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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: 2004-12-15
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 Agrobacterium injection was first successfully applied to cauliflower mosaic virus among dsDNA viruses (Grimsley 1986), and was gradually applied to geminiviruses. It is a simple and effective method, but the plant expression vectors used in the past replicated The amount is low, and it is not easy to carry out molecular biology operations. The present invention is based on the Chinese yellow leaf curl virus genome DNA-A and the new satellite molecule DNAβ, and this method has been improved.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Cloning and Determination of Complete Genome Sequence of DNA-A Component and DNAβ Component of Chinese Tomato Yellow Leaf Curl Virus Yunnan Honghe Y10 Isolate

[0030] 1. Extraction of total plant DNA

[0031] Weigh 0.5-1g geminivirus-infected leaves, add liquid nitrogen and grind to powder, add 15ml extraction buffer (100mM Tris.Cl pH8.0, 50mM EDTA pH8.0, 500mM NaCl, add β-mercaptoethanol 2.3 μl / ml), continue to grind, transfer to a 50ml centrifuge tube. All samples are kept at 4°C. Add 1ml of 20% SDS, mix by inverting gently, and incubate at 65°C for 10min. Add 5ml 5M KAc, mix by inverting gently, and place on ice 20 minutes. Centrifuge at 4000rpm for 30 minutes. Filter the supernatant through gauze and pour it into a pre-cooled 50ml centrifuge tube with 15ml of isopropanol, mix well, and place at -20°C for 30min (or -80°C for 10min). Discard the supernatant, dissolve the precipitate with 0.75ml TE (50mM Tris-Cl pH8.0, 10mM EDTA), and transfer to a 1.5ml ce...

Embodiment 2

[0034] Example 2 Construction of Chinese Tomato Yellow Leaf Curl Virus Yunnan Isolate DNA-A and DNAβ Infectious Vector

[0035] a. Construction of DNA-A component invasive clone

[0036] Agrobacterium-mediated invasive cloning of geminiviruses requires a full-length genome of more than 1.3-2.0 repeats and must include the common region. Using full-length DNA-A clone as template, FL / F(5'-C GTC GAC ACCTGTTTGGGGATATGAGAT-3') and PL / R(5'T GAGCTC GTATTAGTCATAGAGGGTGATAG-3') as primers, the PCR product was cloned into pGEM-T Easy Vector to obtain pGEM-T-Vector-PL, digested with Sal I and EcoR I, recovered about 2kb band and inserted into the corresponding site of Agrobacterium vector pBinPLUS , and obtained 0.7 full-length clone pBINplus-PL. Another full-length copy of DNA-A came from the splicing of pGEM-T-Vector-DNA A and pGEM-T-PL inserted in the same direction, using Bam H and Sac I digested pGEM-T-PL, recovered a fragment of about 1.8kb and ligated it with pGEM-T-Vector-DN...

Embodiment 3

[0039] Embodiment 3. Transformation of recombinant vectors to Agrobacterium

[0040] The transformation of the plant expression vector is to enter the Agrobacterium host cell through the method of triparental mating, and the plant expression vector pBinPLUS needs to be mediated by the auxiliary plasmid pROK203 to enter the Agrobacterium EHA105. pBinPLUS-PL+FL(DH5α) was inoculated with 5ml of LB liquid medium containing Km (50mg / ml), cultured overnight at 200rpm at 37°C; EHA105 was inoculated with 5ml of YEP liquid medium containing Sm (50mg / ml), and cultured at 28°C at 200rpm 48 hours; inoculate 5 ml of LB liquid medium containing Km (50 mg / ml) with the helper plasmid pROK2013, and culture overnight at 200 rpm at 37°C. Take 400μl of pBinPLUS-PL+FL(DH5α), EHA105, and pROK2013 bacteria solution, mix them upside down, collect the bacteria solution at 6000rpm for 30 seconds, and wash the collected bacteria with 200μl ddH 2 O suspension, coated with a glass coating rod on a non-re...

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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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Patent Type & Authority Patents(China)
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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