家蚕BmPTERP和Bm3HCDH基因全长cDNA的克隆与表达特征分析
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摘要
家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrosis virus, BmCPV)是家蚕致病的重要病原之一,往往给蚕业生产造成极大危害和经济损失。以寻找家蚕感染质型多角体病毒相关应答基因为前提,正确理解病毒感染致病的分子机制对家蚕质型多角体病的防御及蚕药的研发具有重要的意义。
我们运用基因芯片技术在感染质型多角体病毒的家蚕中肠中鉴定出大量的差异表达基因。根据基因芯片库中基因的表达序列标签(EST),结合利用生物信息学技术和cDNA末端快速扩增(RACE)技术,从家蚕P50品种的中肠组织克隆获得了家蚕2个基因的全长cDNA序列,对所获得的序列进行生物信息学分析。主要研究结论如下:
一、家蚕磷酸三酯酶相关蛋白(BmPTERP)基因的克隆和表达特性分析
家蚕磷酸三酯酶相关蛋白(BmPTERP)基因全长cDNA序列为1349bp,其中包括131 bp的5’非翻译区(5’UTR)和168bp的3’端非翻译区(3’UTR)。ORF从cDNA的132位起,在1181位终止,编码350个氨基酸,蛋白的分子量为39.03KDa,等电点为5.72。基因结构分析发现该基因由6个外显子和5个内含子组成,该基因属于金属相关的水解酶家族,包含磷酸三酯酶(PTE)特殊区域。利用RT-PCR对生殖体、中肠、血液、脂肪体及丝腺表达量分析,其结果显示该基因在家蚕各组织中均有表达;荧光定量PCR结果表明该基因在BmCPV感染蚕中肠后的表达水平明显降低,正常蚕中肠组织中该基因的表达水平是感染蚕中肠中表达水平的9.8倍。
二、家蚕3 -羟酰辅酶A脱氢酶蛋白(Bm3HCDH)基因的克隆和表达特性分析
家蚕3-羟酰辅酶A脱氢酶蛋白(Bm3HCDH)基因全长cDNA序列为1168bp,包含83 bp的5’端非编码区(5’UTR)和155 bp的3’端非编码区(3’UTR),开放阅读框(ORF)为930 bp,编码310个氨基酸,蛋白分子量为33.6 kD,等电点为9.0;基因结构分析发现该基因由5个外显子和4个内含子组成。利用RT-PCR对生殖体、中肠、血液、脂肪体及丝腺表达量分析,其结果显示该基因在家蚕各组织中均有表达;荧光定量PCR结果表明在BmCPV感染蚕中肠后的表达水平明显降低,在正常蚕中肠组织中的表达水平为感染BmCPV中肠组织的8.2倍。
根据BmPTERP和Bm3HCDH的实验结果推测,BmPTERP和Bm3HCDH可能与BmCPV感染家蚕的病理过程有关。研究结果为从分子水平上阐明BmCPV对家蚕致病分子机制提供新的理论依据,为进一步研究BmCPV感染与其相关基因的的功能和关系奠定了基础。
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), one of the important viral pathogens for the silkworm disease, causes harm to product of silkworm and enormous damages to the sericultural industry. Studies on genes related to silkworm infected with BmCPV and understanding of silkworm molecular mechanism of BmCPV infection is crucial to the prevention of BmCPV-caused silkworm disease and the development of new medicine of silkworm.
In our previous work, large number of differentially expressed genes were identified in the midgut of infected BmCPV by using microarry analysis. Based on the EST information of this microarry analysis, the cDNA of two genes were cloned using the strategy of rapid amplification of cDNA ends. Concurrently bioinformatic methods were applied to analyze the obtained sequences of these genes. The conclusion are as follows:
1. Molecular cloning and characterization analysis of phosphotriesterase-related protein gene of silkworm,Bombyx mori (BmPTERP).
BmPTERP consists of 1349 bp and contains a 131 bp 5’UTR and a 168-bp 3’UTR. The open reading frame which locates in the region from 132 bp to 1181 bp of cDNA encodes a 350 amino acid protein. The molecular weight of the putative BmPTERP was 39.03-KDa and isoelectric point 5.72. The result by GSDS (Gene structure display server) showed that the gene sequence of BmPTERP contains six exons and five introns. BmPTERP belongs to the superfamily of metallo-dependent hydrolases and contains specific hits of PTE. RT-PCR analysis revealed that BmPTERP was expressed in all the tissues tested, including gonad, midgut, hemocyte, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of BmPTERP in the normal midgut was approximately as 9.8 fold of that in the BmCPV-infected midgut.
2 Molecular cloning and characterization analysis of 3-hydroxyacyl-coa dehydrogenase protein gene of silkworm, Bombyx mori (Bm3HCDH).
Bm3HCDH consists of 1168 bp and contains a 83 bp 5’UTR and a 155 bp 3’UTR. A 930 bp open reading frame (ORF) encodes a 310 amino acid protein. The molecular weight of the putative HCDHBm was 33.6-KDa and isoelectric point 9.0. The result by GSDS (Gene structure display server) showed that the gene sequence of HCDHBm contains five exons and four introns. RT-PCR analysis revealed that Bm3HCDH was expressed in all the tissues tested, including gonad, midgut, hemocyte, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of Bm3HCDH in the normal midgut was 8.2 fold of that in the infected midgut.
According to our research results of BmPTERP and Bm3HCDH, it is presumed that BmPTERP and Bm3HCDH might be involved in infection of silkworm with Bombyx mori cytoplasmic polyhedrosis virus. It provided not only new clues for investigating the molecular mechanism of BmCPV infection but also the theoretical basis for further study on the function of BmPTERP and Bm3HCDH of silkworm.
我们运用基因芯片技术在感染质型多角体病毒的家蚕中肠中鉴定出大量的差异表达基因。根据基因芯片库中基因的表达序列标签(EST),结合利用生物信息学技术和cDNA末端快速扩增(RACE)技术,从家蚕P50品种的中肠组织克隆获得了家蚕2个基因的全长cDNA序列,对所获得的序列进行生物信息学分析。主要研究结论如下:
一、家蚕磷酸三酯酶相关蛋白(BmPTERP)基因的克隆和表达特性分析
家蚕磷酸三酯酶相关蛋白(BmPTERP)基因全长cDNA序列为1349bp,其中包括131 bp的5’非翻译区(5’UTR)和168bp的3’端非翻译区(3’UTR)。ORF从cDNA的132位起,在1181位终止,编码350个氨基酸,蛋白的分子量为39.03KDa,等电点为5.72。基因结构分析发现该基因由6个外显子和5个内含子组成,该基因属于金属相关的水解酶家族,包含磷酸三酯酶(PTE)特殊区域。利用RT-PCR对生殖体、中肠、血液、脂肪体及丝腺表达量分析,其结果显示该基因在家蚕各组织中均有表达;荧光定量PCR结果表明该基因在BmCPV感染蚕中肠后的表达水平明显降低,正常蚕中肠组织中该基因的表达水平是感染蚕中肠中表达水平的9.8倍。
二、家蚕3 -羟酰辅酶A脱氢酶蛋白(Bm3HCDH)基因的克隆和表达特性分析
家蚕3-羟酰辅酶A脱氢酶蛋白(Bm3HCDH)基因全长cDNA序列为1168bp,包含83 bp的5’端非编码区(5’UTR)和155 bp的3’端非编码区(3’UTR),开放阅读框(ORF)为930 bp,编码310个氨基酸,蛋白分子量为33.6 kD,等电点为9.0;基因结构分析发现该基因由5个外显子和4个内含子组成。利用RT-PCR对生殖体、中肠、血液、脂肪体及丝腺表达量分析,其结果显示该基因在家蚕各组织中均有表达;荧光定量PCR结果表明在BmCPV感染蚕中肠后的表达水平明显降低,在正常蚕中肠组织中的表达水平为感染BmCPV中肠组织的8.2倍。
根据BmPTERP和Bm3HCDH的实验结果推测,BmPTERP和Bm3HCDH可能与BmCPV感染家蚕的病理过程有关。研究结果为从分子水平上阐明BmCPV对家蚕致病分子机制提供新的理论依据,为进一步研究BmCPV感染与其相关基因的的功能和关系奠定了基础。
Bombyx mori cytoplasmic polyhedrosis virus (BmCPV), one of the important viral pathogens for the silkworm disease, causes harm to product of silkworm and enormous damages to the sericultural industry. Studies on genes related to silkworm infected with BmCPV and understanding of silkworm molecular mechanism of BmCPV infection is crucial to the prevention of BmCPV-caused silkworm disease and the development of new medicine of silkworm.
In our previous work, large number of differentially expressed genes were identified in the midgut of infected BmCPV by using microarry analysis. Based on the EST information of this microarry analysis, the cDNA of two genes were cloned using the strategy of rapid amplification of cDNA ends. Concurrently bioinformatic methods were applied to analyze the obtained sequences of these genes. The conclusion are as follows:
1. Molecular cloning and characterization analysis of phosphotriesterase-related protein gene of silkworm,Bombyx mori (BmPTERP).
BmPTERP consists of 1349 bp and contains a 131 bp 5’UTR and a 168-bp 3’UTR. The open reading frame which locates in the region from 132 bp to 1181 bp of cDNA encodes a 350 amino acid protein. The molecular weight of the putative BmPTERP was 39.03-KDa and isoelectric point 5.72. The result by GSDS (Gene structure display server) showed that the gene sequence of BmPTERP contains six exons and five introns. BmPTERP belongs to the superfamily of metallo-dependent hydrolases and contains specific hits of PTE. RT-PCR analysis revealed that BmPTERP was expressed in all the tissues tested, including gonad, midgut, hemocyte, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of BmPTERP in the normal midgut was approximately as 9.8 fold of that in the BmCPV-infected midgut.
2 Molecular cloning and characterization analysis of 3-hydroxyacyl-coa dehydrogenase protein gene of silkworm, Bombyx mori (Bm3HCDH).
Bm3HCDH consists of 1168 bp and contains a 83 bp 5’UTR and a 155 bp 3’UTR. A 930 bp open reading frame (ORF) encodes a 310 amino acid protein. The molecular weight of the putative HCDHBm was 33.6-KDa and isoelectric point 9.0. The result by GSDS (Gene structure display server) showed that the gene sequence of HCDHBm contains five exons and four introns. RT-PCR analysis revealed that Bm3HCDH was expressed in all the tissues tested, including gonad, midgut, hemocyte, fat body and silk gland. Real-time quantitative polymerase chain reaction analysis indicated that the relative transcript of Bm3HCDH in the normal midgut was 8.2 fold of that in the infected midgut.
According to our research results of BmPTERP and Bm3HCDH, it is presumed that BmPTERP and Bm3HCDH might be involved in infection of silkworm with Bombyx mori cytoplasmic polyhedrosis virus. It provided not only new clues for investigating the molecular mechanism of BmCPV infection but also the theoretical basis for further study on the function of BmPTERP and Bm3HCDH of silkworm.
引文
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