Beet black withered virus as expression carrier of foreigh gene

A technology of exogenous gene and sugar beet, applied in the direction of virus/bacteriophage, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2004-02-11
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

China is the only country that has carried out research on BBSV, but current research on the virus is limited to basic biology such as virus biology, morphology and serology

Method used

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  • Beet black withered virus as expression carrier of foreigh gene
  • Beet black withered virus as expression carrier of foreigh gene
  • Beet black withered virus as expression carrier of foreigh gene

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Effect test

Embodiment 2

[0029] The various test methods and test techniques involved in this embodiment are common knowledge, and those of ordinary skill in the art can implement the basic molecular biology test methods described in the "Molecular Cloning Experiment Guide" and the like. Example 2: Construction of BBSV invasive cDNA clone under the control of T7 promoter, in vitro transcription and infective activity determination

[0030] The primers BB-18 and BB-14 were designed according to the BBSV RNA terminal sequence. The 5′ end of the BB-18 primer introduced the T7 RNA polymerase promoter sequence, and the 5′ end of the BB-14 primer introduced the SmaI restriction site (in BBSV There is no such site in the whole sequence). Using purified BBSV RNA as a template, RT-PCR amplification (denaturation at 94°C for 1 min, annealing at 52°C for 1 min, and extension at 72°C for 4 min) yielded a 3.64 kb full-length BBSV cDNA ( figure 2 ). The PCR product was cut flat by T4 DNA polymerase and then ligated in...

Embodiment 3

[0032] Amaranthus leaves were inoculated with pUBF52 in vitro transcript and viral RNA, and total RNA from the leaves was extracted, separated by 1% denaturing agarose gel electrophoresis, and transferred to Hybond-H via 20×SSC by capillary method. + On nylon membrane. According to the basic experimental method described in the "Molecular Cloning Experiment Guide", the 3'-end 0.3kb non-coding region of digoxigenin-labeled BBSV was used as a probe, and the probe was pre-hybridized (pre-hybridized at 42°C for 5-6 hours) and hybridized (42 ℃ hybridization for more than 12 hours), washing and color development and other steps for Northern blot detection, the results showed that in addition to the BBSV genomic RNA (size 3641bp) main band, there are two small subgenomic bands ( Figure 4 ). It shows that the in vitro transcript of pUBF52 can not only infect the host plant, but can also replicate in the host, just like BBSV RNA. Example 4: Western blot detection of in vitro transcript in...

Embodiment 4

[0033] PUBF52 in vitro transcript and viral RNA were used to inoculate the leaves of Chenopodium arundinacea. After 3-4 days, 0.5-1.0 g of diseased leaves were taken, ground in liquid nitrogen, and 300μl of protein loading buffer (40mM Tris-Cl, pH6.8, 10% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.1% bromophenol blue), shake it in a boiling water bath for 10 minutes, immediately place it on ice to cool, centrifuge to take the supernatant, and separate it with 12.5% ​​SDS-PAGE gel , Using electrotransfer method to transfer protein to nitrocellulose membrane (NC membrane), using the inventor’s self-made BBSV-specific antiserum and alkaline phosphatase-labeled goat anti-rabbit IgG as the primary and secondary antibodies, respectively. The secondary antibody, using NBT and BCIP as chromogenic substrates, was used to detect the in vitro transcripts of pUBF52 and the expression products of viral RNA in host plants by Western blot. The results showed that it was detectable in the amaranth...

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Abstract

The present invention describes the whole nucleotide sequence of beet black scorch virus (BBSV). The cDNA cloning of BBSV whole genome may be transcribed under the control of eukaryotic and prokaryotic promoter to produce viral RNA with infection capacity to host plant. Taking the insertion of medusa green fluorescent protein gene as example, the present invention shows that obtained recombinant BBSV may be used as carrier for carrying virus to express exotic gene in plant.

Description

1. Technical Field [0001] The present invention relates to a vector used in genetic engineering. Specifically, it is a virus-derived vector that can carry foreign genes for expression in plants. 2. Background technology [0002] Since the 1980s, a black scorched beet virus disease has been discovered in the main sugar beet producing areas of Xinjiang, Ningxia, Inner Mongolia, Gansu, Heilongjiang, Jilin, etc., which has brought huge losses to production [Cui Xingming, etc., Shihe Zinong College Journal, 10(1), 73-78(1988)]. The symptoms of the disease on sugar beet are mainly manifested as the leaves stand upright, the black scorch spots between the leaf veins, the leaf margins are curled, and the root hairs are massively necrotic. The damage to the sugar beet has exceeded that of the Beet necrotic yellow vein virus BNYVV), and is getting worse [Liu Jiexian et al., Chinese sugar beet, (3), 30-31 (1995)]. In 1989, the diseased area in Ningxia alone accounted for 69.2% of the sugar ...

Claims

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Application Information

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IPC IPC(8): A01H1/00C12N7/01C12N15/34C12N15/40C12N15/63C12N15/82
Inventor 于嘉林蔡祝南李大伟韩成贵刘仪曹云鹤原雪峰丁群
Owner CHINA AGRI UNIV
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