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Study on Identification and Function Analysis of Two RNA Silencing Suppressors Encoded by BYDVs

Author: DiHao
Tutor: LiuYan
School: Chinese Academy of Agricultural Sciences
Course: Plant Pathology
Keywords: Barley yellow dwarf virus Gene silencing Silencing suppressor Infectious cDNA clone
CLC: S435.121
Type: Master's thesis
Year: 2011
Downloads: 46
Quote: 0
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Abstract


BYDV caused by barley yellow dwarf virus (Barley yellow dwarf viruses, BYDVs) occur widely in the world, in China's wheat-growing gap large threat to wheat production. Order to ultimately achieve the purpose of prevention and treatment of the disease, the scientists of countries of barley yellow dwarf virus gene expression, evolution, spread mediator host interactions, etc. to carry out an in-depth fundamental research. Screening identified the BYDVs genome that may exist in silence suppressor, host interaction and provide new evidence for the study of BYDV gene. Our laboratory has been initially identified the ORF6 of BYDV-GAV and BYDV-GPV P0 silencing suppressor activity in local infection. Filter constructed plasmid vector PGD-GPV-P0 the PGD-GPV-CP the PGD-GPV-MP, pBin-GAV-CP, pBin-GAV-ORF1 and pBin-GAV-ORF6 6 gene a recombinant vector, to increase the rigor of the experiment, the experiment new construction of a the PGD-GPV-UTR the-P0 recombinant vector; followed by experiments proved by repeating silencing phenotype observed the GPV P0 and GAV ORF6 indeed Localized Infection Inhibition of promoter activity. Followed by semi-quantitative PCR infiltration of GFP mRNA levels were tested and found strong inhibition of sub-protein HC-Pro pBin-mGFP5-ER N. benthamiana after infiltration, infiltration of GFP mRNA levels were significantly higher than pBin-GAV-ORF6 or PGD-GPV-P0 and pBin-mGFP5-ER co-infiltration blade, levels of GFP mRNA accumulation level is higher, the vaccination PGD-GPV-P0 and pBin of-mGFP5-ER co-infiltration leaves levels of GFP mRNA accumulation level than pBin-GAV -ORF6 and pBin-mGFP5-ER accumulation level. RNA quantification standard internal reference gene EF-1-alpha of mRNA levels in the plants of the type of infiltration composition are identical. The results indicate that the GAV-ORF6 inhibition of promoter activity is weaker than the GPV-P0. GFP protein by Western blot and GFP mRNA, siRNA of Northern blot analysis further confirmed by GPV-P0 GAV-ORF6 showed inhibition of promoter activity differences. Although GPV-P0, GAV-ORF6 expression with the positive control HC-Pro, it is possible to increase the amount of expression of the GFP protein and GFP mRNA accumulated amount, thus enhancing the GFP fluorescence can be seen from the experimental GPV-P0 such suppressors The cumulative amount of the amount of GFP protein and GFP mRNA was significantly higher than Gav-ORF6. That GPV-P0 suppressor GAV-ORF6 is weak inhibitor. In addition, the PGD-GPV-P0 plant pBin-GAV-the ORF6 containing GFP expression vector pBin-mGFP5-ER four-leaf stage of transgenic tobacco 16c plants of infiltration, and HC-Pro as a control. Non-invasive blade Northern blot detection, it also proves GPV P0 GAV ORF6 and PVY HC-Pro as systemic gene silencing suppression caused by the GFP positive single-stranded RNA in 16 days after infection. In addition, to study the the BYDV genomic structure of the gene mutation (insertion, deletion, repeat, etc.) affect viral replication and expression strategy, to verify viral protein function and to determine important functional domains, create BYDVs infestation cDNA cloning technology platform is particularly important. This article to PAV-06KM14 isolates the object, the successfully constructed pTCK303-06KM14 (Ubi promoter under the control of) and the PGD-06KM14 full-length gene group infectious cloning vectors were transfected gene gun wheat immature embryo or Agrobacterium infection The N. benthamiana get infectious plant. BYDV symptoms like plants experimental, infection activity verification experiment underway.

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CLC: > Agricultural Sciences > Plant Protection > Pest and Disease Control > Crop pests and diseases and their prevention > Cereal crop pests and diseases > Wheat pests and diseases > Disease
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