Molecular Diversity Of Rice Stripe Virus In Anhui Province And The Functional Identification Of NS3 Gene

In recent years, rice stripe disease caused by Rice stripe virus (RSV) has brought serious economic losses to rice production in Anhui province. Rice plants showing stripe symptom were collected from various rice growing areas in Anhui province. Seventeen CP genes and twelve NS3 genes of RSV Anhui isolates were cloned and sequence analysis were carried out to ascertain the origin and evolution of RSV derived from Anhui as well as to provide a theoretical basis for disease resistance of rice varieties in Anhui province. Furthermore plant expression vector containing NS3 gene of RSV Ma'anshan isolate was constructed and the function of NS3 protein as a RNA silencing suppressor was identified.1. Cloning of CP genes and NS3 genes of RSV Anhui isolatesThe total RNA was extracted from rice leaves infected with Rice stripe virus. Two pairs of specific primers were designed to amplify 17 RNA3 partial fragments including CP gene and 12 RNA3 partial fragments including NS3 gene of RSV Anhui isolates by RT-PCR, and then the fragments were cloned and sequenced. Sequence analysis showed that the full-length of 17 RNA3 fragments including CP gene were 1051 nts and 12 RNA3 fragments including NS3 gene were 869-897 nts. The 969 nts CP gene encodes 322 amino acids, and the 869-897 nts NS3 gene encodes 211 amino acids.2. Sequence analysis of CP genes of RSV Anhui isolatesComparing nucleotide sequences of CP genes of 17 RSV Anhui isolates with that of other 75 RSV CP genes, and the results showed that the similarities of nucleotide sequences of 17 RSV Anhui isolates reached 97.9%-99.2%. RSV CP genes of Anhui isolates shared the highest sequence similarities (98.2%-99.7%) with those of Jiangsu, Shandong, Hebei, Henan isolates, whereas shared the lowest sequence similarity (93.4%-94.4%) with RSV CP genes of Yunnan isolates. A phylogenetic tree based on alignment of nucleotide sequences of 92 RSV CP genes was constructed. The result indicated that the 92 RSV CP sequences could be divided into two groups. Isolates from eastern China and Japan, including 17 CP genes of Anhui isolates, clustered into one group, moreover, isolates from Yunnan province clustered into another group.3. Sequence analysis of NS3 genes of RSV Anhui isolatesComparing nucleotide sequences of NS3 genes of 12 RSV Anhui isolates with that of other 58 RSV NS3 genes, and the results showed that the similarities of nucleotide sequences of 12 RSV Anhui isolates reached 96.4%-98.7%. RSV NS3 genes of Anhui isolates shared the highest sequence similarities (97.0%-99.5%) with those of Hebei, Jiangsu, Shandong isolates, whereas shared the lowest sequence similarity (91.0%-93.9%) with RSV NS3 genes of Yunnan isolates. A phylogenetic tree based on alignment of nucleotide sequences of 70 RSV NS3 genes was constructed. The result indicated that the 70 RSV NS3 sequences could be divided into two groups. Isolates from eastern China and Japan including 17 CP genes of Anhui isolates, clustered into one group, moreover, isolates from Yunnan province clustered into another group.4. Construction of plant expression vectors of NS3 gene of RSV Ma'anshan isolateUsing RNA3 partial fragment of Ma'anshan isolate as a template, and specific primers were designed to amplify NS3 fragment by RT-PCR. NS3 fragment was linked to clone vectors. After being transformed and screened, the positive clones were obtained. Moreover the NS3 fragment was sub-cloned into vector pBin438 and TRV RNA2, and the acquired plant expression vectors were named pBin438-NS3 and TRV RNA2-NS3.5. Local silencing experiment were conducted by transforming expression vectors via AgrobacteriumPlant expression vector pBin438-NS3 was transformed into Agrobacterium tumefaciens, and co-infiltration was carried out in the leaves of 16c N. benthamiana plants with A. tumefaciens harboring pBin438-NS3 and 35S: GFP, respectively. GFP green fluorescence could be stimulated at infiltrating site on leaf and could be observed under UV lamp at 6 days post inoculation. The results indicated that NS3 protein is a RNA silencing suppressor.6. Reversing experiment were conducted by transforming expression vectors via AgrobacteriumThe expression vector TRV RNA1 and TRV RNA2-NS3 were transformed into A. tumefaciens, respectively, and co-infiltration was carried out in the leaves of 16c N. benthamiana plants being occurred systemic silencing. Moreover, GFP green fluorescence could not be observed under UV lamp at 30 days post inoculation. The results indicated that NS3 protein could not reverse the GFP silencing being established.