Identification Of Transgenic Rice Resistant To Rice Black-streaked Dwarf Virus And RNA-seq Analysis For Diseased Plants

Rice black-streaked dwarf disease is a serious disease caused by rice black streaked-dwarf virus (RBSDV), which threatens the rice growth in the south countries. The disease not only caused great decrease in rice production, but also brought serious damage on maize and other crops. The main symptoms of this disease in rice are obvious dwarf plants, dark green leaves and tumor-like protrusions appearing on veins. RBSDV belongs to the reovirus Branch (Reoviridae) the Fiji viral genus (Fijivirus). Its genome is consisted of 10 double-stranded RNA (dsRNA). In previously study, we have intrduced the RNAi structures of S7-1 and S8 into a japonica rice variety, Kangtiaowuyujing 3. We have got selectable marker free transgenic lines resistantance to rice black-streaked dwarf virus in present study. And we also analysis the transcriptome of the disease plans. The main results are as follows:1. Marker free transgenic rice selection:11 and 9 transformants without selectable marker were selected from the offspring of 19 independed S7-RNAi transformants and 16 independed S8-RNAi transgenic lines, respectively. The frequency of selectable marker free transgenic rice obtained is 57.89% and 56.25%, respectively. We got homozygotes from the offspring of these transgenic lines.2. Rresistance identification in natural conditions:Resistance to RBSDV of thees transgenic lines are evaluated in natural conditions.18 disease plants were found in 500 control plants, which were veified by qRT-PCR. The frequency of infection is 3.6%, while no disease plant was found in the 1200 S7-RNAi and 1000 S8-RNAi transgenic plants, which indicated that the higher resistance to RBSDV of the transgenic plants.3. Performance of the transgenic plants:plant height, panicle length, productive tillers, fertility,1000-grain brown rice weigth of 10 lines of S7-RNAi transgenic rice and 10 lines of S8-RNAi transgenic rice were surveyed, respectively. We found that exogenous genes had no obvious effects on rice growth in most of the transgenic lines.4. Transcriptom analysis of diseased plants:RNA-seq based on the Illumina GA/HiSeq was used to generate extensive transcriptom maps of three diesased plants and corresponding control rice plants.26482 and 31150 genes expressed were detected in the two control plants, while 23692,28363 and 31853 genes expressed were generated from three diseased plants, respectively. Based on the criterion of fold change>=2 and P value<=0.05, we got 4329 differentially expressed genes (DEGs). Among these DEGs,2225 genes were up-regulated and 1904 genes were down-regulated. Out of these DEGs, nine genes which are related with rice resistance to disease and stress were found by mapping these DEGs to 48 reference biological pathways in the KEGG database.