Abstract:
The nuclear inclusion "a" (NIa) protease gene from three different strains of wheat streak mosaic virus (WSMV) was isolated from WSMV-infected wheat using custom-designed oligonucleotide primers and amplified by polymerase chain reaction. Isolated DNA was cloned into TOP10F' E. coli cells after ligation into the pCR-TOPO plasmid.
Transformed TOP10F' cells were visualized by blue/white screening on LB-cb50 medium and ligation was verified by enzyme digestion with EcoRl. Nucleotide sequences of the Nla gene from the different WSMV strains were determined
from purified pCR-TOPO plasmid DNA with the NIa gene inserted. Identity of the cloned gene was determined by sequence analysis with an on-line gene screening databank. The NIa nucleotide sequence was 1268 bases long and began at base # 5424 in the open reading frame of the WSMV genome. The NIa nucleotide sequences from the three different WSMV strains were 97.3% similar. EcoRl restriction sites were found within the Nla protease gene from strains H88 and H94PM of WSMV but not from PV57.
