家蚕Prosβ1基因在蛾翅及早期发育中的作用
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摘要
家蚕是鳞翅目的代表物种,研究其翅发育相关基因有助于理解影响昆虫翅发育的内在因素,为鳞翅目害虫的遗传防治提供理论基础和靶标。家蚕的蛋白酶体β亚基1(Bombyx mori proteasome subunitβtype-1,BmProsβ1)是家蚕蛋白酶体核心颗粒的组件之一,负责水解折叠错误或细胞不需要的蛋白质,在细胞发育和器官形成过程中发挥重要作用。利用RNAi干扰技术,在家蚕体腔翅原基附近注射体外合成的针对BmProsβ1基因的小RNA,qRT-PCR检测试验组翅原基中BmProsβ1的表达水平得到有效抑制;对干扰后翅表型的统计结果显示,试验组中小翅个体的比率占52.38%,而对照组中未出现小翅个体。表明在BmProsβ1表达受到抑制的情况下,会严重影响家蚕翅的发育。进一步利用CRISPR/Cas9技术对BmProsβ1进行基因敲除实验,结果显示注射sgRNA与Cas9 mRNA的蚕卵孵化率极低并在幼虫期死亡,而对照组孵化率达到37.92%。这表明BmProsβ1基因在家蚕的早期发育过程中也起到重要作用,其功能缺失会导致家蚕胚胎和幼蚕致死。
Bombyx mori is a representative species of Lepidoptera. Studying the genes involved in development of wing helps to understand the intrinsic factors affecting the wing development of insect, and provides a theoretical basis and target for the genetic control of Lepidoptera pests. As one of the core components of proteasome, proteasome subunit β type-1 of Bombyx mori(BmProsβ1) is responsible for hydrolyzing proteins that are misfolded or unwanted in cells, and plays an important role in cell development and organogenesis. Using RNAi interference technology, small RNAs were synthesized and injected near the wing disc to inhibit the expression of BmProsβ1. qRT-PCR results showed that expression level of BmProsβ1 was effectively inhibited in wing disc. It also showed that 52.38% adults had minute wings in the experimental group, while no minute wing individual appeared in the control group. All above indicated that BmProsβ1 severely affected the development of silkworm wing when it was inhibited. Furthermore, based on the construction of knockout mutants of BmProsβ1 by CRISPR/Cas9 tech-nology, we found that few silkworms could hatch from eggs and even died during larva stage in the experi-mental group in contrast to 37.92% eggs hatched in the control group. In conclusion, BmProsβ1 gene plays an important role in early development of embryo, without which the embryo and larva will die.
Bombyx mori is a representative species of Lepidoptera. Studying the genes involved in development of wing helps to understand the intrinsic factors affecting the wing development of insect, and provides a theoretical basis and target for the genetic control of Lepidoptera pests. As one of the core components of proteasome, proteasome subunit β type-1 of Bombyx mori(BmProsβ1) is responsible for hydrolyzing proteins that are misfolded or unwanted in cells, and plays an important role in cell development and organogenesis. Using RNAi interference technology, small RNAs were synthesized and injected near the wing disc to inhibit the expression of BmProsβ1. qRT-PCR results showed that expression level of BmProsβ1 was effectively inhibited in wing disc. It also showed that 52.38% adults had minute wings in the experimental group, while no minute wing individual appeared in the control group. All above indicated that BmProsβ1 severely affected the development of silkworm wing when it was inhibited. Furthermore, based on the construction of knockout mutants of BmProsβ1 by CRISPR/Cas9 tech-nology, we found that few silkworms could hatch from eggs and even died during larva stage in the experi-mental group in contrast to 37.92% eggs hatched in the control group. In conclusion, BmProsβ1 gene plays an important role in early development of embryo, without which the embryo and larva will die.
引文
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[2] FUJII T,ABE H,KATSUMA S,et al.Identification and characterization of the fusion transcript,composed of the apterous homolog and a putative protein phosphatase gene,generated by 1.5-Mb interstitial deletion in the vestigial (Vg) mutant of Bombyx mori[J].Insect Biochem Molec,2011,41(5):306-312
[3] MATSUOKA T,FUJIWARA H.Expression of ecdysteroid-regulated genes is reduced specifically in the wing discs of the wing-deficient mutant (fl) of Bombyx mori[J].Dev Genes Evol,2000,210(3):120-128
[4] ZHOU Q,TANG S,CHEN Y,et al.A scaleless wings mutant associated with tracheal system developmental deficiency in wing discs in the silkworm,Bombyx mori[J].Int J Dev Biol,2004,48(10):1113-1117
[5] ZHOU Q X,LI Y N,SHEN X J,et al.The scaleless wings mutant in Bombyx mori is associated with a lack of scale precursor cell differentiation followed by excessive apoptosis[J].Dev Genes Evol,2006,216(11):721-726
[6] ZECCA M,STRUHL G.A feed-forward circuit linking wingless,fat-dachsous signaling,and the warts-hippo pathway to Drosophila wing growth[J/OL].PLoS Biol,2010,8(6):e1000386[2018-08-20].https://www.ncbi.nlm.nih.gov/pubmed/20532238.DOI:10.1371/journal.pbio.1000386
[7] COUSO J P,BISHOP S A,ARIAS A M.The wingless signalling pathway and the patterning of the wing margin in Drosophila[J].Development,1994,120(3):621-636
[8] MARTíN-CASTELLANOS C,EDGAR B A.A characterization of the effects of Dpp signaling on cell growth and proliferation in the Drosophila wing[J].Development,2002,129(4):1003-1013
[9] BOSCH M,SERRAS F,MARTíN-BLANCO E,et al.JNK signaling pathway required for wound healing in regenerating Drosophila wing imaginal discs[J].Dev Biol,2005,280(1):73-86
[10] MAURANGE C,PARO R.A cellular memory module conveys epigenetic inheritance of hedgehog expression during Drosophila wing imaginal disc development[J].Gene Dev,2002,16(20):2672-2683
[11] DE CELIS J F,BRAY S.Feed-back mechanisms affecting Notch activation at the dorsoventral boundary in the Drosophila wing[J].Development,1997,124(17):3241-3251
[12] PARKER J,STRUHL G.Scaling the Drosophila wing:TOR-dependent target gene access by the Hippo pathway transducer Yorkie[J/OL].PLoS Biol,2015,13(10):e1002274[2018-08-20].https://www.ncbi.nlm.nih.gov/pubmed/26474042.DOI:10.1371/journal.pbio.1002274
[13] ZHANG J,BLESSING D,WU C,et al.Comparative transcriptomes analysis of the wing disc between two silkworm strains with different size of wings[J/OL].PLoS ONE,2017,12(6):e0179560[2018-08-20].https://www.ncbi.nlm.nih.gov/pubmed/28617839.DOI:10.1371/journal.pone.0179560
[14] SMITH D M,CHANG S C,PARK S,et al.Docking of the proteasomal ATPases' carboxyl termini in the 20S proteasome's α ring opens the gate for substrate entry[J].Mol Cell,2007,27(5):731-744
[15] LOWE J,STOCK D,JAP B,et al.Crystal structure of the 20S proteasome from the archaeon T.acidophilum at 3.4 ? resolution[J].Science,1995,268(5210):533-539
[16] ZWICKL P,NG D,WOO K M,et al.An archaebacterial ATPase,homologous to ATPases in the eukaryotic 26 S proteasome,activates protein breakdown by 20 S proteasomes[J].J Biol Chem,1999,274(37):26008-26014
[17] HEINEMEYER W,FISCHER M,KRIMMER T,et al.The active sites of the eukaryotic 20 S proteasome and their involvement in subunit precursor processing[J].J Biol Chem,1997,272(40):25200-25209
[18] VELENTZAS P D,VELENTZAS A D,PANTAZI A D,et al.Proteasome,but not autophagy,disruption results in severe eye and wing dysmorphia:a subunit- and regulator-dependent process in Drosophila[J/OL].PLoS ONE,2013,8(11):e80530[2018-08-20].https://www.ncbi.nlm.nih.gov/pubmed/24282550.DOI:10.1371/journal.pone.0080530
[19] VILELLA A J,SEVERIN J,URETA-VIDAL A,et al.Ensembl Compara GeneTrees:complete,duplication aware phylogenetic trees in vertebrates[J].Genome Res,2009,19(2):327-335
[20] DE CELIS J F.Pattern formation in the Drosophila wing:the development of the veins[J].Bioessays,2003,25(5):443-451
[21] BLAIR S S.Wing vein patterning in Drosophila and the analysis of intercellular signaling[J].Annu Rev Cell Dev Biol,2007,23:293-319
[22] WATTS R J,HOOPFER E D,LUO L.Axon pruning during Drosophila metamorphosis:evidence for local degeneration and requirement of the ubiquitin-proteasome system[J].Neuron,2003,38(6):871-885
[23] 周庆祥,易咏竹,张志芳,等.家蚕翅原基的一些发育规律研究[J].蚕业科学,2004,30(1):44-49
[24] ROBINSON B S,HUANG J,HONG Y,et al.Crumbs regulates Salvador/Warts/Hippo signaling in Drosophila via the FERM-domain protein Expanded[J].Curr Biol,2010,20(7):582-590
[25] KLEIN U,GERNOLD M,KLOETZEL P M.Cell-specific accumulation of Drosophila proteasomes (MCP) during early development[J].J Cell Biol,1990,111(6):2275-2282
[26] L?W P,BUSSELL K,DAWSON S P,et al.Expression of a 26S proteasome ATPase subunit,MS73,in muscles that undergo developmentally programmed cell death,and its control by ecdysteroid hormones in the insect Manduca sexta[J].FEBS Lett,1997,400(3):345-349
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