整联蛋白在家蚕翅发育中的作用研究
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摘要
昆虫翅在昆虫生命活动中起着十分重要的作用,将昆虫的活动范围从平面扩大到立体,使其在躲避敌害、觅食和繁衍等方面增强了竞争优势。对家蚕翅突变表型的研究能够分析翅发育过程中基因调控的分子机制。本研究以家蚕泡状翅突变体w13为材料,分析了整联蛋白在翅原基中的表达和分布。qRT-PCR结果显示,整联蛋白基因家族在野生型和w13突变型翅原基中有着显著的差异表达。对其中差异表达的整联蛋白基因Bmintegrinβ3进行克隆、原核表达并制备了多克隆抗体。免疫组化分析结果显示,整联蛋白Bm INTEGRINβ3在w13突变型翅原基中的表达量极低。在c108野生型中Bm INTEGRINβ3呈微丝状,将翅的背腹侧表皮联系在一起,但在w13突变型中未检测到该蛋白。推测Bm INTEGRINβ3的下降或缺失可能是导致翅背腹表皮间的维系不紧密并形成泡状翅的原因之一。本研究为解释整联蛋白在昆虫翅发育中的功能作用提供了线索。
Wings of insects play an important role in their life in aspects of expanding the range of insects' activities,promoting the competitive advantages of insects in avoiding the enemies,foraging and reproduction. In this study,a w13 blister mutant of silkworm was used to analyze the expression and distribution of integrin genes. The results of qRT-PCR indicated that the genes of the integrin family genes were differentially expressed in the wing discs between the c108 wild-type and w13 mutant silkworm. Bmintegrin β3 cDNA,which had differentially expressed in w13 mutant,was cloned and the recombinant protein was expressed. Polyclonal antibody was prepared and used for immunohistochemistry analysis. The results indicated that the expression of protein was much lower in the w13 mutant than in the wild-type. Bm INTEGRIN β3 was present as microfilament connecting the dorsal and ventral epidermis of the wings in the c108 wild-type. However,the protein was not detected in the w13 mutant. Thedecrease or loss of the expression of Bm INTEGRIN β3 in the blister wing disc may result in failure of connecting the cells between the dorsal and ventral epidermis of the wings,which probably is one of causes for the blister wings. The results of this study provide insights into the functional role of integrins in wing development of insects.
Wings of insects play an important role in their life in aspects of expanding the range of insects' activities,promoting the competitive advantages of insects in avoiding the enemies,foraging and reproduction. In this study,a w13 blister mutant of silkworm was used to analyze the expression and distribution of integrin genes. The results of qRT-PCR indicated that the genes of the integrin family genes were differentially expressed in the wing discs between the c108 wild-type and w13 mutant silkworm. Bmintegrin β3 cDNA,which had differentially expressed in w13 mutant,was cloned and the recombinant protein was expressed. Polyclonal antibody was prepared and used for immunohistochemistry analysis. The results indicated that the expression of protein was much lower in the w13 mutant than in the wild-type. Bm INTEGRIN β3 was present as microfilament connecting the dorsal and ventral epidermis of the wings in the c108 wild-type. However,the protein was not detected in the w13 mutant. Thedecrease or loss of the expression of Bm INTEGRIN β3 in the blister wing disc may result in failure of connecting the cells between the dorsal and ventral epidermis of the wings,which probably is one of causes for the blister wings. The results of this study provide insights into the functional role of integrins in wing development of insects.
引文
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Brabant MC,Fristrom D,Bunch TA,et al.The PS integrins are required for a regulatory event during Drosophila wing morphogenesis[J].Annals of the New York Academy of Sciences,1998,857:99-109.
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Burke RD.Invertebrate integrins:Structure,function,and evolution[J].International Review of Cytology,1999,191:257-284.
Civera M,Arosio D,Bonato F,et al.Investigating the interaction of cyclic RGD peptidomimetics with alphaV beta(6)integrin by biochemical and molecular docking studies[J].Cancers(Basel),2017.9(10).pii:cancers9100128.doi:10.3390/cancers9100128.
Delimont D,Dufek BM,Meehan DT,et al.Lamininα2-mediated focal adhesion kinase activation triggers alport glomerular pathogenesis[J].PLoS ONE,2014,9(6):e99083.
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Green ML,Breite AG,Beechler CA,et al.Effectiveness of different molecular forms of C.histolyticum class I collagenase to recover islets[J].Islets,2017,e1365996.doi:10.1080/19382014.2017.1365996.
Hauck CR,Borisova M,Muenzner P.Exploitation of integrin function by pathogenic microbes[J].Current Opinion in Cell Biology,2012,24(5):637-644.
Hegde S,Raghavan S.A skin-depth analysis of integrins:Role of the integrin network in health and disease[J].Cell Communication and Adhesion,2013,20(6):155-169.
Hirata H,Chiam KH,Lim CT,et al.Actin flow and talin dynamics govern rigidity sensing in actin-integrin linkage through talin extension[J].Journal of the Royal Society Interface,2014,11(99).pii:20140734.doi:10.1098/rsif.2014.0734.
Hughes AL.Evolution of the integrin alpha and beta protein families[J].Journal of Molecular Evolution,2001,52(1):63-72.
Lee SB,Cho KS,Kim E,et al.Blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development[J].Development,2003,130(17):4001-4010.
Liepinsh E,Trexler M,Kaikkonen A,et al.NMR structure of theLCCL domain and implications for DFNA9 deafness disorder[J].The EMBO Journal,2001,20(19):5347-5353.
Martin D,Zusman S,Li X,et al.Wing blister,a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development[J].Journal of Cell Biology,1999,145(1):191-201.
Salem JC,Reviriego-Mendoza MM,Santy LC.ARF-GEF cytohesin-2/ARNO regulates R-Ras and alpha5-integrin recycling through an EHD1-positive compartment[J].Molecular Biology of the Cell,2015,26(23):4265-4279.
Tai YL,Chu PY,Lai IR,et al.An EGFR/Src-dependent beta4integrin/FAK complex contributes to malignancy of breast cancer[J].Scientific Reports,2015,5:16408.doi:10.1038/srep16408.
Teoh CM,Tan SS,Tran T.Integrins as therapeutic targets for respiratory diseases[J].Current Molecular Medicine,2015,15(8):714-734.
Tong X,He S,Chen J,et al.A novel laminin beta gene BmL an B1-w regulates wing-specific cell adhesion in silkworm,Bombyx mori[J].Scientific Reports,2015,5:12562.doi:10.1038/srep12562.
Zervas CG,Gregory SL,Brown NH.Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane[J].Journal of Cell Biology,2001,152(5):1007-1018.
Zhang K,Xu M,Su J,et al.Characterization and identification of the integrin family in silkworm,Bombyx mori[J].Gene,2014,549(1):149-155.
Beumer KJ,Rohrbough J,Prokop A,et al.A role for PS integrins in morphological growth and synaptic function at the postembryonic neuromuscular junction of Drosophila[J].Development,1999,126(24):5833-5846.
Bilousov O,Koval A,Keshelava A,et al.Identification of novel elements of the Drosophila blisterome sheds light on potential pathological mechanisms of several human diseases[J].PLoSONE,2014,9(6):e101133.
Brabant MC,Brower DL.PS2 integrin requirements in Drosophila embryo and wing morphogenesis[J].Developmental Biology,1993,157(1):49-59.
Brabant MC,Fristrom D,Bunch TA,et al.The PS integrins are required for a regulatory event during Drosophila wing morphogenesis[J].Annals of the New York Academy of Sciences,1998,857:99-109.
Brower DL.Platelets with wings:The maturation of Drosophila integrin biology[J].Current Opinion in Chemical Biology,2003,15(5):607-613.
Brower DL,Jaffe SM.Requirement for integrins during Drosophila wing development[J].Nature,1989,342(6247):285-287.
Brown NH,Gregory SL,Rickoll WL,et al.Talin is essential for integrin function in Drosophila[J].Developmental Cell,2002,3(4):569-579.
Burke RD.Invertebrate integrins:Structure,function,and evolution[J].International Review of Cytology,1999,191:257-284.
Civera M,Arosio D,Bonato F,et al.Investigating the interaction of cyclic RGD peptidomimetics with alphaV beta(6)integrin by biochemical and molecular docking studies[J].Cancers(Basel),2017.9(10).pii:cancers9100128.doi:10.3390/cancers9100128.
Delimont D,Dufek BM,Meehan DT,et al.Lamininα2-mediated focal adhesion kinase activation triggers alport glomerular pathogenesis[J].PLoS ONE,2014,9(6):e99083.
Ghatak S,Morgner J,Wickstrom SA.ILK:A pseudokinase with a unique function in the integrin-actin linkage[J].Biochemical Society Transactions,2013,41(4):995-1001.
Giancotti FG,Ruoslahti E.Integrin signaling[J].Science,1999,285(5430):1028-1032.
Green ML,Breite AG,Beechler CA,et al.Effectiveness of different molecular forms of C.histolyticum class I collagenase to recover islets[J].Islets,2017,e1365996.doi:10.1080/19382014.2017.1365996.
Hauck CR,Borisova M,Muenzner P.Exploitation of integrin function by pathogenic microbes[J].Current Opinion in Cell Biology,2012,24(5):637-644.
Hegde S,Raghavan S.A skin-depth analysis of integrins:Role of the integrin network in health and disease[J].Cell Communication and Adhesion,2013,20(6):155-169.
Hirata H,Chiam KH,Lim CT,et al.Actin flow and talin dynamics govern rigidity sensing in actin-integrin linkage through talin extension[J].Journal of the Royal Society Interface,2014,11(99).pii:20140734.doi:10.1098/rsif.2014.0734.
Hughes AL.Evolution of the integrin alpha and beta protein families[J].Journal of Molecular Evolution,2001,52(1):63-72.
Lee SB,Cho KS,Kim E,et al.Blistery encodes Drosophila tensin protein and interacts with integrin and the JNK signaling pathway during wing development[J].Development,2003,130(17):4001-4010.
Liepinsh E,Trexler M,Kaikkonen A,et al.NMR structure of theLCCL domain and implications for DFNA9 deafness disorder[J].The EMBO Journal,2001,20(19):5347-5353.
Martin D,Zusman S,Li X,et al.Wing blister,a new Drosophila laminin alpha chain required for cell adhesion and migration during embryonic and imaginal development[J].Journal of Cell Biology,1999,145(1):191-201.
Salem JC,Reviriego-Mendoza MM,Santy LC.ARF-GEF cytohesin-2/ARNO regulates R-Ras and alpha5-integrin recycling through an EHD1-positive compartment[J].Molecular Biology of the Cell,2015,26(23):4265-4279.
Tai YL,Chu PY,Lai IR,et al.An EGFR/Src-dependent beta4integrin/FAK complex contributes to malignancy of breast cancer[J].Scientific Reports,2015,5:16408.doi:10.1038/srep16408.
Teoh CM,Tan SS,Tran T.Integrins as therapeutic targets for respiratory diseases[J].Current Molecular Medicine,2015,15(8):714-734.
Tong X,He S,Chen J,et al.A novel laminin beta gene BmL an B1-w regulates wing-specific cell adhesion in silkworm,Bombyx mori[J].Scientific Reports,2015,5:12562.doi:10.1038/srep12562.
Zervas CG,Gregory SL,Brown NH.Drosophila integrin-linked kinase is required at sites of integrin adhesion to link the cytoskeleton to the plasma membrane[J].Journal of Cell Biology,2001,152(5):1007-1018.
Zhang K,Xu M,Su J,et al.Characterization and identification of the integrin family in silkworm,Bombyx mori[J].Gene,2014,549(1):149-155.