Research
Development of Baculovirus resistant transgenic silkworms
Double-stranded RNA (dsRNA) is an important regulator of gene expression in many eukaryotes. It triggers different types of gene silencing that are collectively referred to as RNA interference or RNA silencing. A key step in known silencing pathways is the processing of dsRNAs into short RNA duplexes of characteristic size and structure. These short dsRNAs guide RNA silencing by specific and distinct mechanisms. Many components of the RNA interference machinery still need to be identified and characterized, but a more complete understanding of the process is imminent.
Figure 1: Transgenic silkmoth showing red fluorescent protein expression only in eyes.
RNA silencing mechanisms were first recognized as antiviral mechanisms that protect organisms from RNA viruses or which prevent the random integration of transposable elements. RNAi is now believed to be an ancient anti-viral strategy, a part of which the cellular machine also utilizes for gene regulation mechanism. RNAi has been well demonstrated in many arthropod species including the silkworm B. mori and has been demonstrated to suppress AcNPV proliferation in Spodoptera frugiperda, while lef-1 targeting RNAi was not effective to inhibit the Bombyx mori nucleopolyhedrovirus (BmNPV) growth in B. mori completely. In the ongoing study, we report RNAi mediated inhibition of baculovirus proliferation in transgenic silkworms by targeting multiple viral genes simultaneously. We also report the successful transfer of the resistance property to a non-transgenic silkworm strain.
Application of RNAi for baculovirus inhibition
The domesticated silkworm, Bombyx mori, is a major lepidopteron insect with significant economic importance, which is also considered as an ideal lepidopteron model system for genomic analysis. Besides its usefulness as an economic insect and a genetic tool, industrial application of the silkworm is augmented by the successful germline transgenesis using PiggyBac transposon based vectors. This lepidoptera provides livelihood to hundreds of thousands of farmers in India, whose revenue is affected because of a severe baculoviral disease called ‘Grasserie disease’. Nearly 20-50% of the crop losses due to various diseases are accounted for by the baculovirus, BmNPV. The sturdy nature of the polyhedra helps wide spread grassarie infection especially in summer season. Thus development of the BmNPV resistant silkworm strain is utmost priority for improving the silk output and standard of living of the farmers.
In our laboratory, we used RNAi for preventing baculovirus infection in silkworm. We targeted the baculoviral immediate early-1 gene, first in a transformed lepidopteron cell line and then in the transgenic silkworm. Constitutive expression of double stranded RNA was achieved by piggyBac- mediated transformation of Sf9 cell line with a transgene encoding double stranded ie-1 RNA (dsie-1). Strong viral repression was observed at early stages of infection but subsequent recovery of viral proliferation was observed. In contrast, the same transgene inserted into the chromosomes of transgenic silkworms induced long-term inhibition of BmNPV infection, with nearly 40% protection compared to non-transgenic animals. By crossing transgenic individuals with a susceptible strain of silkworm, we demonstrated that the anti-viral trait could be readily transferred to a hybrid background. These results show that RNA interference can be used to protect silkworm strains from baculovirus infection. (Kanginakudru et al. 2007)
Projects being pursued
- Transgenic silkmoth for viral resistance by RNA interference.
- Development of transgenic silkworms to elucidate the function of genes.
- Maintenance of transgenic silkworm stocks
- Introduction of transgene to productive baculovirus breeds
Working group
- Edupalli V Subbaiah
- A Sobhan Babu
- Shiva Prasad APSSRDI, Hindupur, INDIA
- Pierre Couble CGMC. FRANCE
Publications
- Beech CJ, Vasan SS, Quinlan MM, Capurro ML, Alphey L, Bayard V, Bouare M, Kittayapong P, Lavery JV, Lee HL, Marrelli MT, McLeod MC, Nagaraju J, Ombongi K, Othman RY, Pillai V, Ramsey J, Reuben R, Rose RI, Tyagi BK, and Mumford J (2009a) Deployment of innovative genetic vector control strategies: Progress on regulatory and biosafety aspects, capacity building and development of best-practice guidance. Asia Pacific Journal of Molecular Biology and Biotechnology 17: 75-85.PDF
- Beech CJ, Nagaraju J, Vasan SS, Rose RI, Othman RY, Pillai V, and Saraswathy TS (on behalf of the working groups) (2009b) Risk analysis of a hypothetical open field release of a self-limiting transgenic Aedes aegypti mosquito strain to combat dengue. Asia Pacific Journal of Molecular Biology and Biotechnology, 17: 99-111
- Kanginakudru S, Royer C, Edupalli SV, Jalabert A, Mauchamp B, Chandrashekaraiah, Prasad S V, Chavancy G , Couble P, Nagaraju J (2007) Targeting ie-1 gene by RNAi induces baculoviral resistance in lepidopteran cell lines and in the transgenic silkworms. Insect Molecular Biology 16: 635-644.
- Kanginakudru S, Subbaiah EV, Couble P, Nagaraju J (2003) LepidopteraAbstracts. Towards development of baculoviral resistant strains of the silkworm, Bombyx mori. Sixth International Workshop on the Molecular Biology and Genetics of the Lepidoptera. Journal of Insect Science 3: 36.
- Nagaraju J, Kanda T, Tamura T, Coulon M, Couble P (1996) Attempts at transgenesis of the silkworm (Bombyx mori) by egg injection of foreign DNA. Applied Entomology and Zoology 31: 587-596.