Isolation of the NIa protease gene in wheat infected with wheat streak mosaic virus.

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.