Antisense suppression of replicase gene expression recovers
tomato plants from leaf curl virus infection
Shelly Praveena,*, Anil K. Mishraa, Arupratan Dasguptab
Plant Science 168 (2005) 1011–1014
學生 : 林怡芳
Introduction
• 作者要把這 ANTISENE 送進被 ToLCV 感染的蕃茄 , 使 ToLCV 病毒 Rep gene silence
7 days — young leaves start to curl
21 days — leaves curling and early stages of interveinal yellowing
35 days — marked leaf curling and interveinal yellowing 56 days — plant distortion and loss of leaf development
Stages of disease development in tomato
http://www.dpi.qld.gov.au/health/4250.html
Normal tomato leaf
~1600bp
pCAMBIA R-gene plants
R-gene bacteria
Polylinker Reporter
Gene
T-DNA size (bp)
Reading Frame or lacZ Vector
Vector
family
2301 nptII kan pUC18 gusA 5391 - GIS
http://www.cambia.org/daisy/cambia/585.html
利用植物組織培養建立環境污染物檢測技術國立高雄師範大學生物科學研究所 廖 麗 貞
Methods以葉原片為材料
農桿菌為媒介
轉殖基因
以打孔器取葉原片
將葉原片與農桿菌共同培養
移植至植物組織生長培養基培養含特殊營養成分之培養基
葉緣細胞分裂
移至含特殊營養成分之誘芽培養基
含特殊營養成分之誘根培養基
共同培養 2-3 天
共同培養 2-4 週
長出芽體
長出根移出種植於土中
再生植株利用植物組織培養建立環境污染物檢測技術國立高雄師範大學生物科學研究所 廖 麗 貞
Since the only RNA found in a cell should be single stranded, the presence of double stranded RNA signals is an abnormality. The cell has a specific enzyme (in Drosophila it is called Dicer) that recognizes the double stranded RNA and chops it up into small fragments between 21-25 base pairs in length. These short RNA fragments (called small interfering RNA, or siRNA) bind to the RNA-induced silencing complex (RISC). The RISC is activated when the siRNA unwinds and the activated complex binds to the corresponding mRNA using the antisense RNA. The RISC contains an enzyme to cleave the bound mRNA (called Slicer in Drosophila) and therefore cause gene suppression. Once the mRNA has been cleaved, it can no longer be translated into functional protein
http://www.bioteach.ubc.ca/MolecularBiology/AntisenseRNA/
後轉錄基因靜默化 PTGS ( post-transcriptional gene silencing )又名為 RNA 靜默化( RNA silencing ),被認為是真核生物所演化出的種對抗外來基因
http://www.nre.vic.gov.au/agvic/ihd/images/rt-pcr1.gif
Methods
• Analysis of transgenic plants
1. PCR analysis
2. PCR-Southern
Fig. 1. Transgene (antisense-Rep)-induced silencing of Tomato leaf curl virus: (B) phenotypic alterations caused by transgene-induced silencing of virus and recovery of plant from viral infection; (C) molecular analysis of post-transcriptionally silenced (transformedrecovered) and non-silenced (non-transformed-regenerated); designated by _ respectively; PCR using specific primers for ToLCV-CP amplification (_750 bp)in transformed-recovered,non-transformed-regenerated and infected positive control (C) to determine the presence of virions; Southern and Northern blot analyses to demonstrate the presence of viral genome in non-silenced and positive control infected plant and 21–25 ntd long siRNA in silenced plants using different probes indicated in the left. Numbers below indicate individual representatives of silenced and non-silenced plants.
Results
Discussion
• ToLCV-infected tomato plants containing antisense Rep gene construct
• 作者以他設計的實驗證明其 Rep of ToLCV antisense gene 會使 ToLCV 病毒 Rep gene silence