家蚕丝腺细胞系BmSG-SWU1与丝腺组织部分基因的表达差异研究
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摘要
家蚕丝腺不但是蚕丝业的基础，而且其高效的蛋白质合成能力可被利用来开发新的生物产业，这被国际上誉为具有广阔应用前景的研究领域。家蚕丝蛋白合成关键基因的功能研究、家蚕丝蛋白合成机理研究和丝腺生物反应器的应用研究都需要家蚕丝腺细胞系作为研究平台，但至今还没有一个基因表达谱清楚的家蚕丝腺细胞系，它已成为制约这些研究的瓶颈。本论文利用家蚕全基因组芯片数据分析、RT-PCR和荧光定量PCR技术对家蚕丝腺细胞系BmSG-SWU1和丝腺组织的特异基因的表达谱以及细胞分裂周期基因的表达谱进行了分析，结果如下：
1．利用家蚕全基因组芯片对5龄3天雌、雄家蚕的丝腺和其它组织进行了18个丝腺特异表达基因的表达谱分析，芯片数据分析结果显示这些基因在5龄3天的家蚕丝腺组织中的表达量远高于在其它组织中的表达量。这18个基因中包括激素代谢相关蛋白、转运蛋白、蛋白修饰降解类磷酸化酶、脱氢酶及其它几种蛋白，它们可能与丝物质的分泌密切相关。
2．根据家蚕基因组芯片数据，分析了家蚕中与细胞周期调控相关的16个基因，包括周期蛋白基因Cyclin A、Cyclin B、Cyclin D、Cyclin E、Cyclin H及PCNA，周期蛋白依赖性激酶基因cdc2、Cdk2、Cdk5、Cdk6。这些基因大多在家蚕具有分裂增殖能力的幼虫精巢、卵巢中高量表达，而在处于高度分化状态的丝腺组织中低量表达。雌蛹到蛾的变态过程，也是卵巢减数分裂的旺盛期，这些基因的表达量逐渐变大。结果显示这一组基因与周期调控有关。
3．家蚕18个丝腺特异表达的基因的RT-PCR表明：在丝腺细胞系BmSG-SWU1中有部分丝腺特异表达基因表达，由此推测它仍然保留了丝腺组织细胞的部分特征。在18个基因中，有12个基因全部表达或都不表达；6个基因有较为明显的组织、细胞差异，其中有2个基因只在丝腺组织中有大量表达，而有3个基因在丝腺组织和丝腺细胞系BmSG-SWU1中的表达量要比精巢、卵巢高，但在BmN-SWU1中几乎不表达，另外有1个基因虽然在各个组织细胞中有表达差异，但差异不不大。
4．Fib-L和P25在不同组织细胞中的荧光定量PCR结果表明BmSG-SWU1细胞已经丧失了大量表达Fib-L和P25mRNA的能力。丝腺细胞系BmSG-SWU1中Fib-L和P25的表达量仅分别是丝腺组织的0.0013％和0.0048％，卵巢细胞系BmN-SWU1中Fib-L和P25的表达量分别是丝腺组织的0.0016％和0.0055％。两个细胞系表达这两个基因的水平基本一致，均远远低于丝腺组织的表达量。
5．预测的家蚕周期调控基因在家蚕不同组织和细胞系的RT-PCR结果表明：Bmcyclin D2、Bmcyclin EI、BmCyclin A1、BmCyclinx(含有Cyclin box的未知周期蛋白基因)等四个周期蛋白基因在卵巢、丝腺、BmSG-SWU1、BmN-SWU1中都有较强的表达，而BmCyclinB和BmCyclinB3在丝腺组织中不表达。BmPCNA在四个组织、细胞内都有表达。这些结果暗示BmSG-SWU1细胞系重新启动了周期调控基因，以有丝分裂的方式进行细胞增值，而丝腺组织则是核内有丝分裂。
家蚕丝腺细胞系BmSG-SWU1经过长期的体外培养，已经从核内有丝分裂状态突破G2／M关键控制点重新进入了细胞有丝分裂周期，该细胞系虽失去了高量表达丝素基因Fib-L、P25的能力，但仍旧保留着表达原丝腺组织部分特异基因的能力，是一个保留了丝腺组织细胞的部分特征的细胞系。
The silk gland of silkworm is not only the foundation of silk industry, but also provides us its high effective protein-synthesis function which can be developed to be a new biological industry, so it is honored for its broad applification in research field. A silk gland cell line is essential for us to perform series of reseaches such as the functional investigation of key Fibroin synthesizing genes, the investigation of mechanism about Fibroin synthesizing, and the investigation of silk gland as a biological reactor. However, there is not any cell line whose expression profile is very clear, which has become the bottleneck of those studies. This experiment investigate the genic expression profile of cell line BmSG-SWU1, silk gland specific genes and genes of cell spliting cyclin through whole genome microarray, normal RT-PCR, and fluorescent quantitative PCR (FQ-PCR) analysis, we obtain some results as follows:
1. To analysis the expression profile of eighteen genes which are expressed special highly in silk gland, we utilized the whole genome microarray data for silk glands and other tissues of male and female silkworms in day 3 of fifth instar, and the results showed that the expression quantity of 18 genes in silk gland was outclass compared with other tissues. And these eighteen genes contains Hormone-metabolism-related protein, Transporter, Protein-modified degradation phosphorylase, Dehydrogenase and other kinds of proteins, which maybe closely relate to the secretion of Fibroin.
2. According to the microarray data for silk glands, we analysed sixteen genes relating to cell cycle controlling, which contained CyclinA, CyclinB, CyclinD, CyclinE, CyclinH, PCNA and Cyclin-dependent kinase cdc2, Cdk2, Cdk5, Cdk6. These genes will be highly expressed mostly in spermary and ovary which have ability to split and multiply in silkworm, but will be lowly expressed in silk gland which has been highly differentiated. The course from female-pupa to moth, is also the exuberant phase of miosis in ovary, meanwhile the expression quantity of these genes gradually largen. So the result shows that these genes correlated with cycle controlling.
3. An RT-PCR analyse was performed to analysis eighteen silk-gland-specific genes, and it showed that in the silkgland celline BmSG-SWUl,severeal silk-gland-specific genes were detected, it reveal that BmSG-SWU1 may preserve some characteristics of silk gland cell. Among the eighteen genes, twelve of them were all expressed or none were expressed; six genes showed a different expression profile between tissue and cell, two of them were highly expressed only in silk gland tissue, three of them had higher expression quantity in silk gland and BmSG-SWU1 than those in spermary and ovary, however, they were hardly expressed in BmN-SWU1, and another one was Bmb001492, whose expressions were ubiquitous among the tested tissues.
4.The result of FQ-PCR of Fib-L and P25 in different ceils of tissue suggested that, the ceils of BmSG-SWU1 had lost the ability to highly express the Fibroin gene Fib-L and P25. The expression quantity of Fib-L and P25 in silk gland cell line BmSG-SWU1 was only about 0.0013% and 0.0048% respectively compared with that of silk gland; meanwhile that quantity in the ovary cell line BmN-SWU1 was only 0.0016% and 0.0055% respectively. And these suggest that, the expression quantity of these two genes in these two cell lines are nearly consistent, which are far below that of silk gland.
5. Then result of RT-PCR about the forecasted silkworm cycle-controlling genes in different tissues and cell lines showed that, the four cyclin genes BmCyclinD2、BmcyclinEI、BmCyclinAl BmCyclinAl had high expression in ovary, silk gland, BmSG-SWU1 and BmN-SWU1, while BmCyclinB and BmCyclinB3 were not expressed in silk gland. Meanwhile, BmPCNA was expressed in these four cDNAs. All the results above suggest that the cell line BmSG-SWU1 has restarted the cycle-controlling genes, and it performs multiplication with mitosis while that of silk gland tissue is endoreduplication.
In summary, the cell line of silkworm BmSG-SWU1 passed through long-term cultivated in vitro, they have breakthrough the key point in G2/M from endoreduplication, and returned to a mitostic cycle. Meanwhile, this cell line has lost the ability to highly express the Fibroin gene Fib-L、P25, but has reserved the function of expressing some special genes in former silk gland, and it is a cell line which preserves some characteristics of cells in silk gland.
1．利用家蚕全基因组芯片对5龄3天雌、雄家蚕的丝腺和其它组织进行了18个丝腺特异表达基因的表达谱分析，芯片数据分析结果显示这些基因在5龄3天的家蚕丝腺组织中的表达量远高于在其它组织中的表达量。这18个基因中包括激素代谢相关蛋白、转运蛋白、蛋白修饰降解类磷酸化酶、脱氢酶及其它几种蛋白，它们可能与丝物质的分泌密切相关。
2．根据家蚕基因组芯片数据，分析了家蚕中与细胞周期调控相关的16个基因，包括周期蛋白基因Cyclin A、Cyclin B、Cyclin D、Cyclin E、Cyclin H及PCNA，周期蛋白依赖性激酶基因cdc2、Cdk2、Cdk5、Cdk6。这些基因大多在家蚕具有分裂增殖能力的幼虫精巢、卵巢中高量表达，而在处于高度分化状态的丝腺组织中低量表达。雌蛹到蛾的变态过程，也是卵巢减数分裂的旺盛期，这些基因的表达量逐渐变大。结果显示这一组基因与周期调控有关。
3．家蚕18个丝腺特异表达的基因的RT-PCR表明：在丝腺细胞系BmSG-SWU1中有部分丝腺特异表达基因表达，由此推测它仍然保留了丝腺组织细胞的部分特征。在18个基因中，有12个基因全部表达或都不表达；6个基因有较为明显的组织、细胞差异，其中有2个基因只在丝腺组织中有大量表达，而有3个基因在丝腺组织和丝腺细胞系BmSG-SWU1中的表达量要比精巢、卵巢高，但在BmN-SWU1中几乎不表达，另外有1个基因虽然在各个组织细胞中有表达差异，但差异不不大。
4．Fib-L和P25在不同组织细胞中的荧光定量PCR结果表明BmSG-SWU1细胞已经丧失了大量表达Fib-L和P25mRNA的能力。丝腺细胞系BmSG-SWU1中Fib-L和P25的表达量仅分别是丝腺组织的0.0013％和0.0048％，卵巢细胞系BmN-SWU1中Fib-L和P25的表达量分别是丝腺组织的0.0016％和0.0055％。两个细胞系表达这两个基因的水平基本一致，均远远低于丝腺组织的表达量。
5．预测的家蚕周期调控基因在家蚕不同组织和细胞系的RT-PCR结果表明：Bmcyclin D2、Bmcyclin EI、BmCyclin A1、BmCyclinx(含有Cyclin box的未知周期蛋白基因)等四个周期蛋白基因在卵巢、丝腺、BmSG-SWU1、BmN-SWU1中都有较强的表达，而BmCyclinB和BmCyclinB3在丝腺组织中不表达。BmPCNA在四个组织、细胞内都有表达。这些结果暗示BmSG-SWU1细胞系重新启动了周期调控基因，以有丝分裂的方式进行细胞增值，而丝腺组织则是核内有丝分裂。
家蚕丝腺细胞系BmSG-SWU1经过长期的体外培养，已经从核内有丝分裂状态突破G2／M关键控制点重新进入了细胞有丝分裂周期，该细胞系虽失去了高量表达丝素基因Fib-L、P25的能力，但仍旧保留着表达原丝腺组织部分特异基因的能力，是一个保留了丝腺组织细胞的部分特征的细胞系。
The silk gland of silkworm is not only the foundation of silk industry, but also provides us its high effective protein-synthesis function which can be developed to be a new biological industry, so it is honored for its broad applification in research field. A silk gland cell line is essential for us to perform series of reseaches such as the functional investigation of key Fibroin synthesizing genes, the investigation of mechanism about Fibroin synthesizing, and the investigation of silk gland as a biological reactor. However, there is not any cell line whose expression profile is very clear, which has become the bottleneck of those studies. This experiment investigate the genic expression profile of cell line BmSG-SWU1, silk gland specific genes and genes of cell spliting cyclin through whole genome microarray, normal RT-PCR, and fluorescent quantitative PCR (FQ-PCR) analysis, we obtain some results as follows:
1. To analysis the expression profile of eighteen genes which are expressed special highly in silk gland, we utilized the whole genome microarray data for silk glands and other tissues of male and female silkworms in day 3 of fifth instar, and the results showed that the expression quantity of 18 genes in silk gland was outclass compared with other tissues. And these eighteen genes contains Hormone-metabolism-related protein, Transporter, Protein-modified degradation phosphorylase, Dehydrogenase and other kinds of proteins, which maybe closely relate to the secretion of Fibroin.
2. According to the microarray data for silk glands, we analysed sixteen genes relating to cell cycle controlling, which contained CyclinA, CyclinB, CyclinD, CyclinE, CyclinH, PCNA and Cyclin-dependent kinase cdc2, Cdk2, Cdk5, Cdk6. These genes will be highly expressed mostly in spermary and ovary which have ability to split and multiply in silkworm, but will be lowly expressed in silk gland which has been highly differentiated. The course from female-pupa to moth, is also the exuberant phase of miosis in ovary, meanwhile the expression quantity of these genes gradually largen. So the result shows that these genes correlated with cycle controlling.
3. An RT-PCR analyse was performed to analysis eighteen silk-gland-specific genes, and it showed that in the silkgland celline BmSG-SWUl,severeal silk-gland-specific genes were detected, it reveal that BmSG-SWU1 may preserve some characteristics of silk gland cell. Among the eighteen genes, twelve of them were all expressed or none were expressed; six genes showed a different expression profile between tissue and cell, two of them were highly expressed only in silk gland tissue, three of them had higher expression quantity in silk gland and BmSG-SWU1 than those in spermary and ovary, however, they were hardly expressed in BmN-SWU1, and another one was Bmb001492, whose expressions were ubiquitous among the tested tissues.
4.The result of FQ-PCR of Fib-L and P25 in different ceils of tissue suggested that, the ceils of BmSG-SWU1 had lost the ability to highly express the Fibroin gene Fib-L and P25. The expression quantity of Fib-L and P25 in silk gland cell line BmSG-SWU1 was only about 0.0013% and 0.0048% respectively compared with that of silk gland; meanwhile that quantity in the ovary cell line BmN-SWU1 was only 0.0016% and 0.0055% respectively. And these suggest that, the expression quantity of these two genes in these two cell lines are nearly consistent, which are far below that of silk gland.
5. Then result of RT-PCR about the forecasted silkworm cycle-controlling genes in different tissues and cell lines showed that, the four cyclin genes BmCyclinD2、BmcyclinEI、BmCyclinAl BmCyclinAl had high expression in ovary, silk gland, BmSG-SWU1 and BmN-SWU1, while BmCyclinB and BmCyclinB3 were not expressed in silk gland. Meanwhile, BmPCNA was expressed in these four cDNAs. All the results above suggest that the cell line BmSG-SWU1 has restarted the cycle-controlling genes, and it performs multiplication with mitosis while that of silk gland tissue is endoreduplication.
In summary, the cell line of silkworm BmSG-SWU1 passed through long-term cultivated in vitro, they have breakthrough the key point in G2/M from endoreduplication, and returned to a mitostic cycle. Meanwhile, this cell line has lost the ability to highly express the Fibroin gene Fib-L、P25, but has reserved the function of expressing some special genes in former silk gland, and it is a cell line which preserves some characteristics of cells in silk gland.
引文
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