【摘要】：昆蟲在長期的進化過程中對多變的環(huán)境具備了高度的適應(yīng)能力,這與其神奇的變態(tài)發(fā)育過程和高效的先天性免疫體系密不可分。加強昆蟲變態(tài)發(fā)育及免疫機制的研究,對拓展昆蟲資源的利用和害蟲的綠色防控等有重要的現(xiàn)實意義。同時,可為研究人類干細胞分化、組織器官的形成及免疫機制提供重要線索�；|(zhì)金屬蛋白酶家族(Matrix metalloproteinase family,MMPs)是參與昆蟲變態(tài)發(fā)育調(diào)節(jié)和先天性免疫應(yīng)答的一類重要的鋅依賴性內(nèi)肽酶。該家族成員幾乎能夠降解所有種類的細胞外基質(zhì),在生物體多種生理和病理過程中起重要作用,如組織重塑、器官發(fā)育、炎癥調(diào)節(jié)、免疫應(yīng)答、細胞凋亡、創(chuàng)傷修復(fù)及腫瘤的侵襲和轉(zhuǎn)移等。組織金屬蛋白酶抑制劑(Tissue inhibitor of metalloproteinases,TIMPs)是MMPs內(nèi)源性的蛋白抑制因子,能夠結(jié)合MMPs并抑制其降解活性,在維持細胞外基質(zhì)穩(wěn)態(tài)中起重要作用。然而,對果蠅以外其它昆蟲的MMPs和TIMPs功能研究較少。同時,在不同昆蟲中MMPs和TIMPs的數(shù)目和功能也存在極大差異。因此,全面、深入解析這兩個家族在昆蟲中的作用具有重要意義。鑒于此,本研究以家蠶(Bombyx mori)為模式,研究了家蠶BmMMPs家族和BmTIMP表達特征;在體內(nèi)和體外利用基因過表達和CRISPR/Cas9基因敲除技術(shù),探究了BmMMPs家族和BmTIMP在家蠶抵抗家蠶核型多角體病毒(Bombyx mori nuclearpolyhedrovirus,BmNPV)和家蠶變態(tài)發(fā)育中的作用;通過對BmMMPs家族的轉(zhuǎn)錄調(diào)控研究及互作蛋白的鑒定,闡明BmMMPs家族作用的分子機制。通過該研究,可為揭示BmMMPs和BmTIMP在家蠶變態(tài)發(fā)育及先天性免疫中的作用提供實驗依據(jù)和理論支撐。論文的主要實驗結(jié)果和結(jié)論如下:1.BmMMPs和Bm TIMP基因的鑒定及表達特征分析本研究在家蠶基因組中鑒定出3個mmps家族基因,其中一個基因包含兩種剪切體,分別命名為bmmmp1a、bmmmp1b、bmmmp2和bmmmp3。同時鑒定到1個timp基因,命名為bmtimp。通過序列分析發(fā)現(xiàn),家蠶mmp家族基因均包含mmp家族典型的結(jié)構(gòu)特征,前肽區(qū)(pro-peptide)、催化結(jié)構(gòu)域(catalyticdomain)、鉸鏈區(qū)(hingeregion)及血紅素蛋白類似區(qū)域(hemopexin-likec-terminaldomain)。系統(tǒng)發(fā)生分析發(fā)現(xiàn)家蠶mmp家族聚類于昆蟲mmps家族一支。bmtimp包含timp家族典型的ntr結(jié)構(gòu)域,且系統(tǒng)發(fā)生分析顯示其同樣與昆蟲timp家族聚為一支。家蠶各發(fā)育時期的表達特征分析發(fā)現(xiàn),bmmmps和bmtimp在化蛹第一天有高量表達;在五齡三天,bmmmp1、bmmmp3和bmtimp在中腸和脂肪體中高量表達,bmmmp2在血液有高量表達。通過免疫熒光和蛋白截短實驗對家蠶mmps家族和timp的亞細胞定位模式進行分析發(fā)現(xiàn),bmmmp1ab和bmmmp2的全蛋白及預(yù)測跨膜區(qū)域的截短蛋白均定位于細胞質(zhì)中;bmmmp3的全蛋白定位于細胞質(zhì)中,預(yù)測的核定位信號區(qū)域定位于細胞核中,bmmmp3催化結(jié)構(gòu)域的n端影響了其核定位信號正常行使功能;bmtimp具有信號肽區(qū)域,其蛋白部分定位于細胞質(zhì)中,部分分泌到細胞外。探究了家蠶mmps家族基因和timp在不同抗原刺激下的表達模式,結(jié)果顯示,在bmnpv病毒感染家蠶體外細胞系和體內(nèi)中腸、血液、脂肪體的過程中,bmmmps和bmtimp在bmnpv病毒感染前期均出現(xiàn)顯著上調(diào)表達,隨后表達量下調(diào),在感染后期出現(xiàn)極高量表達;在利用lps和大腸桿菌刺激家蠶體外細胞系或體內(nèi)中腸、血液、脂肪體的過程中,bmmmps和bmtimp均有顯著誘導(dǎo)上調(diào)表達趨勢。上述結(jié)果表明,bmmmps和bmtimp在表達模式上具有一定的協(xié)同性;bmmmps和bmtimp可能參與調(diào)控家蠶的變態(tài)發(fā)育及先天性免疫。2.bmmmps和bmtimp的相互作用鑒定本部分通過雙熒光共定位、熒光雙分子互補實驗(bifc)及免疫共沉淀實驗(co-ip),分析了bmmmp1a、bmmmp2和bmmmp3三個蛋白與bmtimp蛋白之間的相互作用關(guān)系。雙熒光共定位結(jié)果顯示,bmmmps均能分別與bmtimp共定位于細胞質(zhì)中;bifc結(jié)果顯示,bmmmps都能與bmtimp發(fā)生相互作用,并且都聚集于細胞質(zhì)中;co-ip實驗結(jié)果顯示,bmmmp1s分別與bmtimp之間同樣存在特異性的相互作用。上述結(jié)果表明,家蠶mmps家族三個蛋白與timp都具有相互作用,推測bmtimp可能通過直接結(jié)合bmmmps抑制其活性。3.bmmmps和bmtimp對bmnpv侵染的影響本部分通過在體外家蠶細胞系bmns-swu1中分別創(chuàng)制bmmmps和bmtimp基因的過表達及crispr/cas9基因敲除模型,在體外水平探索bmmmps和bmtimp對bmnpv侵染的影響。結(jié)果顯示,bmmmps基因過表達和bmtimp基因敲除可以顯著增強bmnpv的增殖復(fù)制;bmmmps基因敲除和bmtimp基因過表達可以顯著抑制bmnpv的增殖復(fù)制。同時,利用iv型膠原(collagen-iv)降解實驗、mmps的廣譜性抑制劑bb-94、gm6001和bmmmps蛋白活性區(qū)域截短實驗,進一步探究了家蠶mmps活性對bmnpv病毒侵染的影響。結(jié)果顯示,過表達bmmmp1a、bmmmp2和bmmmp3的截短活性區(qū)域均能夠增強家蠶細胞對collagen-iv的降解;bb-94和gm6001能夠顯著抑制bmnpv的增殖復(fù)制;相對于bmmmps全蛋白,其截短活性區(qū)域能顯著增強bmnpv的在家蠶細胞中的增殖復(fù)制。上述結(jié)果表明,bmmmps可能以活性形式參與調(diào)控bmnpv病毒的增殖復(fù)制。4.轉(zhuǎn)基因過表達bmmmp3和bmtimp對bmnpv增殖及家蠶變態(tài)發(fā)育的影響利用顯微注射技術(shù)創(chuàng)制了bmmmp3和bmtimp過表達家蠶品系,在體內(nèi)水平探究bmmmp3和bmtimp對bmnpv增殖及家蠶變態(tài)發(fā)育的影響。通過對轉(zhuǎn)基因品系的表型統(tǒng)計和bmnpv感染后的致死率和病毒關(guān)鍵基因分析,發(fā)現(xiàn)bmmmp3過表達品系相對于正常品系表現(xiàn)為體型增大、體重增加,且bmnpv在bmmmp3-oe品系中的感染能力被顯著增強;bmtimp過表達品系具有發(fā)育遲緩、幼蟲期全部致死及不能正常上簇等表型。這些結(jié)果表明,bmmmp3和bmtimp能夠參與調(diào)控家蠶的生長發(fā)育;在體內(nèi)過表達bmmmp3能夠顯著增強bmnpv的增殖復(fù)制。5.轉(zhuǎn)基因敲除bmmmps家族基因?qū)mnpv增殖及家蠶變態(tài)發(fā)育的影響利用crispr/cas9的基因編輯技術(shù)和轉(zhuǎn)基因技術(shù)相結(jié)合的方法,創(chuàng)制了單獨過表達cas9蛋白的轉(zhuǎn)基因家蠶品系cas9-oe,以及單獨過表達sgrna靶標序列的轉(zhuǎn)基因家蠶品系bmmmp1-sgrna、bmmmp2-sgrna和bmmmp3-sgrna共4種轉(zhuǎn)基因品系。隨后,利用cas9-oe與bmmmps-sgrna雜交敲除目的基因的方法,獲得mmps基因特異性敲除的三個雜交敲除品系bmmmp1-ko、bmmmp2-ko和bmmm3-ko。通過對bmmmps-ko品系生長發(fā)育過程的表型統(tǒng)計分析,結(jié)果發(fā)現(xiàn),bmmmp1-ko出現(xiàn)發(fā)育遲緩、幼蟲和蛹期致死,影響氣管的延伸和分枝。bmmmp2-ko具有發(fā)育遲緩、蛾期生存活力不高、不能正常交配,雌蛾下顎和側(cè)胞保持凸出、被毛易脫落、腹部積水、卵發(fā)育異常及馬氏管發(fā)育異常等多種表型,雄蛾除不具有側(cè)胞保持凸出和卵發(fā)育異常外,其它與雌蛾表型一致。bmmmp3-ko品系具有發(fā)育遲緩、幼蟲期致死、蛾期活力不高、雌蛾不能產(chǎn)卵等表型。同時,將bmnpv病毒利用口服和注射的方法感染家蠶bmmmps-ko雜交敲除品系和正常品系,結(jié)果發(fā)現(xiàn),bmmmps-ko雜交敲除品系能夠顯著抑制vp39基因的轉(zhuǎn)錄。上述結(jié)果進一步表明,BmMMPs在家蠶的變態(tài)發(fā)育和調(diào)控BmNPV侵染中起重要作用。6.BmMMPs和BmTIMP作用機制的探究及相互作用蛋白的鑒定通過在家蠶細胞中單獨過表達BmNPV增殖關(guān)鍵基因IE1、FGF、GP64和VP39,檢測對BmMMPs和BmTIMP的轉(zhuǎn)錄水平的影響。結(jié)果發(fā)現(xiàn),除VP39未檢測到顯著誘導(dǎo)外,IE1、FGF、GP64都對BmMMPs和BmTIMP具有一定的誘導(dǎo)活性�；贐mMMPs和BmTIMP在表達模式上具有很高的一致性,通過在家蠶細胞中單獨過表達BmMMPs和BmTIMP各個基因,探究其是否存在相互調(diào)控關(guān)系,結(jié)果發(fā)現(xiàn),BmMMPs和BmTIMP之間均具有相互誘導(dǎo)活性。同時發(fā)現(xiàn)在家蠶細胞中過表達BmMMPs可以顯著誘導(dǎo)Caspase3、7和9的活性,敲除BmMMPs可以抑制Caspase3、7和9的活性。上述結(jié)果進一步暗示了,BmMMPs和BmTIMP參與家蠶抵御BmNPV侵染的調(diào)控;BmMMPs和Bm TIMP之間可能存在相互調(diào)控關(guān)系。通過免疫共沉淀的方法,并借助質(zhì)譜分析技術(shù),分別以BmMMP1a和BmMMP3為誘餌蛋白,從家蠶BmN-SWU1細胞中篩選并鑒定到6個與BmMMP1a互作的蛋白,6個與BmMMP3互作的蛋白。經(jīng)初步驗證發(fā)現(xiàn),ATP synthase與BmMMP1a存在相互作用;PP6與BmMMP3存在相互作用。這些結(jié)果暗示了,BmMMPs可能與能量代謝息息相關(guān),BmMMP3可能與PP6互作共同參與調(diào)控細胞生理活動。綜上所述,BmMMPs和BmTIMP同時在家蠶抵御BmNPV病毒侵染的先天性免疫中和家蠶的變態(tài)發(fā)育過程中起著重要的作用,此研究不僅為全面解析家蠶BmMMPs和BmTIMP基因功能奠定了堅實的基礎(chǔ),同時也為家蠶先天性免疫及變態(tài)發(fā)育研究提供了新的線索。
[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.
【學(xué)位授予單位】：西南大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：Q963

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6 陳鑫磊,高洪娟,高飛,魏鵬,胡召元,劉以訓(xùn);MMP-2,-9,-14及其抑制劑TIMP-1,-2,-3在恒河猴周期黃體中的時空表達[J];中國科學(xué)C輯:生命科學(xué);2005年03期

7 鄭勇;李睿;周婷;楊軍;陸天才;李洪安;;反義TIMP-1對實驗性肝纖維化中TIMP-1及MMP-13表達的影響[J];華夏醫(yī)學(xué);2006年06期

8 卜淑敏;胡增;彭莎;段恩奎;;出生后小鼠睪丸不同發(fā)育期TIMP-4的表達[J];動物學(xué)報;2007年01期

9 卜淑敏;汪建紅;;TIMP-4 mRNA在小鼠子宮中的表達和激素調(diào)節(jié)[J];新疆師范大學(xué)學(xué)報(自然科學(xué)版);2008年01期

10 陳敏霞;郁衛(wèi)東;梁蓉;楊麗君;晁爽;趙賀華;郭靜竹;;TIMP-1基因緘默削弱SH-SY5Y細胞的增殖潛能并誘導(dǎo)其凋亡增強[J];中國生物化學(xué)與分子生物學(xué)報;2008年11期

相關(guān)會議論文 前10條

1 ;Study of TIMP-2 Gene Over-expression Inhibiting the Invasiveness of Ameloblastoma cells in vitro[A];中華口腔醫(yī)學(xué)會老年口腔醫(yī)學(xué)專業(yè)委員會換屆選舉暨第四屆全國老年口腔醫(yī)學(xué)學(xué)術(shù)研討會論文匯編[C];2008年

2 梁清華;湯天鳳;羅徐;;痹腫消湯對活動期RA患者血清MMP-3及TIMP-1的影響[A];全國中西醫(yī)結(jié)合強直性脊柱炎專題研討會論文匯編[C];2007年

3 yuejie yang;;Effect of IL-10 on expression of CTGF、TIMP-1 in Hepatic Stellate Cells mediated by TGFβ1[A];中華醫(yī)學(xué)會第十六次全國病毒性肝炎及肝病學(xué)術(shù)會議論文匯編[C];2013年

4 ;Study of TIMP-2 Gene Over-expression Inhibiting the Invasiveness of Ameloblastoma Xenografts on CAM[A];中華口腔醫(yī)學(xué)會老年口腔醫(yī)學(xué)專業(yè)委員會換屆選舉暨第四屆全國老年口腔醫(yī)學(xué)學(xué)術(shù)研討會論文匯編[C];2008年

5 盧曉梅;陳琦;溫浩;鄭莉;任加強;朱虹光;;金屬蛋白酶組織抑制劑1(TIMP-1)的克隆與序列分析[A];第七屆全國腫瘤生物治療學(xué)術(shù)會議論文集[C];2001年

6 王虹;曾位森;陳金華;鄒耀東;劉丹;楊艷妮;;組織金屬蛋白抑制因子-1(TIMP-1)單克隆抗體的制備及ELISA檢測試劑盒的研制[A];第6次全國微生物學(xué)與免疫學(xué)大會論文摘要匯編[C];2004年

7 薛博瑜;李q詮，

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