卵样本匹配获得14342个基因,匹配基因数已能够充分代表样本的基因表达信息。
在家蚕黑卵及第2白卵品系间转色卵检出859个表达差异的基因,其中白卵品系上调表达基因595个、下调表达基因264个。有93个匹配基因只在黑卵或白卵一方检出,认为可能包含了第2白卵性状表达相关的基因。
379个已知功能的差异表达基因涉及161个代谢通路,其中新陈代谢途径的基因89个,核糖体通路的基因19个,参与嘌呤代谢基因16个、参与氧化磷酸化的基因13个、参与嘧啶代谢的基因11个等。
与家蚕眼色素合成原料——色氨酸代谢通路有5个差异基因参与,蚕卵多巴脱羧酶(EC：4.1.1.28)基因和色氨酰-tRNA合成酶(EC：6.1.1.2)在第2白卵中的表达调控保持了卵内色氨酸代谢的动态平衡。
绝大部分基因实时荧光定量RT-PCR定量检测结果与表达谱测序结果倾向一致,黑卵和白卵之间差异表达基因的最大差异倍数达到16158倍。
上述研究获得了大量第2白卵性状相关的差异表达基因信息,通过这些信息的深入挖掘,进一步开展基因克隆和功能验证分析,将促进家蚕第2白卵和卵色性状相关分子机制的解析。
The white egg2mutant (w-2) of the silkworm, Bombyx mori, is suitable as a screening marker for screening of transgenic silkworm because of its features, such as egg and eye color, regular hereditary. However, the biochemical processe and molecular mechanism of the w-2mutant still remains unclear so far. In this study, the multidisciplinary approach combined with cDNA microarray based on SSH library, silkworm genome microarray, expression sequencing, and Real-time quantitative PCR, was employed to explore the differentially expressed genes in wild type strain Jingsong A and its near-isogenic line w-2mutant Jingsong A white, the current study provide useful data for further studying of the molecular mechanism of the white egg2mutant.
1. The differentially expressed genes identified by SSH and cDNA microarray analysis
Based on construction of suppression subtractive hybridization cDNA library from wild type strain Jingsong A and its near-isogenic line w-2mutant,300clones were randomly selected for further analysis of cDNA microarray, and finally11differentially expressed genes were identified.
J241exhibited the most different expression level between wild type Jingsong A and mutant Jingsong A white among these11genes. The expression difference between wild type and mutant reached5752.60and7951.40folds at24hour point and48hour point after normally incubated respectively. In the early development stage of egg, J241was found to be abundantly expressed in wild type, but no expression was observed in w-2mutant.. The result of RACE shows that J241is specific sequence splicing only in wild type Jingsong A.
After microinjection of double-stranded RNA (dsRNA) of J241into the wild type eggs, no inhibitory effect was observed on the ommochrome pigment formation, indicating that J241is not the core gene on the course of ommochrome biosynthesis.
2. The differentially expressed genes identified by silkworm genome microarray
At the early stage of embryonic development of wild type Jingsong A and mutant Jingsong A white,163and186differentially expressed genes were identified by23K silkworm genome microarray at24hour and48hour respectively. The maximal up-regulation in mutant was33.00folds higher, and maximal down-regulation was32.47folds lower compared to wild type at24hour point, whereas the maximal up-regulation reached14.52folds, and maximal down-regulation22.83folds at48hour point.
26genes were identified to differentially express between wild type and w-2mutant, representing8%of total differentially expressed genes, among them, there are17genes with known function including8metabolic enzymes,3intercellular or intracellular transport-related proteins, and3regulatory and receptor proteins.
Analysis of GO and metabolic pathway has demonstrated that differential expressions of genes involve in cellular components, biological processes, and molecular functions, including involvement of eight metabolic pathways at24hour point and23metabolic pathways at48hour point respectively.
The further validation using real-time quantitative RT-PCR indicated that the degree of concordance between data generated by the two methods was high, and the maximal differential expression reached826folds between wild type and w-2mutant.
3. The differentially expressed genes identified by expression profile sequencing
Solexa sequencing, a high-throughput expression profile sequencing technology, was employed to explore the differentially expressed genes during certain development stage in egg of silkworm. In this experiment, we got3493817clean tags and112384distinct clean tags from the time course mixture sample of w-2mutant, and2497077clean tags and95089distinct clean tags from wild type respectively. Saturation analysis showed that the sequence data of this experiment was a true reflection of the expressed genes in these two wild type and mutant samples.
All these distinct tags of wild type were mapped to13501silkworm genes, and the matching ratio is up to35.30%. These distinct tags of w-2mutant were mapped to14342genes, and the matching ratio is up to39.46%, suggesting that the data of sole sequence have been fully representative of gene expression profile in wild type and mutant.
In this study,859differentially expressed genes were identified by sole sequencing between wild type and white egg2mutant.595genes of them are up-regulated, and264genes are down-regulated in w-2mutant. Interestingly, there are93genes specifically expressed only in wild type or w-2mutant. It is most likely that among these differentially expressed genes, some are involved in the regulation of w-2mutant phenotype, and worth to be further investigated.
379differentially expressed genes are annotated to161metabolic pathways, including in89genes in metabolic pathway,19genes in ribosomal channels,16genes in purine metabolism,13genes in oxidative phosphorylation, and11genes in pyrimidine metabolism.
There are five differentially expressed genes involved in tryptophan pathway, which metabolites are the source of eye pigment synthesis. Among them, dopa decarboxylase (EC:4.1.1.28) gene and L-tryptophanyl-tRNA synthetase gene (EC:6.1.1.2) expressed in w-2mutant are likely required to maintain the in vivo dynamic balance of the tryptophan metabolism.
The results obtained from the expression profile sequencing analysis are perfectly consistent with data of real-time quantitative RT-PCR. The maximal differential expression level is up to16158times tested by real time quantitative RT-PCR between wild type and w-2mutant.
The present study has harvested tons of information on genes differentially expressed in wild type or w-2mutant, and will be helpful to elucidate the molecular mechanism of the white egg2mutant.
引文
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