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Gene transfer vector comprising papaya ringspot virus auxiliary component protease gene and application thereof for providing broad-spectrum virus resistance in crops

An auxiliary component, the technology of ringspot virus, is applied in the direction of plant gene improvement, application, genetic engineering, etc. It can solve the problems of inability to function, failure of transgenic papaya, etc., and achieve the effect of not relying on homologous resistance

Active Publication Date: 2011-12-14
薛富盛
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

HC-Pro is a gene silencing suppressor (suppressor), which has the ability to inhibit post-transcriptional gene silencing (Anadalakshmi et al., 1998; Brigneti et al., 1998; Shi et al., 1997), the applicant speculated that PRSV 5-19 HC-Pro has a strong ability to inhibit gene silencing, which can make the gene silencing mechanism of PRSV YK CP transgenic papaya inhibited by 5-19HC-Pro, so that it cannot function, thus suppressing the resistance of transgenic papaya and fail

Method used

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  • Gene transfer vector comprising papaya ringspot virus auxiliary component protease gene and application thereof for providing broad-spectrum virus resistance in crops
  • Gene transfer vector comprising papaya ringspot virus auxiliary component protease gene and application thereof for providing broad-spectrum virus resistance in crops
  • Gene transfer vector comprising papaya ringspot virus auxiliary component protease gene and application thereof for providing broad-spectrum virus resistance in crops

Examples

Experimental program
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Effect test

Embodiment 1

[0051] Embodiment 1. Preparation of the non-translational construct of the auxiliary component protein HC-Pro gene

[0052] The HC-Pro gene (SEQ ID NO: 8) of PRSV 5-19 was amplified by RT-PCR and constructed in Vector (Invitrogen), constructed in a non-translational manner, designed a set of specific primers: 519HCStopA (5'-CGCATGAACATGC TCTAGA TGAACGATTGATGAGAAAAAATTTCTG-3') (SEQ ID NO: 1) and 519HCSacB (5'-GTGGTTGGATCAAA GAGCTC ACCGACAATGTAGTGTTTCATTTC-3′) (SEQ ID NO: 2), two stop codons (TGATGA), XbaI restriction site ( TCTAGA ) and add one more base (T) to shift the entire protein reading frame, and the downstream primer (519HCSacB) designed a SacI restriction site ( CTCGAG ), the HC-Pro full-length gene (after 1371bp) on the TOPO carrier was amplified by PCR to 1372bp (SEQ ID NO: 5) by adding a base T, and loaded with the same restriction enzyme after digestion with XbaI and SacI restriction enzymes Digested pBI121 (Clontech, CA), and finally obtained the non-tran...

Embodiment 2

[0056] Embodiment 2. Preparation of transgenic papaya

[0057] In order to shorten the time for transfer and screening, a set of rapid papaya transgenic methods (Kung et al., 2009) developed by the applicant's laboratory in recent years was used. Papaya bottle seedling propagation in laboratory tissue culture system. In Murashige-Skoog medium containing 0.02mg / lα-naphthaleneacetic acid and 0.2mg / l benzylaminopurine (MSNB ) were cultured for two weeks, transferred to MS medium containing indole-3-butyric acid (IBA), and induced root primordia to occur after one week of dark culture at 28°C. At this time, it was in the root induction period (root induction period) [with Plant growth hormone dependent (Auxin dependent)], and then moved to pearlite containing 1 / 2 volume of MS to prolong root development, at this time in the root development period (Root development period) [has nothing to do with plant growth hormone (Auxin independent )], two weeks later, the root tip was excis...

Embodiment 3

[0059] Example 3. Detection of quasi-transgenic plants by polymerase chain reaction

[0060] Extract the genomic DNA of the plant to be transgenic as a template, and use the pBI-HCF, pBI-HCN and pBI-HCC constructs obtained above as the positive control group, and use the specific primer pair designed according to the sequence of the target gene HC-Pro , which includes 519HCStopA / 519HCSacB, 519HCStopA / 519HCSacA, and 519HCStopB / 519HCSacB primer pairs, respectively performing polymerase chain reaction (PCR) amplification on the HC-Pro full-length gene, N-terminal fragment and C-terminal fragment. On the other hand, the nptII gene was amplified with the primer pair nptII 5' primer 5'-CCCCTCGGTATCCAATTAGAG-3' (SEQ ID NO: 9) and nptII 3' primer 5'-CTGGAGTTCTTCGCCA-3' (SEQ ID NO: 10), and expressed as PCR products were analyzed by gel electrophoresis. According to the analysis of the results of gel electrophoresis, the plants with the expected fragments amplified by the aforemention...

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Abstract

The invention discloses a recombinant vector comprising a control sequence and a coded sequence fragment of papaya ringspot virus (PRSV) helper component protease gene (HC-Pro gene) in manipulatable connection with the control sequence. The invention also provides a recombinant microorganism, a method for endowing a plant with resistance to viruses, and application of a full-length gene or a genefragment thereof of the PRSV HC-Pro gene to culture of plants with resistance to the viruses. In the invention, by adopting the strategy of attacking the virus by taking the PRSV HC-Pro gene as a target, the capability of the PRSV to resist plant host defense responses is collapsed, the problem that the resistance of genetically modified genes subjected to gene silencing is lost is solved, the wide-spectrum resistance of a genetically modified plant to different PRSV strain viruses is provided, and the cultured genetically modified plant is endowed with an application value of crossing geographical regions.

Description

technical field [0001] The invention relates to a recombinant vector for providing plant resistance to viruses, which comprises a coding sequence fragment of a papaya ringspot virus helper-component protease gene (PRSV HC-Pro gene). The invention also relates to a recombinant microorganism. The invention also relates to a method for rendering plants resistant to viruses. The invention also relates to the use of the full-length gene of the auxiliary component protease or a gene fragment thereof for producing plants resistant to viruses. Background technique [0002] Papaya (Carica papaya L.) is a crop with high economic value. There is no medicine to treat its viral diseases. In recent years, due to the improvement of plant tissue culture technology, the breakthrough of plant transgenic technology, and the theory of pathogen-induced resistance (pathogen- Inspired by derived resistance (PDR) (Sanford and Johnson 1985), transferring a part of the viral genome to the chromosom...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N1/21C12N15/57C12N5/10A01H5/00C12R1/01
Inventor 叶锡东龚怡蓉王惠菁王馨兰
Owner 薛富盛
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