Transcript Antisense suppression of replicase gene expression recovers

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
Stages of disease development in tomato
Normal tomato leaf
7 days — young leaves start to
curl
35 days — marked leaf curling and interveinal yellowing
21 days — leaves curling and early
stages of interveinal yellowing
56 days — plant distortion and loss of leaf development
http://www.dpi.qld.gov.au/health/4250.html
~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
以打孔器取葉原片
以葉原片為材料
Methods
將葉原片與農
桿菌共同培養
農桿菌為媒介
轉殖基因
含特殊營
養成分之
培養基
葉緣細
胞分裂
移植至植物組織生長培養基培養
共同培養2-3天
移至含特殊營養成分之誘芽培養基
共同培養2-4週
長出芽體
含特殊營養成分之誘根培養基
長出根
移出種植
於土中
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
再生植株
後轉錄基因靜默化 PTGS（posttranscriptional 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
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/
http://www.nre.vic.gov.au/agvic/ihd/images/rt-pcr1.gif
Methods
• Analysis of transgenic plants
1. PCR analysis
2. PCR-Southern
Results
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 posttranscriptionally silenced (transformedrecovered) and non-silenced (non-transformed-regenerated); designated
by _ respectively; PCR using specific primers for ToLCV-CP amplification (_750 bp)in transformedrecovered,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.
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