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