利用体腔显微注射法实现非编码RNA在家蚕头部稳定过表达
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摘要
【目的】通过体腔显微注射非编码RNA(non-coding RNA,ncRNA)过表达载体,研究注射后ncRNA在家蚕Bombyx mori幼虫不同组织中的表达情况,探索研究家蚕ncRNA功能的便捷方法。【方法】利用显微注射仪(玻璃显微注射针)在家蚕5龄第3天幼虫第7-8腹节褶皱处分别注射家蚕ncRNA Bm-15和Bm-152过表达载体piggyBac[A3-EGFP-A3-Bm-15]和piggyBac[A3-EGFP-A3-Bm-152],注射48 h后提取家蚕幼虫头、胸、尾部、腹部背板、腹部腹板和丝腺总RNA,荧光定量PCR检测Bm-15和Bm-152在家蚕幼虫不同组织中的表达情况。【结果】采用玻璃显微注射针造成的创伤面小,注射后家蚕出现感染和死亡的比例低于10%。注射ncRNA Bm-15过表达载体后,在家蚕幼虫头部和尾部都检测到Bm-15的显著性过表达,头部中Bm-15表达量较注射piggyBac[A3-EGFP]空载体时上升了17倍,在其他4个组织中Bm-15表达量与正常饲养组家蚕对照相比变化不显著。注射Bm-152过表达载体后,同样在家蚕幼虫头部和尾部都检测到Bm-152的显著过表达,其中头部中的表达量较注射piggyBac[A3-EGFP]空载体时上升了6. 6倍。腹部背板和丝腺中Bm-152过表达效果也较明显,分别是注射piggyBac[A3-EGFP]空载体时表达量的3. 4和4. 5倍。【结论】体腔显微注射ncRNA过表达载体可以实现ncRNA在家蚕头部中稳定过表达。本研究为后续研究神经相关ncRNA的功能提供了较好的思路和方法。
【Aim】 To investigate the convenient method for functional study of non-coding RNAs( ncRNAs) in the silkworm Bombyx mori,via injection of the overexpression vector of ncRNAs into the coelom of B. mori larva and detection of the expression changes of ncRNAs in different tissues of the larvae after injection. 【Methods 】 The overexpression vectors piggyBac [A3-EGFP-A3-Bm-15] and piggyBac[A3-EGFP-A3-Bm-152] of ncRNA Bm-15 and Bm-152,respectively,were injected into the junction of the 7 th-8 th abdominal segments of the day-3 5 th instar larvae of B. mori by microinjector( glass microinjection needle). The total RNA was extracted from larval head,thorax,caudal end,abdominal tergum,abdominal sternum and silk gland after 48 h of injection,and the expression levels of Bm-15 and Bm-152 in different larval tissues were detected by q PCR. 【Results】The wound surface caused by glass microinjection needle was small,and the infection and mortality rates of B. mori larvae were less than 10%. Bm-15 was significantly overexpressed in the larval head and caudal end after injection of the overexpression vector,with the expression level increased by 17-fold in the head as compared to that in the injection group of the blank vector piggyBac [A3-EGFP]. However,the expression levels of Bm-15 in the other four tissues of B. mori larvae after injection of overexpression vector showed little change as compared to those of the uninjected control group. Bm-152 was also significantly overexpressed in larval head and caudal end of the silkworm after injection of the overexpression vector,with the expression level being 6. 6-fold higher than that in the injection group of the blank vector piggyBac [A3-EGFP] in larval head. Bm-152 also showed obvious overexpression in larval abdominal tergum and silk gland,with the expression level being 3. 4 and 4. 5 times as high as that in the injection group of the blank vector piggyBac [A3-EGFP ],respectively. 【Conclusion 】 By coelomic microinjection of overexpression vectors of ncRNAs,ncRNAs can be stably overexpressed in the larval head of B. mori. This study provides a good strategy and method for the subsequent functional study of neuro-related ncRNAs.
【Aim】 To investigate the convenient method for functional study of non-coding RNAs( ncRNAs) in the silkworm Bombyx mori,via injection of the overexpression vector of ncRNAs into the coelom of B. mori larva and detection of the expression changes of ncRNAs in different tissues of the larvae after injection. 【Methods 】 The overexpression vectors piggyBac [A3-EGFP-A3-Bm-15] and piggyBac[A3-EGFP-A3-Bm-152] of ncRNA Bm-15 and Bm-152,respectively,were injected into the junction of the 7 th-8 th abdominal segments of the day-3 5 th instar larvae of B. mori by microinjector( glass microinjection needle). The total RNA was extracted from larval head,thorax,caudal end,abdominal tergum,abdominal sternum and silk gland after 48 h of injection,and the expression levels of Bm-15 and Bm-152 in different larval tissues were detected by q PCR. 【Results】The wound surface caused by glass microinjection needle was small,and the infection and mortality rates of B. mori larvae were less than 10%. Bm-15 was significantly overexpressed in the larval head and caudal end after injection of the overexpression vector,with the expression level increased by 17-fold in the head as compared to that in the injection group of the blank vector piggyBac [A3-EGFP]. However,the expression levels of Bm-15 in the other four tissues of B. mori larvae after injection of overexpression vector showed little change as compared to those of the uninjected control group. Bm-152 was also significantly overexpressed in larval head and caudal end of the silkworm after injection of the overexpression vector,with the expression level being 6. 6-fold higher than that in the injection group of the blank vector piggyBac [A3-EGFP] in larval head. Bm-152 also showed obvious overexpression in larval abdominal tergum and silk gland,with the expression level being 3. 4 and 4. 5 times as high as that in the injection group of the blank vector piggyBac [A3-EGFP ],respectively. 【Conclusion 】 By coelomic microinjection of overexpression vectors of ncRNAs,ncRNAs can be stably overexpressed in the larval head of B. mori. This study provides a good strategy and method for the subsequent functional study of neuro-related ncRNAs.
引文
Bachiller D,Schellander K,Peli J,Rüther U,1991.Liposomemediated DNA uptake by sperm cells.Mol.Reprod.Dev.,30(3):194-200.
Chen M,Li DD,Qiao HL,Lu N,Wen ZZ,Kan YC,2012.RNAinterference on the Ago genes in silkworm(Bombyx mori).J.Henan Agric.Sci.,41(9):148-151,157.[陈敏,李丹丹,乔惠丽,卢楠,文祯中,阚云超,2012.RNA干涉家蚕细胞Ago蛋白家族基因的表达研究.河南农业科学,41(9):148-151,157]
Cheng YJ,Li ZX,2018.Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae.Arch.Insect Biochem.Physiol.,100(3):e21530.
Choi MY,Vander Meer RK,Coy M,Scharf ME,2012.Phenotypic impacts of PBAN RNA interference in an ant,Solenopsis invicta,and a moth,Helicoverpa zea.J.Insect Physiol.,58(8):1159-1165.
Eckermann KN,Ahmed HMM,Karami NejadRanjbar M,Dippel S,Ogaugwu CE,Kitzmann P,Isah MD,Wimmer EA,2018.Hyperactive piggy Bac transposase improves transformation efficiency in diverse insect species.Insect Biochem.Molec.Biol.,98:16-24.
Gregory M,Alphey L,Morrison NI,Shimeld SM,2016.Insect transformation with piggy Bac:getting the number of injections just right.Insect Mol.Biol.,25(3):259-271.
Guo W,Ren D,Zhao L,Jiang F,Song J,Wang X,Kang L,2018.Identification of odorant-binding proteins(OBPs)and functional analysis of phase-related OBPs in the migratory locust.Front.Physiol.,9:984.
Kennerdell JR,Carthew RW,1998.Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.Cell,95(7):1017-1026.
Li D,Wang Y,Zhang K,Jiao Z,Zhu X,Skogerboe G,Guo X,Chinnusamy V,Bi L,Huang Y,Dong S,Chen R,Kan Y,2011.Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori.Nucleic Acids Res.,39(9):3792-3805.
Li Z,Zeng B,Ling L,Xu J,You L,Aslam AF,Tan A,Huang Y,2015.Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.Int.J.Biol.Sci.,11(2):176-185.
Ling L,Ge X,Li Z,Zeng B,Xu J,Aslam AF,Song Q,Shang P,Huang Y,Tan A,2014.MicroRNA Let-7 regulates molting and metamorphosis in the silkworm,Bombyx mori.Insect Biochem.Molec.Biol.,53:13-21.
Liu C,Yamamoto K,Cheng TC,Kadono-Okuda K,Narukawa J,Liu SP,Han Y,Futahashi R,Kidokoro K,Noda H,Kobayashi I,Tamura T,Ohnuma A,Banno Y,Dai FY,Xiang ZH,Goldsmith MR,Mita K,Xia QY,2010.Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm,Bombyx mori.Proc.Natl.Acad.Sci.USA,107(29):12980-12985.
Liu S,Li K,Gao Y,Liu X,Chen W,Ge W,Feng Q,Palli SR,Li S,2018.Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila.Proc.Natl.Acad.Sci.USA,115(1):139-144.
Liu X,Qiu W,Li X,Kan Y,Li D,2018.Preliminary study on the function of non-coding RNA Bm-152 of silkworm,Bombyx mori.J.Xinyang Normal Univ.(Nat.Sci.Ed.),31(1):40-45.[刘晓,邱妩洁,李新梅,阚云超,李丹丹,2018.家蚕非编码RNABm-152功能初探.信阳师范学院学报(自然科学版),31(1):40-45]
Liu X,Zhang S,Shen Z,Liu Y,Li Z,Liu X,2019.Vrille is required for larval moulting and metamorphosis of Helicoverpa armigera(Lepidoptera:Noctuidae).Insect Mol.Biol.,28(3):355-371.
Liu XS,Hu QH,Zheng SC,Feng QL,Deng HM,2016.Analysis of apoptosis and autophagy in wing discs of the silkworm,Bombyx mori.Acta Entomol.Sin.,59(12):1317-1324.[刘学术,胡启豪,郑思春,冯启理,邓惠敏,2016.家蚕翅原基细胞的凋亡和自噬分析.昆虫学报,59(12):1317-1324]
Port F,Chen HM,Lee T,Bullock SL,2014.Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.Proc.Natl.Acad.Sci.USA,111(29):E2967-E2976.
Robinson KO,Ferguson HJ,Cobey S,Vaessin H,Smith BH,2000.Sperm-mediated transformation of the honey bee,Apis mellifera.Insect Mol.Biol.,9(6):625-634.
Rubin GM,Spradling AC,1982.Genetic transformation of Drosophila with transposable element vectors.Science,218(4570):348-353.
Schulte C,Theilenberg E,Muller-Borg M,Gempe T,Beye M,2014.Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee(Apis mellifera).Proc.Natl.Acad.Sci.USA,111(24):9003-9008.
Suzuki MG,Suzuki K,Aoki F,Ajimura M,2012.Effect of RNAimediated knockdown of the Bombyx mori transformer-2 gene on the sex-specific splicing of Bmdsx pre-mRNA.Int.J.Dev.Biol.,56(9):693-699.
Terenius O,Papanicolaou A,Garbutt JS,Eleftherianos I,Huvenne H,Kanginakudru S,Albrechtsen M,An C,Aymeric JL,Barthel A,Bebas P,Bitra K,Bravo A,Chevalier F,Collinge DP,Crava CM,de Maagd RA,Duvic B,Erlandson M,Faye I,Felfldi G,Fujiwara H,Futahashi R,Gandhe AS,Gatehouse HS,Gatehouse LN,Giebultowicz JM,Gómez I,Grimmelikhuijzen CJ,Groot AT,Hauser F,Heckel DG,Hegedus DD,Hrycaj S,Huang L,Hull JJ,Iatrou K,Iga M,Kanost MR,Kotwica J,Li C,Li J,Liu J,Lundmark M,Matsumoto S,Meyering-Vos M,Millichap PJ,Monteiro A,Mrinal N,Niimi T,Nowara D,Ohnishi A,Oostra V,Ozaki K,Papakonstantinou M,Popadic A,Rajam MV,Saenko S,Simpson RM,Soberón M,Strand MR,Tomita S,Toprak U,Wang P,Wee CW,Whyard S,Zhang W,Nagaraju J,ffrench-Constant RH,Herrero S,Gordon K,Swevers L,Smagghe G,2011.RNAinterference in Lepidoptera:an overview of successful and unsuccessful studies and implications for experimental design.J.Insect Physiol.,57(2):231-245.
Wu W,Gu D,Yan S,Li Z,2019.RNA interference of endoglucanases in the formosan subterranean termite Coptotermes formosanus shiraki(Blattodea:Rhinotermitidae)by dsRNA injection or ingestion.J.Insect Physiol.,112:15-22.
Xu J,Dong Q,Yu Y,Niu B,Ji D,Li M,Huang Y,Chen X,Tan A,2018.Mass spider silk production through targeted gene replacement in Bombyx mori.Proc.Natl.Acad.Sci.USA,115(35):8757-8762.
Zhang H,Li HC,Miao XX,2013.Feasibility,limitation and possible solutions of RNAi-based technology for insect pest control.Insect Sci.,20(1):15-30.
Zhang K,2017.Identification of Hemocyte-specific Molecular Markers and Study on Hematopoietic Regulation Mechanism in Silkworm,Bombyx mori.PhD Dissertation,Southwest University,Chongqing.[张奎,2017.家蚕血细胞特异性分子标记鉴定及造血调控机制研究.重庆:西南大学博士学位论文]
Zhang WN,Ma L,Liu C,Chen L,Xiao HJ,Liang GM,2018.Dissecting the role of Krüppel homolog 1 in the metamorphosis and female reproduction of the cotton bollworm,Helicoverpa armigera.Insect Mol.Biol.,27(4):492-504.
Zhang Z,Niu B,Ji D,Li M,Li K,James AA,Tan A,Huang Y,2018.Silkworm genetic sexing through W chromosome-linked,targeted gene integration.Proc.Natl.Acad.Sci.USA,115(35):8752-8756.
Zheng Y,Hu Y,Yan S,Zhou H,Songm D,Yi M,Shen J,2019.Apolymer/detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybean aphid Aphis glycines.Pest Manag.Sci.,75(7):1993-1999.
Zhou CY,Zha XF,Liu HW,Xia QY,2017.Zinc finger protein rotund deficiency affects development of the thoracic leg in Bombyx mori.Insect Sci.,24(3):385-396.
Zhu B,Shan J,Li R,Liang P,Gao X,2019.Identification and RNAibased function analysis of chitinase family genes in diamondback moth,Plutella xylostella.Pest Manag.Sci.,75(7):1951-1961.
Chen M,Li DD,Qiao HL,Lu N,Wen ZZ,Kan YC,2012.RNAinterference on the Ago genes in silkworm(Bombyx mori).J.Henan Agric.Sci.,41(9):148-151,157.[陈敏,李丹丹,乔惠丽,卢楠,文祯中,阚云超,2012.RNA干涉家蚕细胞Ago蛋白家族基因的表达研究.河南农业科学,41(9):148-151,157]
Cheng YJ,Li ZX,2018.Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae.Arch.Insect Biochem.Physiol.,100(3):e21530.
Choi MY,Vander Meer RK,Coy M,Scharf ME,2012.Phenotypic impacts of PBAN RNA interference in an ant,Solenopsis invicta,and a moth,Helicoverpa zea.J.Insect Physiol.,58(8):1159-1165.
Eckermann KN,Ahmed HMM,Karami NejadRanjbar M,Dippel S,Ogaugwu CE,Kitzmann P,Isah MD,Wimmer EA,2018.Hyperactive piggy Bac transposase improves transformation efficiency in diverse insect species.Insect Biochem.Molec.Biol.,98:16-24.
Gregory M,Alphey L,Morrison NI,Shimeld SM,2016.Insect transformation with piggy Bac:getting the number of injections just right.Insect Mol.Biol.,25(3):259-271.
Guo W,Ren D,Zhao L,Jiang F,Song J,Wang X,Kang L,2018.Identification of odorant-binding proteins(OBPs)and functional analysis of phase-related OBPs in the migratory locust.Front.Physiol.,9:984.
Kennerdell JR,Carthew RW,1998.Use of dsRNA-mediated genetic interference to demonstrate that frizzled and frizzled 2 act in the wingless pathway.Cell,95(7):1017-1026.
Li D,Wang Y,Zhang K,Jiao Z,Zhu X,Skogerboe G,Guo X,Chinnusamy V,Bi L,Huang Y,Dong S,Chen R,Kan Y,2011.Experimental RNomics and genomic comparative analysis reveal a large group of species-specific small non-message RNAs in the silkworm Bombyx mori.Nucleic Acids Res.,39(9):3792-3805.
Li Z,Zeng B,Ling L,Xu J,You L,Aslam AF,Tan A,Huang Y,2015.Enhancement of larval RNAi efficiency by over-expressing Argonaute2 in Bombyx mori.Int.J.Biol.Sci.,11(2):176-185.
Ling L,Ge X,Li Z,Zeng B,Xu J,Aslam AF,Song Q,Shang P,Huang Y,Tan A,2014.MicroRNA Let-7 regulates molting and metamorphosis in the silkworm,Bombyx mori.Insect Biochem.Molec.Biol.,53:13-21.
Liu C,Yamamoto K,Cheng TC,Kadono-Okuda K,Narukawa J,Liu SP,Han Y,Futahashi R,Kidokoro K,Noda H,Kobayashi I,Tamura T,Ohnuma A,Banno Y,Dai FY,Xiang ZH,Goldsmith MR,Mita K,Xia QY,2010.Repression of tyrosine hydroxylase is responsible for the sex-linked chocolate mutation of the silkworm,Bombyx mori.Proc.Natl.Acad.Sci.USA,107(29):12980-12985.
Liu S,Li K,Gao Y,Liu X,Chen W,Ge W,Feng Q,Palli SR,Li S,2018.Antagonistic actions of juvenile hormone and 20-hydroxyecdysone within the ring gland determine developmental transitions in Drosophila.Proc.Natl.Acad.Sci.USA,115(1):139-144.
Liu X,Qiu W,Li X,Kan Y,Li D,2018.Preliminary study on the function of non-coding RNA Bm-152 of silkworm,Bombyx mori.J.Xinyang Normal Univ.(Nat.Sci.Ed.),31(1):40-45.[刘晓,邱妩洁,李新梅,阚云超,李丹丹,2018.家蚕非编码RNABm-152功能初探.信阳师范学院学报(自然科学版),31(1):40-45]
Liu X,Zhang S,Shen Z,Liu Y,Li Z,Liu X,2019.Vrille is required for larval moulting and metamorphosis of Helicoverpa armigera(Lepidoptera:Noctuidae).Insect Mol.Biol.,28(3):355-371.
Liu XS,Hu QH,Zheng SC,Feng QL,Deng HM,2016.Analysis of apoptosis and autophagy in wing discs of the silkworm,Bombyx mori.Acta Entomol.Sin.,59(12):1317-1324.[刘学术,胡启豪,郑思春,冯启理,邓惠敏,2016.家蚕翅原基细胞的凋亡和自噬分析.昆虫学报,59(12):1317-1324]
Port F,Chen HM,Lee T,Bullock SL,2014.Optimized CRISPR/Cas tools for efficient germline and somatic genome engineering in Drosophila.Proc.Natl.Acad.Sci.USA,111(29):E2967-E2976.
Robinson KO,Ferguson HJ,Cobey S,Vaessin H,Smith BH,2000.Sperm-mediated transformation of the honey bee,Apis mellifera.Insect Mol.Biol.,9(6):625-634.
Rubin GM,Spradling AC,1982.Genetic transformation of Drosophila with transposable element vectors.Science,218(4570):348-353.
Schulte C,Theilenberg E,Muller-Borg M,Gempe T,Beye M,2014.Highly efficient integration and expression of piggyBac-derived cassettes in the honeybee(Apis mellifera).Proc.Natl.Acad.Sci.USA,111(24):9003-9008.
Suzuki MG,Suzuki K,Aoki F,Ajimura M,2012.Effect of RNAimediated knockdown of the Bombyx mori transformer-2 gene on the sex-specific splicing of Bmdsx pre-mRNA.Int.J.Dev.Biol.,56(9):693-699.
Terenius O,Papanicolaou A,Garbutt JS,Eleftherianos I,Huvenne H,Kanginakudru S,Albrechtsen M,An C,Aymeric JL,Barthel A,Bebas P,Bitra K,Bravo A,Chevalier F,Collinge DP,Crava CM,de Maagd RA,Duvic B,Erlandson M,Faye I,Felfldi G,Fujiwara H,Futahashi R,Gandhe AS,Gatehouse HS,Gatehouse LN,Giebultowicz JM,Gómez I,Grimmelikhuijzen CJ,Groot AT,Hauser F,Heckel DG,Hegedus DD,Hrycaj S,Huang L,Hull JJ,Iatrou K,Iga M,Kanost MR,Kotwica J,Li C,Li J,Liu J,Lundmark M,Matsumoto S,Meyering-Vos M,Millichap PJ,Monteiro A,Mrinal N,Niimi T,Nowara D,Ohnishi A,Oostra V,Ozaki K,Papakonstantinou M,Popadic A,Rajam MV,Saenko S,Simpson RM,Soberón M,Strand MR,Tomita S,Toprak U,Wang P,Wee CW,Whyard S,Zhang W,Nagaraju J,ffrench-Constant RH,Herrero S,Gordon K,Swevers L,Smagghe G,2011.RNAinterference in Lepidoptera:an overview of successful and unsuccessful studies and implications for experimental design.J.Insect Physiol.,57(2):231-245.
Wu W,Gu D,Yan S,Li Z,2019.RNA interference of endoglucanases in the formosan subterranean termite Coptotermes formosanus shiraki(Blattodea:Rhinotermitidae)by dsRNA injection or ingestion.J.Insect Physiol.,112:15-22.
Xu J,Dong Q,Yu Y,Niu B,Ji D,Li M,Huang Y,Chen X,Tan A,2018.Mass spider silk production through targeted gene replacement in Bombyx mori.Proc.Natl.Acad.Sci.USA,115(35):8757-8762.
Zhang H,Li HC,Miao XX,2013.Feasibility,limitation and possible solutions of RNAi-based technology for insect pest control.Insect Sci.,20(1):15-30.
Zhang K,2017.Identification of Hemocyte-specific Molecular Markers and Study on Hematopoietic Regulation Mechanism in Silkworm,Bombyx mori.PhD Dissertation,Southwest University,Chongqing.[张奎,2017.家蚕血细胞特异性分子标记鉴定及造血调控机制研究.重庆:西南大学博士学位论文]
Zhang WN,Ma L,Liu C,Chen L,Xiao HJ,Liang GM,2018.Dissecting the role of Krüppel homolog 1 in the metamorphosis and female reproduction of the cotton bollworm,Helicoverpa armigera.Insect Mol.Biol.,27(4):492-504.
Zhang Z,Niu B,Ji D,Li M,Li K,James AA,Tan A,Huang Y,2018.Silkworm genetic sexing through W chromosome-linked,targeted gene integration.Proc.Natl.Acad.Sci.USA,115(35):8752-8756.
Zheng Y,Hu Y,Yan S,Zhou H,Songm D,Yi M,Shen J,2019.Apolymer/detergent formulation improves dsRNA penetration through the body wall and RNAi-induced mortality in the soybean aphid Aphis glycines.Pest Manag.Sci.,75(7):1993-1999.
Zhou CY,Zha XF,Liu HW,Xia QY,2017.Zinc finger protein rotund deficiency affects development of the thoracic leg in Bombyx mori.Insect Sci.,24(3):385-396.
Zhu B,Shan J,Li R,Liang P,Gao X,2019.Identification and RNAibased function analysis of chitinase family genes in diamondback moth,Plutella xylostella.Pest Manag.Sci.,75(7):1951-1961.