家蚕基质金属蛋白酶家族MMPs及组织金属蛋白酶抑制剂TIMP在家蚕中的功能研究
发布时间：2018-08-29 19:17
【摘要】：昆虫在长期的进化过程中对多变的环境具备了高度的适应能力,这与其神奇的变态发育过程和高效的先天性免疫体系密不可分。加强昆虫变态发育及免疫机制的研究,对拓展昆虫资源的利用和害虫的绿色防控等有重要的现实意义。同时,可为研究人类干细胞分化、组织器官的形成及免疫机制提供重要线索。基质金属蛋白酶家族(Matrix metalloproteinase family,MMPs)是参与昆虫变态发育调节和先天性免疫应答的一类重要的锌依赖性内肽酶。该家族成员几乎能够降解所有种类的细胞外基质,在生物体多种生理和病理过程中起重要作用,如组织重塑、器官发育、炎症调节、免疫应答、细胞凋亡、创伤修复及肿瘤的侵袭和转移等。组织金属蛋白酶抑制剂(Tissue inhibitor of metalloproteinases,TIMPs)是MMPs内源性的蛋白抑制因子,能够结合MMPs并抑制其降解活性,在维持细胞外基质稳态中起重要作用。然而,对果蝇以外其它昆虫的MMPs和TIMPs功能研究较少。同时,在不同昆虫中MMPs和TIMPs的数目和功能也存在极大差异。因此,全面、深入解析这两个家族在昆虫中的作用具有重要意义。鉴于此,本研究以家蚕(Bombyx mori)为模式,研究了家蚕BmMMPs家族和BmTIMP表达特征;在体内和体外利用基因过表达和CRISPR/Cas9基因敲除技术,探究了BmMMPs家族和BmTIMP在家蚕抵抗家蚕核型多角体病毒(Bombyx mori nuclearpolyhedrovirus,BmNPV)和家蚕变态发育中的作用;通过对BmMMPs家族的转录调控研究及互作蛋白的鉴定,阐明BmMMPs家族作用的分子机制。通过该研究,可为揭示BmMMPs和BmTIMP在家蚕变态发育及先天性免疫中的作用提供实验依据和理论支撑。论文的主要实验结果和结论如下:1.BmMMPs和Bm TIMP基因的鉴定及表达特征分析本研究在家蚕基因组中鉴定出3个mmps家族基因,其中一个基因包含两种剪切体,分别命名为bmmmp1a、bmmmp1b、bmmmp2和bmmmp3。同时鉴定到1个timp基因,命名为bmtimp。通过序列分析发现,家蚕mmp家族基因均包含mmp家族典型的结构特征,前肽区(pro-peptide)、催化结构域(catalyticdomain)、铰链区(hingeregion)及血红素蛋白类似区域(hemopexin-likec-terminaldomain)。系统发生分析发现家蚕mmp家族聚类于昆虫mmps家族一支。bmtimp包含timp家族典型的ntr结构域,且系统发生分析显示其同样与昆虫timp家族聚为一支。家蚕各发育时期的表达特征分析发现,bmmmps和bmtimp在化蛹第一天有高量表达;在五龄三天,bmmmp1、bmmmp3和bmtimp在中肠和脂肪体中高量表达,bmmmp2在血液有高量表达。通过免疫荧光和蛋白截短实验对家蚕mmps家族和timp的亚细胞定位模式进行分析发现,bmmmp1ab和bmmmp2的全蛋白及预测跨膜区域的截短蛋白均定位于细胞质中;bmmmp3的全蛋白定位于细胞质中,预测的核定位信号区域定位于细胞核中,bmmmp3催化结构域的n端影响了其核定位信号正常行使功能;bmtimp具有信号肽区域,其蛋白部分定位于细胞质中,部分分泌到细胞外。探究了家蚕mmps家族基因和timp在不同抗原刺激下的表达模式,结果显示,在bmnpv病毒感染家蚕体外细胞系和体内中肠、血液、脂肪体的过程中,bmmmps和bmtimp在bmnpv病毒感染前期均出现显著上调表达,随后表达量下调,在感染后期出现极高量表达;在利用lps和大肠杆菌刺激家蚕体外细胞系或体内中肠、血液、脂肪体的过程中,bmmmps和bmtimp均有显著诱导上调表达趋势。上述结果表明,bmmmps和bmtimp在表达模式上具有一定的协同性;bmmmps和bmtimp可能参与调控家蚕的变态发育及先天性免疫。2.bmmmps和bmtimp的相互作用鉴定本部分通过双荧光共定位、荧光双分子互补实验(bifc)及免疫共沉淀实验(co-ip),分析了bmmmp1a、bmmmp2和bmmmp3三个蛋白与bmtimp蛋白之间的相互作用关系。双荧光共定位结果显示,bmmmps均能分别与bmtimp共定位于细胞质中;bifc结果显示,bmmmps都能与bmtimp发生相互作用,并且都聚集于细胞质中;co-ip实验结果显示,bmmmp1s分别与bmtimp之间同样存在特异性的相互作用。上述结果表明,家蚕mmps家族三个蛋白与timp都具有相互作用,推测bmtimp可能通过直接结合bmmmps抑制其活性。3.bmmmps和bmtimp对bmnpv侵染的影响本部分通过在体外家蚕细胞系bmns-swu1中分别创制bmmmps和bmtimp基因的过表达及crispr/cas9基因敲除模型,在体外水平探索bmmmps和bmtimp对bmnpv侵染的影响。结果显示,bmmmps基因过表达和bmtimp基因敲除可以显著增强bmnpv的增殖复制;bmmmps基因敲除和bmtimp基因过表达可以显著抑制bmnpv的增殖复制。同时,利用iv型胶原(collagen-iv)降解实验、mmps的广谱性抑制剂bb-94、gm6001和bmmmps蛋白活性区域截短实验,进一步探究了家蚕mmps活性对bmnpv病毒侵染的影响。结果显示,过表达bmmmp1a、bmmmp2和bmmmp3的截短活性区域均能够增强家蚕细胞对collagen-iv的降解;bb-94和gm6001能够显著抑制bmnpv的增殖复制;相对于bmmmps全蛋白,其截短活性区域能显著增强bmnpv的在家蚕细胞中的增殖复制。上述结果表明,bmmmps可能以活性形式参与调控bmnpv病毒的增殖复制。4.转基因过表达bmmmp3和bmtimp对bmnpv增殖及家蚕变态发育的影响利用显微注射技术创制了bmmmp3和bmtimp过表达家蚕品系,在体内水平探究bmmmp3和bmtimp对bmnpv增殖及家蚕变态发育的影响。通过对转基因品系的表型统计和bmnpv感染后的致死率和病毒关键基因分析,发现bmmmp3过表达品系相对于正常品系表现为体型增大、体重增加,且bmnpv在bmmmp3-oe品系中的感染能力被显著增强;bmtimp过表达品系具有发育迟缓、幼虫期全部致死及不能正常上簇等表型。这些结果表明,bmmmp3和bmtimp能够参与调控家蚕的生长发育;在体内过表达bmmmp3能够显著增强bmnpv的增殖复制。5.转基因敲除bmmmps家族基因对bmnpv增殖及家蚕变态发育的影响利用crispr/cas9的基因编辑技术和转基因技术相结合的方法,创制了单独过表达cas9蛋白的转基因家蚕品系cas9-oe,以及单独过表达sgrna靶标序列的转基因家蚕品系bmmmp1-sgrna、bmmmp2-sgrna和bmmmp3-sgrna共4种转基因品系。随后,利用cas9-oe与bmmmps-sgrna杂交敲除目的基因的方法,获得mmps基因特异性敲除的三个杂交敲除品系bmmmp1-ko、bmmmp2-ko和bmmm3-ko。通过对bmmmps-ko品系生长发育过程的表型统计分析,结果发现,bmmmp1-ko出现发育迟缓、幼虫和蛹期致死,影响气管的延伸和分枝。bmmmp2-ko具有发育迟缓、蛾期生存活力不高、不能正常交配,雌蛾下颚和侧胞保持凸出、被毛易脱落、腹部积水、卵发育异常及马氏管发育异常等多种表型,雄蛾除不具有侧胞保持凸出和卵发育异常外,其它与雌蛾表型一致。bmmmp3-ko品系具有发育迟缓、幼虫期致死、蛾期活力不高、雌蛾不能产卵等表型。同时,将bmnpv病毒利用口服和注射的方法感染家蚕bmmmps-ko杂交敲除品系和正常品系,结果发现,bmmmps-ko杂交敲除品系能够显著抑制vp39基因的转录。上述结果进一步表明,BmMMPs在家蚕的变态发育和调控BmNPV侵染中起重要作用。6.BmMMPs和BmTIMP作用机制的探究及相互作用蛋白的鉴定通过在家蚕细胞中单独过表达BmNPV增殖关键基因IE1、FGF、GP64和VP39,检测对BmMMPs和BmTIMP的转录水平的影响。结果发现,除VP39未检测到显著诱导外,IE1、FGF、GP64都对BmMMPs和BmTIMP具有一定的诱导活性。基于BmMMPs和BmTIMP在表达模式上具有很高的一致性,通过在家蚕细胞中单独过表达BmMMPs和BmTIMP各个基因,探究其是否存在相互调控关系,结果发现,BmMMPs和BmTIMP之间均具有相互诱导活性。同时发现在家蚕细胞中过表达BmMMPs可以显著诱导Caspase3、7和9的活性,敲除BmMMPs可以抑制Caspase3、7和9的活性。上述结果进一步暗示了,BmMMPs和BmTIMP参与家蚕抵御BmNPV侵染的调控;BmMMPs和Bm TIMP之间可能存在相互调控关系。通过免疫共沉淀的方法,并借助质谱分析技术,分别以BmMMP1a和BmMMP3为诱饵蛋白,从家蚕BmN-SWU1细胞中筛选并鉴定到6个与BmMMP1a互作的蛋白,6个与BmMMP3互作的蛋白。经初步验证发现,ATP synthase与BmMMP1a存在相互作用;PP6与BmMMP3存在相互作用。这些结果暗示了,BmMMPs可能与能量代谢息息相关,BmMMP3可能与PP6互作共同参与调控细胞生理活动。综上所述,BmMMPs和BmTIMP同时在家蚕抵御BmNPV病毒侵染的先天性免疫中和家蚕的变态发育过程中起着重要的作用,此研究不仅为全面解析家蚕BmMMPs和BmTIMP基因功能奠定了坚实的基础,同时也为家蚕先天性免疫及变态发育研究提供了新的线索。
[Abstract]:Insects have a high adaptability to the changeable environment in the long-term evolution process, which is closely related to their magical metamorphosis and efficient innate immune system.Strengthening the research on the metamorphosis and immune mechanism of insects has important practical significance for expanding the utilization of insect resources and green pest control. Matrix metalloproteinase family (MMPs) is an important class of zinc-dependent Endopeptidases involved in the regulation of insect metamorphosis and innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of MMPs. Factors, which bind to and inhibit the degradation of MMPs, play an important role in maintaining the homeostasis of ECM. However, few studies have been done on the functions of MMPs and TIMPs in insects other than Drosophila melanogaster. In view of this, we studied the expression characteristics of BmMMPs family and BmTIMP in silkworm (Bombyx mori) and explored the resistance of BmMMPs family and BmTIMP to Bombyx mori nuclear polyhedrosis virus (Bombyx mori) by gene overexpression and CRISP/Cas9 knockout in vivo and in vitro. The roles of BmMMPs and BmNPV in the metamorphosis of silkworm, Bombyx mori, and the molecular mechanism of BmMMPs were elucidated through the study of their transcriptional regulation and the identification of their interacting proteins. The study may provide experimental and theoretical basis for revealing the roles of BmMMPs and BmTIMP in the metamorphosis and innate immunity of silkworm. The results and conclusions are as follows: 1. Identification and expression characteristics of BmMMPs and Bm TIMP genes were analyzed. Three MMPs family genes were identified in the silkworm genome. One of the genes contained two splicers, named bmmmp1a, bmmmp1b, bmmmp2 and bmmmp3. A TIMP gene named bmtimp was identified. MMP family genes in silkworm all contain typical structural features of MMP family, including pro-peptide, catalytic domain, hingeregion and hemopexin-like c-terminaldomain. phylogenetic analysis revealed that the MMP family of silkworm clustered in an insect MMPs family. bmtimp contains TIMP family canon. The expression of bmmmps and bmtimp was overexpressed on the first day of pupation, bmmmp1, bmmmp3 and bmtimp were overexpressed on the third day of pupation, and bmmmp2 was overexpressed in the midgut and fat, and in the blood. Immunofluorescence and protein truncation assay showed that the whole protein of bmmmp1ab and bmmmp2 and truncated protein of predicting transmembrane region were located in cytoplasm, the whole protein of bmmmp3 was located in cytoplasm, the predicted nuclear localization signal region was located in nucleus and bmmmp3 catalyzed junction. The N-terminus of the domain affects the normal function of the nuclear localization signal; bmtimp has a signal peptide region, part of its protein is localized in the cytoplasm and part is secreted out of the cell. Bmmmps and bmtimp were significantly up-regulated during the early stage of BmNPV infection, and then down-regulated during the late stage of infection. bmmmps and bmtimp were significantly up-regulated during the stimulation of silkworm cell lines in vitro or midgut, blood and fat body by LPS and Escherichia coli. These results indicated that bmmmps and bmtimp were synergistic in the expression pattern; bmmmps and bmtimp may be involved in the regulation of silkworm metamorphosis and innate immunity. 2. The interaction between bmmmps and bmtimp was identified by double fluorescence co-localization, fluorescence bimolecular complementarity (bifc) and immunoprecipitation (co-ip). The interaction of bmmmp1a, bmmmp2 and bmmmp3 with bmtimp was analyzed. These results suggest that all three proteins of the MMPs family interact with timp, suggesting that bmtimp may inhibit its activity by directly binding to bmmmps. 3. The effect of bmmmps and bmtimp on the infection of bmnpv. In this part, bmmmps and bmtimp were synthesized in vitro in the silkworm cell line bmns-swu1, respectively. Overexpression of bmmmps gene and knockout of bmtimp gene could significantly enhance the proliferation and replication of bmnpv, while knockout of bmmmps gene and overexpression of bmtimp gene could significantly inhibit the proliferation and replication of bmnpv. The effect of MMPs activity on the infection of BmNPV was further investigated by collagen-IV degradation test, B b-94, GM6001 and bmmmps activity region truncation test. the results showed that over-expression of bmmmp1a, bmmmp2 and bmmmp3 could enhance the degradation of collagen-IV by silkworm cells. B-94 and GM6001 could significantly inhibit the proliferation and replication of bmnpv, and the truncated active region could significantly enhance the proliferation and replication of BmNPV in silkworm cells compared with the whole protein of bmmmps. These results suggested that bmmmps might participate in the regulation of the proliferation and replication of BmNPV in an active form. 4. The overexpression of bmmmp3 and bmtimp on BmNPV and the proliferation of BmNPV in silkworm. The effects of bmmmp3 and bmtimp on the proliferation and metamorphosis of BmNPV were studied in vivo. The phenotypic statistics of transgenic strains and the analysis of the lethality and key genes of BmNPV infection showed that the overexpression of bmmmp3 strains was related to the growth and metamorphosis of bmnpv. The normal strains showed increased somatotype and weight, and the infection ability of BmNPV in bmmmp3-oe strains was significantly enhanced. bmtimp overexpression strains had phenotypes of retardation, lethal larval stage and abnormal clustering. these results showed that bmmmp3 and bmtimp could participate in the regulation of the growth and development of silkworm. MMP3 can significantly enhance the proliferation and replication of bmnpv. 5. the effects of knockout of bmmmps family genes on the proliferation and metamorphosis of BmNPV in silkworm. a transgenic silkworm strain cas9-oe overexpressing cas9 protein and a single overexpressing sgRNA target sequence were created by combining CRISPR / cas9 gene editing technology with transgenic technology. Four transgenic silkworm strains, namely, bmmmp1-sgrna, bmmmp2-sgrna and bmmmp3-sgrna, were identified. Subsequently, three MMPs gene-specific knockout lines, bmmmp1-ko, bmmmp2-ko and bmmm3-ko, were obtained by cas9-oe and bmmmps-sgrna hybridization. Statistical analysis showed that bmmmp1-ko developed slowly, larvae and pupae died, affecting the extension and branching of trachea. bmmmp2-ko had many phenotypes, such as slow development, low viability in moth stage, abnormal development of martensitic duct and so on. Bmmmp3-ko strain was found to be developmentally retarded, larval lethal, moth inactive and female unable to lay eggs. These results further suggest that BmMMPs play an important role in the allergic development and regulation of BmNPV infection in silkworm. 6. Exploration of the mechanism of BmMMPs and BmTIMP and identification of the interacting proteins by overexpressing IE1, the key gene for BmNPV proliferation, in silkworm cells. The results showed that IE1, FGF and GP64 had certain inductive activity on BmMMPs and BmTIMP except VP39. BmMMPs and BmTIMP had high consistency in expression patterns, and were overexpressed separately in silkworm cells. We also found that overexpression of BmMMPs in silkworm cells could significantly induce the activity of Caspase 3, 7 and 9, and knockout of BmMMPs could inhibit the activity of Caspase 3, 7 and 9. These results further suggest that BmMMPs and BmTIMP participators are involved. BmMMP1a and BmMMP3 were used as bait proteins to screen and identify six interacting proteins with BmMMP1a and six interacting proteins with BmMMP1a in BmN-SWU1 cells. These results suggest that BmMMPs may be closely related to energy metabolism, and BmMMP3 may participate in the regulation of cell physiological activities together with PP6. In summary, BmMMPs and BmTIMP are both involved in innate immunity against BmNPV infection in silkworm and home. This study not only lays a solid foundation for the comprehensive analysis of the functions of BmMMPs and BmTIMP genes in silkworm, but also provides a new clue for the study of innate immunity and metamorphosis of silkworm.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q963
[Abstract]:Insects have a high adaptability to the changeable environment in the long-term evolution process, which is closely related to their magical metamorphosis and efficient innate immune system.Strengthening the research on the metamorphosis and immune mechanism of insects has important practical significance for expanding the utilization of insect resources and green pest control. Matrix metalloproteinase family (MMPs) is an important class of zinc-dependent Endopeptidases involved in the regulation of insect metamorphosis and innate immune response. Tissue inhibitors of metalloproteinases (TIMPs) are endogenous inhibitors of MMPs. Factors, which bind to and inhibit the degradation of MMPs, play an important role in maintaining the homeostasis of ECM. However, few studies have been done on the functions of MMPs and TIMPs in insects other than Drosophila melanogaster. In view of this, we studied the expression characteristics of BmMMPs family and BmTIMP in silkworm (Bombyx mori) and explored the resistance of BmMMPs family and BmTIMP to Bombyx mori nuclear polyhedrosis virus (Bombyx mori) by gene overexpression and CRISP/Cas9 knockout in vivo and in vitro. The roles of BmMMPs and BmNPV in the metamorphosis of silkworm, Bombyx mori, and the molecular mechanism of BmMMPs were elucidated through the study of their transcriptional regulation and the identification of their interacting proteins. The study may provide experimental and theoretical basis for revealing the roles of BmMMPs and BmTIMP in the metamorphosis and innate immunity of silkworm. The results and conclusions are as follows: 1. Identification and expression characteristics of BmMMPs and Bm TIMP genes were analyzed. Three MMPs family genes were identified in the silkworm genome. One of the genes contained two splicers, named bmmmp1a, bmmmp1b, bmmmp2 and bmmmp3. A TIMP gene named bmtimp was identified. MMP family genes in silkworm all contain typical structural features of MMP family, including pro-peptide, catalytic domain, hingeregion and hemopexin-like c-terminaldomain. phylogenetic analysis revealed that the MMP family of silkworm clustered in an insect MMPs family. bmtimp contains TIMP family canon. The expression of bmmmps and bmtimp was overexpressed on the first day of pupation, bmmmp1, bmmmp3 and bmtimp were overexpressed on the third day of pupation, and bmmmp2 was overexpressed in the midgut and fat, and in the blood. Immunofluorescence and protein truncation assay showed that the whole protein of bmmmp1ab and bmmmp2 and truncated protein of predicting transmembrane region were located in cytoplasm, the whole protein of bmmmp3 was located in cytoplasm, the predicted nuclear localization signal region was located in nucleus and bmmmp3 catalyzed junction. The N-terminus of the domain affects the normal function of the nuclear localization signal; bmtimp has a signal peptide region, part of its protein is localized in the cytoplasm and part is secreted out of the cell. Bmmmps and bmtimp were significantly up-regulated during the early stage of BmNPV infection, and then down-regulated during the late stage of infection. bmmmps and bmtimp were significantly up-regulated during the stimulation of silkworm cell lines in vitro or midgut, blood and fat body by LPS and Escherichia coli. These results indicated that bmmmps and bmtimp were synergistic in the expression pattern; bmmmps and bmtimp may be involved in the regulation of silkworm metamorphosis and innate immunity. 2. The interaction between bmmmps and bmtimp was identified by double fluorescence co-localization, fluorescence bimolecular complementarity (bifc) and immunoprecipitation (co-ip). The interaction of bmmmp1a, bmmmp2 and bmmmp3 with bmtimp was analyzed. These results suggest that all three proteins of the MMPs family interact with timp, suggesting that bmtimp may inhibit its activity by directly binding to bmmmps. 3. The effect of bmmmps and bmtimp on the infection of bmnpv. In this part, bmmmps and bmtimp were synthesized in vitro in the silkworm cell line bmns-swu1, respectively. Overexpression of bmmmps gene and knockout of bmtimp gene could significantly enhance the proliferation and replication of bmnpv, while knockout of bmmmps gene and overexpression of bmtimp gene could significantly inhibit the proliferation and replication of bmnpv. The effect of MMPs activity on the infection of BmNPV was further investigated by collagen-IV degradation test, B b-94, GM6001 and bmmmps activity region truncation test. the results showed that over-expression of bmmmp1a, bmmmp2 and bmmmp3 could enhance the degradation of collagen-IV by silkworm cells. B-94 and GM6001 could significantly inhibit the proliferation and replication of bmnpv, and the truncated active region could significantly enhance the proliferation and replication of BmNPV in silkworm cells compared with the whole protein of bmmmps. These results suggested that bmmmps might participate in the regulation of the proliferation and replication of BmNPV in an active form. 4. The overexpression of bmmmp3 and bmtimp on BmNPV and the proliferation of BmNPV in silkworm. The effects of bmmmp3 and bmtimp on the proliferation and metamorphosis of BmNPV were studied in vivo. The phenotypic statistics of transgenic strains and the analysis of the lethality and key genes of BmNPV infection showed that the overexpression of bmmmp3 strains was related to the growth and metamorphosis of bmnpv. The normal strains showed increased somatotype and weight, and the infection ability of BmNPV in bmmmp3-oe strains was significantly enhanced. bmtimp overexpression strains had phenotypes of retardation, lethal larval stage and abnormal clustering. these results showed that bmmmp3 and bmtimp could participate in the regulation of the growth and development of silkworm. MMP3 can significantly enhance the proliferation and replication of bmnpv. 5. the effects of knockout of bmmmps family genes on the proliferation and metamorphosis of BmNPV in silkworm. a transgenic silkworm strain cas9-oe overexpressing cas9 protein and a single overexpressing sgRNA target sequence were created by combining CRISPR / cas9 gene editing technology with transgenic technology. Four transgenic silkworm strains, namely, bmmmp1-sgrna, bmmmp2-sgrna and bmmmp3-sgrna, were identified. Subsequently, three MMPs gene-specific knockout lines, bmmmp1-ko, bmmmp2-ko and bmmm3-ko, were obtained by cas9-oe and bmmmps-sgrna hybridization. Statistical analysis showed that bmmmp1-ko developed slowly, larvae and pupae died, affecting the extension and branching of trachea. bmmmp2-ko had many phenotypes, such as slow development, low viability in moth stage, abnormal development of martensitic duct and so on. Bmmmp3-ko strain was found to be developmentally retarded, larval lethal, moth inactive and female unable to lay eggs. These results further suggest that BmMMPs play an important role in the allergic development and regulation of BmNPV infection in silkworm. 6. Exploration of the mechanism of BmMMPs and BmTIMP and identification of the interacting proteins by overexpressing IE1, the key gene for BmNPV proliferation, in silkworm cells. The results showed that IE1, FGF and GP64 had certain inductive activity on BmMMPs and BmTIMP except VP39. BmMMPs and BmTIMP had high consistency in expression patterns, and were overexpressed separately in silkworm cells. We also found that overexpression of BmMMPs in silkworm cells could significantly induce the activity of Caspase 3, 7 and 9, and knockout of BmMMPs could inhibit the activity of Caspase 3, 7 and 9. These results further suggest that BmMMPs and BmTIMP participators are involved. BmMMP1a and BmMMP3 were used as bait proteins to screen and identify six interacting proteins with BmMMP1a and six interacting proteins with BmMMP1a in BmN-SWU1 cells. These results suggest that BmMMPs may be closely related to energy metabolism, and BmMMP3 may participate in the regulation of cell physiological activities together with PP6. In summary, BmMMPs and BmTIMP are both involved in innate immunity against BmNPV infection in silkworm and home. This study not only lays a solid foundation for the comprehensive analysis of the functions of BmMMPs and BmTIMP genes in silkworm, but also provides a new clue for the study of innate immunity and metamorphosis of silkworm.
【学位授予单位】：西南大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：Q963
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