| In recent years, Rice black-streaked dwarf disease and Maize rough dwarf diseaseinduced by Rice black-streaked dwarf virus (RBSDV) and Southern riceblack-streaked dwarf virus (SRBSDV) caused increasingly serious damage in the riceand maize planting areas of Anhui province. However, it was very difficult todistinguish the two viruses in the field samples only by the symptoms. RT-PCRmethod utilized in our research work can realize one-time detection and identificationof two virus species of RBSDV and SRBSDV. To further realize the origin andevolution relationship between RBSDV and SRBSDV, and ascertain the molecularvariation and the characteristics of genetic evolution, two isolates of RBSDV and oneisolate of SRBSDV in Anhui province containing segment S1-S10, respectively, werecloned and sequenced. n addition, it is the key point of establishing system infectionsuccessfully that the plant virus radial move to adjacent cells from sites of infection,while movement proteins (MPs) encoded by virus involved in the transport of thevirus. At present, it is still unclear that MPs which assist SRBSDV transport of cell tocell in plant. Plant expression vectors of three genes of SRBSDV were constructed inthis study to preliminaryly identify MP protein probably encoded by SRBSDV. Itcould lays the foundation to ascertain the molecular mechanisms of SRBSDVinfection and other gene function analysis.1Molecular identification of RBSDV and SRBSDV in Anhui provinceThree special primers were designed based on the conserved sequence of S9segment of RBSDV and SRBSDV. Not only one different size specific band can beamplified respectively from the rice samples infected with single RBSDV orSRBSDV, but also two different size specific bands can be amplified from the mixedrice samples infected with RBSDV and SRBSDV. The rice samples showed obviousdwarf symptoms were collected from the paddy fields of4areas of Anhui province.The detection results of RT-PCR showed that the rice samples from Lujiang andLangxi were infected with RBSDV, and the rice samples from Huaining andXuanzhou were infected with SRBSDV. Specific bands were amplified by RT-PCR,and then each one was cloned and sequenced respectively. Sequence analysisindicated that the nucleotide sequence of two samples from Lujiang and Langxishared the highest sequence similarity (99.3%-99.8%) with S9partial segment ofRBSDV-Shangdong and RBSDV-Zhijr, and the two nucleotide sequences were most closely related to RBSDV. It illustrated that viruses which infected samples fromLujaing and Langxi were two isolates of RBSDV. The nucleotide sequence of twosamples from Huaining and Xuanzhou shared the highest sequence similarity (99.5%)with S9partial segment of SRBSDV-Shangdong and RBSDV-2, and the twonucleotide sequences were most closely related to SRBSDV. It illustrated that viruseswhich infected samples from Huaining and Xuanzhou were two isolates of SRBSDV.2Determination and sequence analysis of complete genome of S1-S10ofRBSDV and SRBSDV Anhui isolatesThe total RNA was extracted from the samples of maize leaves infected with maizerough dwarf disease from Xiaoxian county and rice leaves infected with riceblack-streaked dwarf disease from Lujiang and Huaining county, Anhui provine. Thespecific primers were designed to amplify complete genome sequence of two RBSDVAnhui isolates and one SRBSDV Anhui isolate (RBSDV-MX8， RBSDV-LJ，SRBSDV-HN2). Complete sequences of S1-S10of3isolates are29141nts,29142nts and29121nts, respectively. Comparing nucleotide sequences of S1-S10ofRBSDV and SRBSDV Anhui isolates with those of the isolates of identical speciesand other members of Fijivirus. It showed that nucleotide sequence similarity betweenfragment S1-S10of RBSDV and SRBSDV and that of other members of Fijivirusisolates reached26.3%~84.8%,20.7%~74.8%,20.9%~99.5%,21.1%~99.5%,20.0%~99.5%,20.5%~99.6%,21.6%~100.0%,21.3%~99.8%,21.9%~99.7%and21.3%~99.8%, and the similarities of nucleotide sequences of S1-S10of each isolateof RBSDV reached53.1%~68.4%,65.3%~74.8%,72.2%~90.5%,94.6%~98.6%,93.3%~98.5%,91.6%~98.6%,93.7%~99.4%,94.0%~99.1%,88.6%~99.7%and93.2%~99.0%, and the similarities of nucleotide sequences of S1-S10of each isolateof SRBSDV reached83.3%~85.3%,67.8%~70.1%,80.5%~99.4%,97.5%~99.5%,98.3%~99.5%,97.4%~99.6%,99.1%~100.0%,98.9%~99.8%,98.6%~99.7%and98.7%~99.8%, and the similarities of nucleotide sequences of S1-S10betweenisolates of RBSDV and SRBSDV reached56.8%~75.3%,58.3%~68.9%,21.2%~23.6%,20.5%~22.1%,63.2%~65.8%,59.6%~62.8%,66.3%~67.2%,65.0%~67.8%,67.7%~70.2%and73.5%~75.9%. A phylogenetic tree based onalignment of nucleotide sequences of S1-S10was constructed. The RBSDV andSRBSDV isolates could be divided into each a group of independent, the resultindicated that the relationship between RBSDV and SRBSDV farther. 3Preliminary identification of gene mp of SRBSDVGene S9-2, S7-2and S5-2of SRBSDV were cloned, and then the recombinationexpression vectors pBin-S9-2, pBin-S7-2and pBin-S5-2were constructed andco-bombarded with a PVX movement-defective vector (PVX-GUS-BSP) into N.benthamiana leaves respectively. Histochemical stain showed that blue dot could beobserved on N. benthamiana leaves co-bombard with pBin-S7-2andpPVX-GUS-BSP, and it could be found that GUS have spreaded into the neighboringcells by microscope. But blue dot could not be observed on N. benthamiana leavesco-bombard pBin-S9-2and pBin-S5-2with PVX-GUS-BSP, respectively. Itillustrated that the movement function of PVX-GUS-BSP could be complementedwith pBin-S7-2driven by35S promoter. Therefor, it preliminaryly suggested thatSRBSDV S7-2probably encode a movement protein. |
PDF Full Text Request