Browse > Article
http://dx.doi.org/10.5656/KSAE.2015.01.1.055

Construction of a Transgenic Tobacco Expressing a Polydnaviral Cystatin  

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)
Publication Information
Korean journal of applied entomology / v.54, no.1, 2015 , pp. 7-15 More about this Journal
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.
Keywords
Cystatin; Transgenic line; Insect resistance; Polydnavirus; Helicoverpa assulta;
Citations & Related Records
Times Cited By KSCI : 2  (Citation Analysis)
연도 인용수 순위
1 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.   DOI
2 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.   DOI
3 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.   DOI
4 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.   DOI
5 Dubin, G., 2005. Proteinaceous cysteine protease inhibitors. Cell Mol. Life Sci. 62, 653-669.   DOI
6 Haunerland, N.H., Shirk, P.D., 1995. Regional and functional differentiation in the insect fat body. Annu. Rev. Entomol. 40, 121-145.   DOI
7 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.   DOI
8 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.   DOI
9 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.   DOI
10 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.   DOI   ScienceOn
11 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.   DOI   ScienceOn
12 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.   DOI
13 Murashige, T., Skoog, F., 1962. A revised medium for rapid growth and bioassays with tobacco cultures. Physiol. Plant. 15, 473-497.   DOI
14 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.   DOI
15 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.   DOI
16 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.   DOI
17 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.   DOI
18 Rawlings, N.D., Barrett, A.J., 1990. Evolution of proteins of the cystatin superfamily. J. Mol. Evol. 30, 60-71.   DOI
19 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.   DOI   ScienceOn
20 Sambrook, J., Fritsh, E.F., Maniatis, T., 1989. Molecular cloning. A laboratory manual. 2nd Ed. Cold Spring Harbor Laboratory Press, NY.
21 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.
22 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.   DOI
23 Tabashnik, B.E., Brevault, T., Carriere, Y., 2013. Insect resistance to Bt crops: lessons from the first billion acres. Nat. Biotech. 31, 510-521.   DOI   ScienceOn
24 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.   DOI   ScienceOn
25 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.   DOI
26 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.
27 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.   DOI
28 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.   DOI
29 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.   DOI
30 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.
31 Burke, G.R., Strand, M.R., 2014. Systematic analysis of a wasp parasitism arsenal. Mol. Ecol. 23, 890-901.   DOI
32 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.   DOI
33 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.
34 Kim, Y., 2006. Polydnavirus and its novel application to insect pest control. Kor. J. Appl. Entomol. 45, 241-259.
35 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.   DOI
36 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.   DOI
37 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.   DOI   ScienceOn
38 Kurata, S., Saito, H. Natori, S., 1992. The 29-kDa hemocyte proteinase dissociates fat body at metamorphosis of Sarcophaga. Dev. Biol. 153, 115-121.   DOI
39 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.   DOI