家蚕感染BmCPV相关未知功能蛋白LOC101742613的基因克隆及表征特征分析
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摘要
在感染家蚕质型多角体病毒(Bombyx mori cytoplasmic polyhedrovirus,BmCPV)的家蚕中肠组织中发现一个表达量明显下调的未知蛋白LOC101742613,与棉铃虫(Helicoverpa armigera)的未知蛋白LOC110378285的序列一致性为51%。利用PCR技术克隆获得了编码该蛋白质的cDNA,其序列全长为1 114 bp,最大开放阅读框(ORF)为993 bp,编码的蛋白质由330个氨基酸残基组成,预测蛋白质分子质量为36.6 kD,等电点为4.31。实时荧光定量PCR分析发现目的基因主要在家蚕的脂肪体、精巢、卵巢和头部组织中表达,且家蚕中肠感染BmCPV后48 h和72 h其mRNA的表达量显著下调,分别为对照组的7.3%和2.1%。通过构建重组表达载体pET28a/BGIBMGA014203对去掉信号肽的目的蛋白进行原核表达,表达产物经SDS-PAGE电泳分析发现重组蛋白主要在包涵体中,分子质量约为37 kD。研究结果为进一步阐明目的基因的生物学功能奠定了基础。
A down-regulated uncharacterized protein LOC101742613 was found in Bm CPV-infected Bombyx mori midgut.This protein had 51% identity with Helicoverpa armigera uncharacterized protein LOC110378285. A 1 114 bp cDNA encoding LOC101742613 was cloned by PCR. It had the longest open reading frame( ORF) of 993 bp and encoded a protein of 330 amino acids with predicted molecular weight of 36. 6 k D and theoretical isoelectric point of 4. 31. Fluorescent quantitative real-time PCR showed that the mRNA transcripts of target gene were mainly expressed in fat body,testicle,ovary and head. At 48 h and 72 h post-inoculation of Bm CPV,the relative expression levels of this gene in Bm CPV-infected midgut were significantly down-regulated, which were7. 3% and 2. 1% of that in control group. Through construction of recombinant expression vector pET28 a/BGIBMGA014203, the target protein without signal peptide sequence was expressed in E. coli. SDS-PAGE result showed that the recombinant protein with a molecular weight of 37 k D was mainly expressed in inclusion body. These results provided a basis for future study on its biological function.
A down-regulated uncharacterized protein LOC101742613 was found in Bm CPV-infected Bombyx mori midgut.This protein had 51% identity with Helicoverpa armigera uncharacterized protein LOC110378285. A 1 114 bp cDNA encoding LOC101742613 was cloned by PCR. It had the longest open reading frame( ORF) of 993 bp and encoded a protein of 330 amino acids with predicted molecular weight of 36. 6 k D and theoretical isoelectric point of 4. 31. Fluorescent quantitative real-time PCR showed that the mRNA transcripts of target gene were mainly expressed in fat body,testicle,ovary and head. At 48 h and 72 h post-inoculation of Bm CPV,the relative expression levels of this gene in Bm CPV-infected midgut were significantly down-regulated, which were7. 3% and 2. 1% of that in control group. Through construction of recombinant expression vector pET28 a/BGIBMGA014203, the target protein without signal peptide sequence was expressed in E. coli. SDS-PAGE result showed that the recombinant protein with a molecular weight of 37 k D was mainly expressed in inclusion body. These results provided a basis for future study on its biological function.
引文
[1] 向仲怀.家蚕遗传育种学[M].北京:中国农业出版社,1994:34-52
[2] 吕鸿声.昆虫病毒分子生物学[M].北京:中国农业科学技术出版社,1998:421-423
[3] 徐安英,李木旺,林昌麒,等.家蚕品种资源对质型多角体病毒抵抗性的初步比较试验[J].蚕业科学,2002,28(2):157-159
[4] 张志芳.家蚕对质型多角体病毒病抵抗性的遗传和育种方法研究:Ⅰ.家蚕对质型多角体病毒病抵抗性的遗传规律初探[J].蚕业科学,1986,12(4):211-214
[5] 邵汝莉.家蚕对CPV抗病性和茧质的选择效应[J].蚕业科学,1981,7(3):155-161
[6] GAO K,DENG X Y,QIAN H Y,et al.Digital gene expression ana-lysis in the midgut of 4008 silkworm strain infected with cytoplas-mic polyhedrosis virus[J].J Invertebr Pathol,2014,115(1):8-13
[7] GAO K,DENG X Y,QIAN H Y,et al.Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility[J].Gene,2014,539(2):230-237
[8] 高坤,邓祥元,裘智勇,等.家蚕感染质型多角体病毒(BmCPV)后中肠组织差异蛋白质分析[J].中国农业科学,2013,46(13):2796-2807
[9] GAO K,DENG X Y,SHANG M K,et al.iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus[J].J Proteomics,2017,152:300-311
[10] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408
[11] 尚梦珂,高坤,郭锡杰,等.家蚕蜕皮激素22激酶2(BmEcK2)的序列及基因表达特征分析[J].蚕业科学,2016,42(6):997-1003
[12] FUJII-KAWATA I,MIURA K I,FUKE M.Segments of genome of viruses containing double-stranded ribonucleic acid[J].J Mol Biol,1970,51(2):247-253
[13] RUBINSTEIN R,HARLEY E H,LOSMAN M,et al.The nucleic acids of viruses infecting Heliothis armigera[J].Virology,1976,69(1):323-326
[14] YUE Y J,TANG X D,XU L,et al.Early responses of silkworm midgut to microsporidium infection:a digital gene expression analysis[J].J Invertebr Pathol,2015,124:6-14
[15] MAGNOLER A.Effects of a cytoplasmic polyhedrosis on larval and postlarval stages of the gypsy moth,Porthetria dispar[J].J Invertebr Pathol,1974,23(3):263
[16] ZHANG L,WANG Y W,LU Z Q.Midgut immune responses induced by bacterial infection in the silkworm,Bombyx mori[J].J Zhejiang Univ Sci,B,2015,16(10):875-882
[17] 孙京臣,陈冬妮,杨艺峰,等.质型多角体病毒在家蚕体内入侵与复制研究[J].中山大学学报(自然科学版),2006,45(2):78-82
[18] OU J,DENG H M,ZHENG S C,et al.Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis[J/OL].BMC Genomics,2014,15:820[2018-05-15].https://www.ncbi.nlm.nih.gov/pubmed/25261999.DOI:10.1186/1471-2164-15-820
[2] 吕鸿声.昆虫病毒分子生物学[M].北京:中国农业科学技术出版社,1998:421-423
[3] 徐安英,李木旺,林昌麒,等.家蚕品种资源对质型多角体病毒抵抗性的初步比较试验[J].蚕业科学,2002,28(2):157-159
[4] 张志芳.家蚕对质型多角体病毒病抵抗性的遗传和育种方法研究:Ⅰ.家蚕对质型多角体病毒病抵抗性的遗传规律初探[J].蚕业科学,1986,12(4):211-214
[5] 邵汝莉.家蚕对CPV抗病性和茧质的选择效应[J].蚕业科学,1981,7(3):155-161
[6] GAO K,DENG X Y,QIAN H Y,et al.Digital gene expression ana-lysis in the midgut of 4008 silkworm strain infected with cytoplas-mic polyhedrosis virus[J].J Invertebr Pathol,2014,115(1):8-13
[7] GAO K,DENG X Y,QIAN H Y,et al.Cytoplasmic polyhedrosis virus-induced differential gene expression in two silkworm strains of different susceptibility[J].Gene,2014,539(2):230-237
[8] 高坤,邓祥元,裘智勇,等.家蚕感染质型多角体病毒(BmCPV)后中肠组织差异蛋白质分析[J].中国农业科学,2013,46(13):2796-2807
[9] GAO K,DENG X Y,SHANG M K,et al.iTRAQ-based quantitative proteomic analysis of midgut in silkworm infected with Bombyx mori cytoplasmic polyhedrosis virus[J].J Proteomics,2017,152:300-311
[10] LIVAK K J,SCHMITTGEN T D.Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method[J].Methods,2001,25(4):402-408
[11] 尚梦珂,高坤,郭锡杰,等.家蚕蜕皮激素22激酶2(BmEcK2)的序列及基因表达特征分析[J].蚕业科学,2016,42(6):997-1003
[12] FUJII-KAWATA I,MIURA K I,FUKE M.Segments of genome of viruses containing double-stranded ribonucleic acid[J].J Mol Biol,1970,51(2):247-253
[13] RUBINSTEIN R,HARLEY E H,LOSMAN M,et al.The nucleic acids of viruses infecting Heliothis armigera[J].Virology,1976,69(1):323-326
[14] YUE Y J,TANG X D,XU L,et al.Early responses of silkworm midgut to microsporidium infection:a digital gene expression analysis[J].J Invertebr Pathol,2015,124:6-14
[15] MAGNOLER A.Effects of a cytoplasmic polyhedrosis on larval and postlarval stages of the gypsy moth,Porthetria dispar[J].J Invertebr Pathol,1974,23(3):263
[16] ZHANG L,WANG Y W,LU Z Q.Midgut immune responses induced by bacterial infection in the silkworm,Bombyx mori[J].J Zhejiang Univ Sci,B,2015,16(10):875-882
[17] 孙京臣,陈冬妮,杨艺峰,等.质型多角体病毒在家蚕体内入侵与复制研究[J].中山大学学报(自然科学版),2006,45(2):78-82
[18] OU J,DENG H M,ZHENG S C,et al.Transcriptomic analysis of developmental features of Bombyx mori wing disc during metamorphosis[J/OL].BMC Genomics,2014,15:820[2018-05-15].https://www.ncbi.nlm.nih.gov/pubmed/25261999.DOI:10.1186/1471-2164-15-820