Nucleotide Sequence Analysis Of Genomic RNAs Of Two Cucumoviruses Infecting Peanut And Transgenic Resistance To The Viruses

To understand the genetic variation and evolution of two Cucumoviruses at nucleic acid level, we completed the sequence analysis of full genome of Peanut stunt virus mild strain (PSV-Mi), Cucumber mosaic virus common strain (CMV-CA) and CMV virulent strain (CMV-CS). Plant expression vectors were developed by inverted repeat style with single PStV cp gene or multiple gene fragments of replicase gene of CMV-CA and PSV-Mi and cp gene of PStV. Mediated by Agrobacterium tumefaciens, these vectors were transformed Nicotiana benthamiana leaf discs to obtain transgenic virus resistance. The detail results were shown below:PSV-Mi RNA1.2 and 3 are 3347 nucleotides (nt), 2966nt and 2170nt in length.The size and structure of PSV-Mi RNAs were similar with other strains of PSV. Alignment of nucleotide sequence of RNAs showed that similarity of RNA1, 2 and 3 of PSV-Mi with PSV-ER or PSV-W is below 80%, compared with CMV and Tomato aspermy virus (TAV), the similarity is below 70%. The results indicated that PSV-Mi represents a new PSV subgroup from China, designated as subgroup III. Phylogenetic analysis of 4 ORFs of PSV-Mi with strains of PSV, CMV and TAV showed that PSV-Mi, PSV-ER and PSV-W form three different evolution branches, which also supports above result.CMV-CA and CS share the same size and frame in RNA1 and RNA2, little difference was found in RNA3. RNA1 and 2 are 3356nt and 3045nt in length. CMV-CA RNA3 was 2219nt in length compared with 2212nt of CMV-CS. The sequence similarity of CMV-CA RNA1, 2 and 3 with corresponding RNAs of CS is 98%, 99% and 97%, with those strains of CMV subgroup I were 90%, around 80% with those strains of CMV subgroup II, and less than 70% with PSV-ER. The results showed that the genetic relationship between CMV-CA and CS is very close, with strains of CMV subgroup I is closer than subgroup II and PSV-ER. The similarity of 5 ORFs and their deduced proteins compared with the corresponding ORFs and proteins of CMV subgroup I, II and PSV-ER also showed the same results.Alignment of nucleotide sequence of RNA3 5'NTR showed that structure of CMV-CA and CS is similar with those strains of CMV subgroup IB, but different from strains of subgroup LA and II. Phylogenetic trees of 5 ORFs showed that the relationship of CMV-CA with CS was very close, they were in CMV IB. The above results showed that CMV-CA and CS which infecting peanut in China were classified in CMV IB subgroup. Reassortment was not found between CMV and PSV in CMV-CA, CS and PSV-Mi by similarity alignment, though PSV and CMV were found in the same infecting area. From this result, we deduced that CMV-CA and CS which infecting peanut in China were came from CMV which was genetic modified to adopt the special bioecological environment of China.To control peanut viral disease caused by PStV, PSV and CMV, RNA silencing strategy was tried to use to induce resistance to these viruses. pKcp, pK450 and pK1500 were constructed with single cp gene or fusion fragments by cp gene and replicase gene. Mediated by Agrobacterium tumefaciens GV3101(pMP90), Leaf discs of Nicotiana bethamiana were transformed by these 3 constucts. Tested by PCR, transgenic plants were obtained for each construct. Resistance was obtained in T0-T2 transgenic plants of pKcp against PStV inoculation. One line of T1 transgenic plants of pK450 and pK1500 was found symptom recovery to CMV inoculation,separately. All lines of pK450 and pK.1500 were found susceptible to PSV inoculation. SiRNA was found in all transgenic plants by Northern-blot test.Three constructs were succeeded in inducing resistance to PStV, it meant that fragment size from 150bp to full cp gene of PStV was efficiently to induce resistance to PStV. One lines of pK450 andpK1500 showed resistance to PStV and CMV, it meant using RNA silencing strategy to induce multiple viruses resistance is reasonable. The project proceed a good trial for single and multiple resistance to viruses infecting peanut, some measures should be modified to improve the resistance efficiency. Now new plant expression vectors are under constructed to improve transgenic resistance.