The Research On Turnip Mosaic Virus By RNAI-Mediated And Anaylsis Of Sequencing Whole Genome And Virus Detction

Turnip mosaic virus(TuMV)is the most serious and widely distributed virus in Crucifers.Traditional method of breeding was longer,and some resistant genes were recessive and not easy to use,and TuMV usually produce variation.We are devoting to find valid and stable resistant way.According to the alignment of 126 sequences of TuMV in GenBank,we choose partial conserved region of CI,VPg and HC-Pro gene to make the RNAi construction respectively.There Were C1?C5 strains in Beijing,and C4 was the main strain.The recombinant plasmids were transformed into wild-type Arabidopsis using the Agrobacterium-mediated inflorescence dipping methord.The isolate of TuMV-C4 was identified and inoculated on the transgenic Arabidopsis to identify the efficiency resistance.The resistance of transgenic plants was increased about 80%than control.This work is the foundation for the TuMV resistance breeding of Chinese cabbage.Up to 2011,there are 126 TuMV complete sequences in GeneBank.In China,TuMV research mainly focus on some genes,such as CP,HC-Pro and N1a,and the complete genomic sequences are less reported.Complete sequence research is the foundation of the study of spreading,evolution and recombination.We amplified the complete sequence of TuMV isolates BJ-R01,BJ-B01,BJ-B02,BJ-B03.The four isolates are 9833nt and analyze their phylogeny.The result of phylogenic analysis shown these four isolates belong to TuMV world-B group.The pathogenic analysis reveals that all the four isolates belong to BR pathogenic type.They all have strong symptomatology to the Brassica,but show apparent difference to the Raphanus.Using of M-MLV and general Taq enzyme,we developed a rapid,convenience and specificity method for detecting of TuMV by one-step multiple RT-PCR.This simple method does not need the step of purification of total RNA.After grinding the samples directly in extraction buffer at room temperature,the crude extract or concentrated DNA-RNA mixture can be used as detection template.In oreder to ensure the accuracy of the detection,two sets of primers were employed in this method.