| A typical symptom recovery appears on tobacco (Nicotiana tabacum cv. white Burley) plants infected with Cucumber mosaic virus strain M (CMV-M). It is a significant step for the finding of symptom recovery mechanism of virus infection if the variation of virus concentration in the tobacco plants infected with CMV-M is demonstrated. A quantitative method for detection of CMV-M was established by purified CMV particles and DAS-ELISA. The results showed that the virus concentration is positively related to the severity of the symptoms, with the highest up to 790μg/g tissue in the first appeared yellow symptom leaves, up to 508μg/ g tissue in the upper mosaic symptom leaves; only 6μg/ g tissue in the recovery symptomless middle leaves, which was about 85 to 135 times less than that in the mosaic or yellow symptom leaves and also much lower than that in the stems and roots. RT-PCR and biological experiment showed that infectious virus did exist in the recovered leaves, but somehow the virus can not reach higher concentration and cause obvious symptom, which indicated that an efficient resistant mechanism exists in symptom recovery leaves.symptom recovery appears on tobacco (Nicotiana tabacum cv. white Burley) plants infected with Cucumber mosaic virus strain M (CMV-M) ,which is liable to gentic of diferent strain,so it is vital to test the geonme sequence of CMV-M. RNA2 of Cucumber mosaic virus encode RdRp and 2b protein,2a gene encode RdRp is a important factor in RISC,2b gene encode 2b protein is supress of RNA silencing.we test part sequence of RNA2 and get the whole of RNA2,We find the Identification is very high.the genetic identification to Fny strain and Y strain is 98%, 2a amino acid identification to Fny strain is 97%,to Y strain is 98%.2b amino acid identification to Fny strain is 97%,to Y strain is 100%. which indicated that symptom recovery affected not only by virus,but by tobacco plant too.Because the two gene is critical to our research,we should research them fully basing on getting the two gene firstly. we clone three segment of RNA2 ORF to pMD18-T Vector,and titled it P2b-T,P2a.3-T,P2a.5-T, Identified by restrictive enzyme,and we prepare probe by P2b-T,which can prepare for analysis existing siRNA come from RNA2 or not, distributing and quantity in tobacco plant,and clone the two gene to GFP,all of this can help us further research symptom recovery relatived to RNA silencing. To recognize 2a gene expression in different tissue in difreent times and location,and it is function in symptom recovery mechanism,needing analysis technology of serology,so it is key to prepare good antiserum.2a gene encode protein is not configuration protein,so can get antiserum by purified CMV particles.We clone segments of 2a gene to pET-30a(+),titled them P2a.3-30a,P2a.5-30a,and express P2a.3-30a in E.coli basing on comparing Expression in E.coli and yeast,get high efficiency expression protein about 63kDa and it's antiserum, we found it's efficacy is 1:8192(1:213) by ACP-ELISA and specialties is good, which indicated that 1200bp of 2a gene 3'is one of antigen region.The Pathogenic of virus to plant is affected by satellite virus drastictly,and different satellite virus have different result.We found that there is no Satellite virus in M-CMV by dsRNA technology, which deny directly symptom recovery relative to Satellite virus.Cucumber mosaic virus is RNA virus, so the ration of mutant is higher and there are many strain. Finding symptom recovery mechanism need research some gene of effective mutant.however,it hard to find the factor relativing to symptom recovery basing on camparing other strain directly,which experinced much time. So the best stragic is to get mutant from M-CMV because it come from other strain.We get a mutant strain of M-CMV by separate in single speckle,which the symptom can't recovery 35 days after inoculation,it afford us good material in further research symptom recovery mechanism. at the same time,it indicated that symptom recovery possible relative to some nucleic acid mutant.
|
PDF Full Text Request