Screening Of Rice Genes Interacted With P9a Of Southern Rice Black-Streaked Dwarf Virus

Southern Rice black-streaked dwarf virus is a new member within the second group of the genus Fijivirus, family Reoviridae. It is transmitted to rice and maize plants by white-backed planthopper, Sogatella furcifera, in a persistent manner. Infected plants show symptoms similar to that of black-streaked dwarf disease and rough dwarf diseases. Its genome consists of10genome segments of double-stranded RNA and at least encodes13viral polypeptides. Among these genome segments, the genome segment S9contains two non-overlapping open reading frames (ORFs). Based on homological analysis, the protein p9a, encoded by the first ORF (ORF-1) on S9, were thought to be a component of viroplasm and play a important role in viral infection and replication. To analysis the function of p9a, we designed a pair of primers according to the sequence of SRBSDV-GD S9(Accession number: EU784843) and amplified ORF-1of genome segment S9using a SRBSDV cDNA library, as template. Amplified products were purified and ligated into plasmid pGEM-T Vector. The ligated plasmid was transformed into E. coli DH5a strain. The target insertion was confirmed to contain the intact ORF-1of SRBSDV S9by PCR and sequencing. The target fragmet was digested with Nde I and BamH from the pGEMR-T Vector and ligated into the bait plasmid pGBKT7. The recombinant plasmid, named pGBKT7-p9a, was transformed into the yeast Gold strain. It was confirmed that the p9a protein has no toxicity and self-activated activity in yeast. The yeast strain harboring the plasmid pGBKT7-p9a were mated with another yeast strain, Y187, containing rice cDNA library and were screened for rice genes interacted with p9a protein on SD/-Ade/-His/-Leu/-Trp and SD/-Ade/-His/-Leu/-Trp/X-a-gal medium. The selected clones were cultured for extraction of plasmids and sequencing, sequence analysis and self-activation experiments.The results showed that total8genes were screened from rice cDNA library and interacted with SRBSDV p9a in yeast cell. These results laid a foundation for further study on the function of SRBSDV p9a protein.