Korean journal of applied entomology (한국응용곤충학회지)

Korean Society of Applied Entomology (한국응용곤충학회)
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Construction of a Transgenic Tobacco Expressing a Polydnaviral Cystatin

폴리드나바이러스 유래 시스타틴 유전자 발현 형질전환 담배 제작

  • Kim, Yeongtae (Department of Bioresource Sciences, Andong National University) ;
  • Kim, Eunsung (Department of Bioresource Sciences, Andong National University) ;
  • Park, Youngjin (Department of Bioresource Sciences, Andong National University) ;
  • Kim, Yonggyun (Department of Bioresource Sciences, Andong National University)
  • 김영태 (안동대학교 생명자원과학과) ;
  • 김은성 (안동대학교 생명자원과학과) ;
  • 박영진 (안동대학교 생명자원과학과) ;
  • 김용균 (안동대학교 생명자원과학과)
  • Received : 2014.10.08
  • Accepted : 2015.01.10
  • Published : 2015.03.01
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Abstract

CpBV (Cotesia plutellae bracovirus) is a polydnavirus and encodes a cystatin (CpBV-CST1) gene. Its overexpression suppresses insect immunity and alters insect developmental processes. This study aimed to construct a genetically modified (GM) tobacco to further explore the physiological function of the viral cystatin and to apply to control insect pests. To this end, the transgenic tobacco lines were screened in expression of the target gene and assessed in insecticidal activity. A recombinant vector (pBI121-CST) was prepared and used to transform a bacterium, Agrobacterium tumefasciens. The transformed bacteria were used to generate transgenic tobacco lines, which were induced to grow callus and resulted in about 92% of shoot regeneration. The regenerated plants were screened by PCR analysis to confirm the insertion of the target gene in the plant genome. In addition, the expression of the target gene was assessed in the regenerated plants by quantitative real-time PCR (qRT-PCR). The qRT-PCR analysis showed that the transgenic line plant expressed the target gene about 17 times more than the control tobacco, indicating a stable insertion and expression of the target gene in the transgenic tobacco line. The insecticidal activity was then analyzed using the screened transgenic tobacco lines against the teneral 1st instar larvae of the oriental tobacco budworm, Helicoverpa assulta. Though there was a variation in the insecticidal efficacy among transgenic lines, T9 and T12 lines exhibited more than 95% mortality at 7 days after feeding treatment. These results suggest that CpBV-CST1 is a useful genetic resource to be used to generate GM crop against insect pests.

폴리드나바이러스의 일종인 CpBV (Cotesia plutellae bracovirus) 바이러스 게놈에 포함된 시스타틴(CpBV-CST1) 유전자의 과발현이 곤충의 면역 및 발육을 교란한다. 이 연구는 바이러스 유래 시스타틴의 생물적 기능의 심화 연구와 해충저항성 작물 개발을 위해 담배형질전환체를 구축하는 데 목적을 두었다. 이를 위해 형질전환체를 대상으로 목표유전자의 발현분석과 곤충에 대한 발육억제에 대한 생물검정을 수행했다. 시스타틴 유전자를 pBI121 운반체에 재조합한 pBI121-CST를 제작하고, 이를 아그로박테리움(Agrobacterium tumefasciens) 세균 매개에 의한 담배 형질전환 및 재분화를 유도하여 약 92%의 높은 신초 재분화율을 나타냈다. 이들 재분화된 개체 가운데 담배 genomic DNA에 시스타틴 유전자가 삽입된 형질전환 추정 개체를 PCR 분석법으로 선발하였다. 다시 quantitative real-time PCR (qRT-PCR) 분석을 통해 이들 목표유전자의 발현을 분석하였다. qRT-PCR 결과는 형질전환 추정 개체가 비형질전환체에 비해 유전자 발현이 약 17 배 높게 나타나 형질전환계통에서 목표유전자가 안정적으로 발현되고 있음을 확인했다. 선발된 형질전환담배를 대상으로 갓 부화한 담배나방(Helicoverpa assulta) 1령 유충에 대한 살충효과를 확인하였다. 살충력에 있어서 형질전환계통간의 차이가 있었다. 특히 T9와 T12계통은 섭식 후 7 일차 조사에서 95% 이상의 살충효과를 보였다. 이상의 결과들은 CpBV-CST1이 해충저항성 작물 개발에 필요한 유용 유전자 자원으로서 활용될 수 있음을 제시하고 있다.

Keywords

References

  1. Agarwala, K.L., Kawabata, S., Hirata, M., Miyagi, M., Tsunasawa, S., Iwanaga, S., 1996. A cysteine protease inhibitor stored in the large granules of horseshoe crab hemocytes: purification, characterization, cDNA cloning, and tissue localization. J. Biochem. 119, 85-94. https://doi.org/10.1093/oxfordjournals.jbchem.a021220
  2. Amani, J., Kazemi, R., Ali Reza Abbasi, A.R., Ali Hatef Salmanian, A.H., 2011. A simple and rapid leaf genomic DNA extraction method for polymerase chain reaction analysis. Iran. J. Biotech. 9, 69-71.
  3. Attardo, G.M., Strickler-Dinglasan, P., Perkin, S.A.H., Caler, E., Bonaldo, M.F., Soareo, M.B., El-Sayeed, N., Aksoy, S., 2006. Analysis of fat body transcriptome from the adult tsetse fly, Glossina morsitans. Insect Mol. Biol. 15, 411-424. https://doi.org/10.1111/j.1365-2583.2006.00649.x
  4. Burke, G.R., Strand, M.R., 2014. Systematic analysis of a wasp parasitism arsenal. Mol. Ecol. 23, 890-901. https://doi.org/10.1111/mec.12648
  5. Carrillo, L., Martinez, M., Ramessar, K., Cambra, I., Castanera, P., Ortego, F., Diaz, I., 2011. Expression of a barley cystatin gene in maize enhances resistance against phytophagous mites by altering their cysteine-proteases. Plant Cell Rep. 30, 101-112. https://doi.org/10.1007/s00299-010-0948-z
  6. Chan, Y., Yang, A., Chen, J., Yeh, K., Chan, M., 2010. Heterologous expression of taro cystatin protects transgenic tomato against Meloidogyne incognita infection by means of interfering sex determination and suppressing gall formation. Plant Cell Rep. 29. 231-238. https://doi.org/10.1007/s00299-009-0815-y
  7. Cho, W.L., Tsao, S.M Hays, A.R., Walter, R., Chen, J.S., Snigirevskaya, E.S., Raikhel, A.S., 1999. Mosquito cathepsin B-like protease involved in embryonic degradation of vitellin is produced as a latent extraovarian precursor. J. Biol. Chem. 274, 13311-13321. https://doi.org/10.1074/jbc.274.19.13311
  8. De Gregorio, E., Spellman, P.T., Rubin, G.M., Lemaitre, B., 2001. Genome-wide analysis of the Drosophila immune response by using oligo nucleotide microarray. Proc. Natl. Acad. Sci. USA 98, 12590-12595. https://doi.org/10.1073/pnas.221458698
  9. Dubin, G., 2005. Proteinaceous cysteine protease inhibitors. Cell Mol. Life Sci. 62, 653-669. https://doi.org/10.1007/s00018-004-4445-9
  10. Haunerland, N.H., Shirk, P.D., 1995. Regional and functional differentiation in the insect fat body. Annu. Rev. Entomol. 40, 121-145. https://doi.org/10.1146/annurev.en.40.010195.001005
  11. Hegedus, D., O'Grady, M., Chamankhah, M., Baldwin, D., Gleddie, S., Braun, L., Erlandson, M., 2002. Changes in cysteine protease activity and localization during midgut metamorphosis in the crucifers root maggots (Delia radicum). Insect Biochem. Mol. Biol. 32, 1585-1596. https://doi.org/10.1016/S0965-1748(02)00099-1
  12. Herrera-Estrella, L., Van den Broeck, G., Maenhaut, R., Van Montagu, M., Schell, J., 1984. Light-inducible and chloroplast associated expression of a chimeric gene introduced into Nicotiana tabacum using a Ti plasmid vector. Nature 310, 115-120. https://doi.org/10.1038/310115a0
  13. Homma, K., Kurata, S., Natori, S., 1994. Purification, characterization, and cDNA cloning of procathepsin L from the culture medium of NIH-Sape-4, an embryonic cell line of Sarcophaga peregrina (flesh fly) and its involvement in the differentiation of imaginal disc. J. Biol. Chem. 269, 15258-15264.
  14. Kim, Y., 2006. Polydnavirus and its novel application to insect pest control. Kor. J. Appl. Entomol. 45, 241-259.
  15. Kim, Y., Choi, J., Je, Y., 2007. Cotesia plutellae bracovirus genome and its function in altering insect physiology. J. Asia Pac. Entomol. 10, 181-191. https://doi.org/10.1016/S1226-8615(08)60351-9
  16. Kim, Y., Hepat, R., Kim, Y., 2013. A copy of cystatin from the diamondback moth Plutella xylostella is encoded in the polydnavirus Cotesia plutellae bracovirus. J. Asia Pac. Entomol. 16, 449-455. https://doi.org/10.1016/j.aspen.2013.06.006
  17. Kim, Y., Eom, S., Park, J., Kim, Y., 2014. Inhibitory effects of a recombinant viral cystatin protein on insect immune and development. Kor. J. Appl. Entomol. 53, 331-338. https://doi.org/10.5656/KSAE.2014.09.0.041
  18. Koo, Y.D., Ahn, J.E., Salzman, R.A., Moon, J., Chi, Y.H., Yun, D.J., Lee, S.Y., Koiwa, H., Zhu-salzman, K., 2008. Functional expression of an insect cathespin B-like counter-defence protein. Insect Mol. Biol. 17, 235-245. https://doi.org/10.1111/j.1365-2583.2008.00799.x
  19. Kurata, S., Saito, H. Natori, S., 1992. The 29-kDa hemocyte proteinase dissociates fat body at metamorphosis of Sarcophaga. Dev. Biol. 153, 115-121. https://doi.org/10.1016/0012-1606(92)90096-Y
  20. Lecaille, F., Kaleta, J., Bromme, D., 2002. Human and parasitic papain-like cysteine proteases: their role in physiology and pathology and recent developments in inhibitor design. Chem. Rev. 102, 4459-4488. https://doi.org/10.1021/cr0101656
  21. Lecardonnel, A., Chauvin, L., Jouanin, L., Beaujean, A., Prevosta, G., Sangwan-Norreeld, B., 1999. Effects of rice cystatin I expression in transgenic potato on Colorado potato beetle larvae. Plant Sci. 140, 71-79. https://doi.org/10.1016/S0168-9452(98)00197-6
  22. Levy, F., Rabel, D., Charlet, M., Bulet, P., Hoffmann, J.A., Ehret-Sabatier, L., 2004. Peptidomic and proteomic analysis of the systemic immune response of Drosophila. Biochimie 86, 607-616. https://doi.org/10.1016/j.biochi.2004.07.007
  23. Liu, J., Shi, G.P., Zhang, W.Q., Zhang, G.R., Xy, W.H., 2006. Cathepsin L function in insect moulting: moleculoar cloning and functional analysis in cotton bollworm, Helicoverpa armigera. Insect Mol. Biol. 15, 823-834. https://doi.org/10.1111/j.1365-2583.2006.00686.x
  24. Livak, K.J., Schmittgen, T.D., 2001. Analysis of relative gene expression data using real-time quantitative PCR and the $2^{-{\Delta}{\Delta}CT}$ method. Methods 25, 402-408. https://doi.org/10.1006/meth.2001.1262
  25. Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15, 473-497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
  26. Olsson, S.L., Ek, B., Bjork, I., 1999. The affinity and kinetics of inhibition of cyteine proteinases by intact recombinant bovine cystatin C. Biochim. Biophys. Acta 1432, 73-81. https://doi.org/10.1016/S0167-4838(99)00090-4
  27. Park, Y., Ahn, S., Vogel, H., Kim, Y., 2014. Integrin ${\beta}$ subunit and its RNA interference in immune and developmental processes of the Oriental tobacco budworm, Helicoverpa assulta. Dev. Comp. Immunol. 47, 59-67. https://doi.org/10.1016/j.dci.2014.06.017
  28. Porta, H., Jimenez, G., Cordoba, E., Leon, P., Soberon, M., Bravo, A., 2011. Tobacco plants expressing the Cry1AbMod toxin suppress tolerance to Cry1Ab toxin of Manduca sexta cadherinsilenced larvae. Insect Biochem. Mol. Biol. 41, 513-519. https://doi.org/10.1016/j.ibmb.2011.04.013
  29. Rawlings, N.D., Barrett, A.J., 1990. Evolution of proteins of the cystatin superfamily. J. Mol. Evol. 30, 60-71. https://doi.org/10.1007/BF02102453
  30. Saito, H., Kurata, S., Natori, S., 1992. Purification and characterization of a hemocyte proteinase of Sarcophaga, possibly participating in elimination of foreign substances. Eur. J. Biochem. 209, 939-944. https://doi.org/10.1111/j.1432-1033.1992.tb17366.x
  31. Sambrook, J., Fritsh, E.F., Maniatis, T., 1989. Molecular cloning. A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, NY.
  32. Senthilkumar, R., Cheng, C.P., Yeh, K.W., 2010. Genetically pyramiding protease-inhibitor genes for dual broad-spectrum resistance against insect and phytopathogens in transgenic tobacco. Plant Biotech. J. 8, 65-75. https://doi.org/10.1111/j.1467-7652.2009.00466.x
  33. Shindo, T., Van Der Hoorn, R.A., 2008. Papain-like cysteine protease: key players at molecular battlefields employed by both plants and their invaders. Mol. Plant Pathol. 9, 119-125.
  34. Smid, I., Gruden, K., Gasparic, M. B., Koruza, K., Petek, M., Pohleven, J., Brzin, J., Kos, J., Zel, J., Sabotic, J., 2013. Inhibition of the growth of Colorado potato beetle larvae by macrocypins, protease inhibitors from the parasol mushroom. J. Agric. Food Chem. 61, 12499-12509. https://doi.org/10.1021/jf403615f
  35. Tabashnik, B.E., Brevault, T., Carriere, Y., 2013. Insect resistance to Bt crops: lessons from the first billion acres. Nat. Biotech. 31, 510-521. https://doi.org/10.1038/nbt.2597
  36. Uchida, K., Ohmori, D., Ueno, T., Nishizuka, M., Eshita, Y., Fukunaga, A., Kominami, E., 2001. Preoviposition activation of cathepsin-like proteinases in degenerating ovarian follicles of the mosquito Culex pipiens pallens. Dev. Biol. 237, 68-78. https://doi.org/10.1006/dbio.2001.0357
  37. Yamamoto, Y., Watabe, S., Kageyama, T., Takahashi, S., 1999. Purification and characterization of Bombyx cysteine proteinase specific inhibitors from the hemolymph of Bombyx mori. Arch. Insect Biochem. Physiol. 41, 119-129.
  38. Zhang, Y., Lu, Y.X., Liu, J., Feng, Q.L., Xu, W.H., 2013. A regulatory pathway, ecdysone-transcription factor Relish-cathepsin L, is involved in insect fat body dissociation. PLOS Genetics 9, e1003273. https://doi.org/10.1371/journal.pgen.1003273
  39. Zhou, J., Ueda, M., Umemiya, R., Battsetseg, B., Boldbaatar, D., Xuan, X., Fujisaka, K., 2006. A secreted cystatin from the tick Haemaphysalis longicornis and its distinct expression patterns in relation to innate immunity. Insect Biochem. Mol. Biol. 36, 527-535. https://doi.org/10.1016/j.ibmb.2006.03.003

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