| Wheat yellow dwarf disease caused by barley yellow dwarf viruses (BYDVs) takes place worldwide, and occurs intermittently in china's main wheat-growing area, threatening wheat production. To achieve the final purpose of preventing and curing this disease, scientists have carried out in-depth researches on gene expression, evolution, and the interaction between transmission vector and host in barley yellow dwarf virus. Screening out silencing suppressors possibly present in BYDV genome can provide new evidence for the study of interaction between BYDV genes and the host.Through preliminary detection, ORF6 in BYDV-GAV and P0 in BYDV-GPV have been identified that they have silencing suppressor activity. On this basis, experimental materials are optimized firstly, and six recombinant vectors P0, CP, MP of GPV with pGD as vector and CP, ORF1, ORF6 of GAV with pBinPLUS as vector are screened out and a new vector pGD-UTR-P0 of GPV is reconstructed to make the experiment more precise and accurate. Besides, through repeating silence phenotype observation, GPV-P0 and GAV-ORF6 is proved to possess silencing suppressor activity in local infection. Then semi-quantitative PCR is used to detect mRNA level of GFP in infiltrating area, and it is found that GFP mRNA accumulation level when strong suppressor protein HC-Pro and pBin-mGFP5-ER are altogether infiltration Nicotiana benthamiana is higher than pBin-GAV-ORF6 or pGD-GPV-P0 and pBin-mGFP5-ER are altogether infiltrated blades, and GFP mRNA accumulation level infiltrated with pGD-GPV-P0 and pBin-mGFP5-ER is higher than that infiltrated with pBin-GAV-ORF6 and pBin-mGFP5- ER. mRNA level of reference genes EF-1-α, quantitative criteria of RNA, is the same in different infiltrating combination of plants. The result indicates that the activity of suppressor GAV-ORF6 is weaker than GPV-P0. The analysis results from Western blot of GFP protein and Northern blot of GFP mRNA and siRNA further confirm the differences in the suppressor activity expressed by GPV-P0 and GAV-ORF6. Though the expression of GPV- P0 and GAV-ORF6, the same as the positive control, can increase the expression and accumulation of GFP protein, the GFP mRNA accumulation, and further strengthens GFP fluorescence. It can also be seen from the experiment that the expression, accumulation and GFP mRNA accumulation of GFP protein of the suppressor GPV-P0 is obviously higher than that of GAV-ORF6. In addition, pGD-GPV-P0, pBin-GAV-ORF6 and the plant expression vector containing GFP (pBin-mGFP5- ER) are respectively used to infiltrate tobacco 16c plant at four-leaf stage with HC-Pro as a control sample. 16 days after the infiltration, Northern blot test to the non-infection leaves proves that GPV-P0, GAV-ORF6, the same as PVY HC-Pro, can restrain the systemic gene silencing caused by single-strand RNA of GFP.In addition, to study the effect of gene mutation (insertion, loss, repetition) of BYDV genome structure on virus replication and its expression strategy, verify the protein function of virus, and confirm the important function domains, it is particularly important to establish BYDVs infectious cDNA clone technology platform. This paper takes isolate PAV-06KM14 as the subject, successfully constructs full-length genome infectious clone vectors pTCK303-06KM14(under control of ubi promoter) and pGD-06KM14, and respectively transfects wheat embryo with Particle gun or infect Nicotiana benthamiana with agrobacterium infiltration to obtain the infective plants. Recently plants with similar BYDV symptoms have been received, and the verification test of the infective activity is underway.
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