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Plant DNA virus satellite silent vector and method for constructing and using same

A DNA virus and silencing technology, applied in the field of plant DNA virus satellite silencing vector, can solve the problem of narrow virus host range, and achieve the effects of small workload, simple construction and high silencing efficiency

Inactive Publication Date: 2010-10-13
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because some viruses have a narrow host range, some produce severe virus symptoms on plants, and some RNA viruses are easily degraded by plants and cannot produce sustained gene silencing phenotypes, etc., it can really be used in a wide range of functional gene research. Viral vectors are still rare, and finding good viral gene silencing vectors that can overcome these shortcomings has always been a hotspot of botanist research (Robertson D, 2004, Annu Rev Plant Biol55: 495-519)

Method used

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  • Plant DNA virus satellite silent vector and method for constructing and using same
  • Plant DNA virus satellite silent vector and method for constructing and using same
  • Plant DNA virus satellite silent vector and method for constructing and using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Construction of TbCSV DNA1 viral satellite silencing vector with a single multiple cloning site

[0027] Extract the total DNA of tobacco plants infected with TbCSV [Murray MG et al., 1987, EMBO J, 6: 3901-3907], because the infectious clone of geminivirus requires 1.3-2.0 direct repeats of the full-length genome (including common domain sequences of two geminiviruses). With TbCSV tobacco plant total DNA as a template, the specific primer designed according to the gene sequence of SEQID NO.1 is DNA1XSB

[0028] (5'-TCTAGACCCGGGATCCGAGTATAAATACGTTAATTTTGC-3' introduced XbaI, SmaI and BamHI restriction sites) and DNA1K

[0029] (5'-GGTACCGTATTTAGTCCAAATACTCGTCGC-3'introduce KpnI enzyme cutting site) carry out PCR amplification to TbCSV tobacco plant total DNA, design another pair of specific primers according to SEQ ID NO.1 gene sequence simultaneously, DNA1S (5'-GTCGACGAGTATAAATACGTTAATTTTGC- 3' introduces SalI restriction site) and DNA1X (5'-TCTAGAGTATTTAGTC...

Embodiment 2

[0032] Embodiment 2 Plant transgene or the cloning of endogenous gene cDNA fragment

[0033] Total RNA was extracted from 16C plants (transformed to GFP Nicotiana benthamiana, see Voinnet and Baulcombe, 1997, Nature, 389:553), Nicotiana benthamiana, tomato and petunia (Johansen, LK, and Carrington, JC, 2001, Plant Physiol , 126:930-938), using the mRNA in the total RNA as a template and the specific primers of each gene to carry out two-step RT-PCR amplification of the cDNA fragment of the target gene. The specific method is as follows: the first-strand cDNA was synthesized using the ReverseTranscription System kit (Promega Company). RNA 5 μl in DEPC-treated Eppendorf tube, Olige dT primer 1 μl, RNase free H 2 O5μl, 70℃ water bath for 10min; add 5×cDNA buffer4μl, 0.1mol / L DTT2μl, 10mM dNTP1μl, Rnase inhibitor1μl, 42℃ water bath for 2min, add 1μl M-MLV42℃50min, then 70℃15min inactivate reverse transcriptase, That is, the first strand of cDNA of a synthetic transgenic plant ge...

Embodiment 3

[0034] Example 3 Construction of DNA1 Recombinant Virus Satellite Silencing Vector Containing Plant Transgene and Plant Target Gene Fragment

[0035] The 170bp GFP cDNA fragment GFP170 (SEQ ID NO.2) amplified from 16C plant mRNA and verified by sequencing, and the 351bp Nicotiana benthamiana Su cDNA amplified from Nicotiana benthamiana and tomato mRNA and verified by sequencing Fragment NbSu351 (SEQ ID NO.3) and tomato Su cDNA fragment ToSu351 (SEQ ID NO.4), the 350bp PCNA cDNA fragment PCNA350 (SEQ ID NO.5) and The 351bp CHS cDNA fragment CHS351 (SEQ ID NO.6) amplified from petunia mRNA and verified by sequencing was inserted into the viral satellite silencing vector pBINPLUS-2mDNA1 after double digestion with XbaI and BamHI, and constructed to induce 16C plants , tobacco, tomato and petunia plant gene silencing pBINPLUS-2mDNA1-GFP170, pBINPLUS-2mDNA1-NbSu351, pBINPLUS-2mDNA1-ToSu351, pBINPLUS-2mDNA1-PCNA350 and pBINPLUS-2mDNA1-CHS351 recombinant virus satellite silencing vec...

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Abstract

The invention discloses a plant DNA virus satellite silencing vector and a construction and use method for the vector. The plant DNA virus satellite silencing vector comes from a satellite molecule incidental with tobacco curly shoot virus-DNA 1, and introduces a single multi-cloning site at the downstream of a Rep open reading frame of the DNA 1. The invention also provides a method for studyingthe functional gene group of plants by taking use of the plant DNA virus satellite silencing vector. The recombinant virus satellite silencing vector containing the target gene of plant is constructed by cloning a cDNA section containing plant target gene by RT-PCR method and inserting the multi-cloning site of DNA 1 virus satellite silencing vector. The recombinant virus satellite silencing vector and DNA-A of China tomato yellowing leaf curling virus are injected and inoculated into a plant by agrobacterium to induce and silence the target gene of the plant so that the surface of the plant will change, and real-time fluorescent quantitative RT-PCR technology is used to detect the expression volume of the target gene so as to determine the function of the gene.

Description

technical field [0001] The field of the invention belongs to the field of biotechnology, and in particular relates to a plant DNA virus satellite silencing vector and its construction and use method. Background technique [0002] Gene silencing is a highly conserved specific degradation mechanism based on nucleic acid level discovered in recent years. Virus induced gene silencing (VIGS) is a powerful research tool for studying plant genomes using the principle of gene silencing. It uses the method of reverse genetics to induce plant gene silencing and phenotypic mutations after the virus carrying the plant gene cDNA infects the plant, so that the changes in the plant phenotype or physiological indicators can reflect the change of the gene. Function. The main mechanism is that the plant gene inserted into the viral vector forms a dsRNA intermediate with the replication of the virus, and is degraded into 21-23nt RNA by an enzyme called Dicer, and combines with RNAase to form...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/33C12Q1/68
Inventor 周雪平黄昌军谢艳吴建祥
Owner ZHEJIANG UNIV
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