Method for establishing carrier by plant DNA virus infestation

A DNA virus and construction method technology, applied in the field of genetic engineering, can solve the problems of low replication volume of plant expression vector, difficult molecular biological operation, high cost of gold powder, etc., and achieve the effects of small replication volume, simple inoculation method, and enhanced efficiency

Inactive Publication Date: 2003-06-18
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The friction inoculation method is only suitable for viruses that can be transmitted mechanically, and most geminiviruses cannot infect host plants through friction inoculation, so this method has great limitations
When using the gene gun method, the construction of clones is simple, but it is highly dependent on experimental equipment, and the cost of gold powder for each gene gun bombardment is expensive, which will be limited by the high cost in large-scale experiments
The use of Agrob

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0028]Example 1 Cloning and determination of the complete genome sequence of the DNA-A component and DNA β component of the Yunnan Honghe Y10 isolate of Chinese tomato yellow leaf curl virus

[0029] 1. Extraction of total plant DNA

[0030] Weigh 0.5-1g of the infected leaves of Gemini virus, add liquid nitrogen to grind to powder, add 15ml extraction buffer (100mM Tris.Cl pH8.0, 50mM EDTA pH8.0, 500mM NaCl, add β-mercaptoethanol 2.3 before use μl / ml), continue grinding, transfer to a 50ml centrifuge tube and keep all samples at 4°C. Add 1ml of 20% SDS, gently mix upside down, and incubate at 65°C for 10 minutes. Add 5ml 5M KAc, gently mix upside down, and place on ice for 20 minutes. Centrifuge at 4000 rpm for 30 minutes, and pour the supernatant through gauze into a pre-cooled 50 ml centrifuge tube of 15 ml isopropanol, mix well, and place at -20°C for 30 minutes (or at -80°C for 10 minutes). Centrifuge at 13000rpm for 15min, discard the supernatant, dissolve the pellet with 0....

Example Embodiment

[0033] Example 2 Construction of DNA-A and DNAβ infectious vector of Chinese tomato yellow leaf curl virus Yunnan isolate a. Construction of invasive clone of DNA-A component

[0034] Agrobacterium-mediated invasive cloning of geminivirus requires a full-length genome with more than 1.3-2.0 repeats and must include a common region. Using full-length DNA-A clone as template, FL / F(5’-C GTCGACAC CTGTTTGGGGATATGAGAT-3’) and PL / R(5’-T GAGCTC GTATTAGTCATAGAGGGTGATAG-3') is the primer, the PCR product is cloned into pGEM-T Easy Vector to obtain pGEM-T-Vector-PL, and after digestion with Sal I and EcoRI, about 2kb band is recovered and inserted into the corresponding site of Agrobacterium vector pBinPLUS , Get 0.7 full-length clone pBINplus-PL. Another full-length copy of DNA-A comes from the splicing of pGEM-T-Vector-DNA A and pGEM-T-PL inserted in the same direction. The pGEM-T-PL is digested with Bam H and Sac I and recovered After the about 1.8kb fragment is ligated with pGEM-T-Vecto...

Example Embodiment

[0036] Example 3. Transformation of Agrobacterium by recombinant vector

[0037] The plant expression vector is transformed into Agrobacterium host cells through three-parent mating. The plant expression vector pBinPLUS needs to be mediated by the helper plasmid pROK2013 to enter Agrobacterium EHA105. pBinPLUS-PL+FL (DH5α) was inoculated with 5ml of Km (50mg / ml) LB liquid medium and incubated overnight at 37°C at 200 rpm; EHA105 was inoculated with 5ml of Sm (50mg / ml) YEP liquid medium and cultured at 28°C at 200 rpm 48 hours; the helper plasmid pROK2013 was inoculated with 5ml of Km (50mg / ml) LB liquid medium and cultured overnight at 37°C at 200rpm. Take 400μl of pBinPLUS-PL+FL(DH5α), EHA105, pROK2013 bacteria solution, invert and mix well, collect the bacteria solution at 6000rpm for 30 seconds, and use 200μl ddH for the collected bacteria 2 O suspended, coated with a glass coated rod on a non-resistant YEP solid medium plate for three-parent mating. After incubating at 28°C fo...

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Abstract

A process for configuring plant DNA virus infections carrier includes such steps as infectious cloning to genom DNA-A and satellite modecule DNA beta by means of the separated yellowing-leaf-curing virus of tomato, overlap-PCR and enzyme severing to obtain two directly repeated genoms, inserting them to high-cloning plant expression carrier, and introducing high-infection Agrobacterium strain by triparental cross.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing a plant DNA virus infective vector. Background technique [0002] Infectious vectors of plant viruses are a crucial platform for the study of viral genomes. To study the structure and function of viral genomes and the interaction between hosts and viruses at the level of molecular biology, it is necessary to obtain infectious clones of viruses. Only on this basis can a uniform population of wild-type molecules or mutant molecules be obtained, and then use DNA recombination technology to perform mutations, deletions, insertions, substitutions, and complementation experiments to locate the functional regions of the viral genome, so that the host plant phenotype level can Reflect and evaluate the expression of gene function. This technology is an effective means for in-depth study of the gene function of viruses and the interact...

Claims

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Application Information

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IPC IPC(8): C12N15/70C12N15/74C12N15/83C12Q1/68C12Q1/70
Inventor 周雪平陶小荣崔晓峰谢艳李正和
Owner ZHEJIANG UNIV
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