[Construction of recombinant Bombyx mori nucleopolyhedrovirus by zero background Tn7 transposition].
OBJECTIVE
In order to construct a recombinant Bombyx mori Nucleopolyhedrovirus by Tn7-mediated transposition in Escherichia coli efficiently, a new zero background transposition system was developed.


METHODS
The new system consisted of a conditional replication donor vector pRADM and an attTn7 site blocked E. coli containing BmNPV-Bacmid. The donor transposon vector pRADM with the replication origin derived from R6K gamma required the factor pi encoded by the pir gene to propagate in host cells. Another conditional replication plasmid pBlockA was constructed to block the attTn7 site in host E. coli genome.


RESULTS
Compared with the original vector with ColE1 origin, the transposition efficiency increased from 5.7% to 66% when using conditional replication vector pRADM transposition into original BmDH10Bac. The attTn7 site blocked strain BmDH10BacdeltaTn7 resulted in a significant increase from 5.7% to 23% in the efficacy of generating recombinant BmNPV Bacmid by transposition. Furthermore, the transposition of BmDH10BacdeltaTn7 with pRADM resulted in 100% white colonies.


CONCLUSIONS
This highly efficient and zero background transposition system provides a simple and rapid way of construction of recombinant BmNPV to express target genes or produce gene-delivery virus particles in silkworm.
Yao
Lunguang
L
China-United Kingdom Nanyang Normal University-Rothamsted Research Joint Laboratory of Insect Biology, Henan Provincial Key Laboratory of Funiu Mountain Insect Biology, Nanyang Normal University, Nanyang 473064, China. lunguangyao@163.com
Zhang
Hongling
H
Feng
Juan
J
Zhang
Erhui
E
Wen
Zhenzhong
Z
D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000821:virology; D003001:Cloning, Molecular; D004251:DNA Transposable Elements / Q000235:genetics*; D017924:Nucleopolyhedroviruses / Q000235:genetics*; D014161:Transduction, Genetic
D004251:DNA Transposable Elements