Potyvirus virus-induced gene silencing (VIGS) system and application thereof

A gene silencing, potato technology, applied in the field of plant genetic engineering, can solve problems such as the limitation of virus host range, and achieve the effects of rich species, significant effect and obvious silencing effect

Active Publication Date: 2020-04-03
INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, each VIGS vector is limited by the host range of the virus, and it is usually necessary to construct different viral VIGS vectors for different plants
The genus Potyvirus (Potyvirus) contains 175 species of viruses, which is the largest genus among plant RNA viruses, and also the genus that causes economic losses and damage to agricultural production on a global scale. However, Potyvirus Y is used in VIGS Research on carrier modification has not been reported yet

Method used

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  • Potyvirus virus-induced gene silencing (VIGS) system and application thereof
  • Potyvirus virus-induced gene silencing (VIGS) system and application thereof
  • Potyvirus virus-induced gene silencing (VIGS) system and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0048] Embodiment 1: Construction and application method of gene silencing vector (pG35S-PLDMV-CaPDS) induced by papaya deformed mosaic virus of silencing papaya PDS gene (CaPDS)

[0049] Experimental reagents: primers were synthesized by Invitrogen; high-speed high-fidelity DNA polymerase (PrimerSTAR MaxDNA Polymerase) and reverse transcription kit (PrimeScript RT reagent Kit with gDNA Eraser (Perfect Real Time)) fluorescent quantitative RT-PCR kit (TB Green Premix DimerEraser (PerfectReal Time)) was purchased from Takara Company; TRIZOL Reagent was purchased from Invitrogen; 2×Rapid Taq MasterMix and Gel Extraction Kit (FastPure Gel DNA Extraction Mini Kit) were purchased from Nanjing Novozyme Biotechnology Co., Ltd., Gibson Assembly Master Mix was purchased from NEB Company, and the competent cells of Agrobacterium strain GV3101 (pSoup) were self-preserved by our laboratory, and the rest of the required reagents can be purchased through commercial channels.

[0050] The pre...

Embodiment 2

[0063] Embodiment 2 The silencing effect of different papaya CaPDS gene fragment lengths and regions

[0064] Using the same method as in Example 1, insert fragments of different lengths and regions from the papaya CaPDS gene at corresponding positions in the pG35S-PLDMV vector ( Figure 6 ), the inserted fragments were 4~105bp, 4~303bp, 4~600bp, 601~1200bp, 1201~1749bp of the CaPDS gene, and the silencing vectors constructed were pG35S-PLDMV-CaPDS102, pG35S-PLDMV-CaPDS300, pG35S - PLDMV-CaPDS597, pG35S-PLDMV-CaPDS600, pG35S-PLDMV-CaPDS549. The results showed that using PLDMV virus as the gene silencing vector, regardless of the length of the inserted segment or the selection of the region, a good silencing effect could be achieved, and the top leaves all showed different degrees of albinism. The relative expression of CaPDS in papaya plants infected with Agrobacterium carrying different vectors was detected, and the results were as follows: Figure 7 , the expression of the...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and discloses a potyvirus virus-induced gene silencing (VIGS) system with a papaya leaf distortion mosaic virus (PLDMV) as avirus vector. According to the system, a target gene is inserted between Nib and coat protein (CP) of the PLDMV, and a NIa-Pro digestion site is introduced between the target gene and the CP. By in-vitro splicing methods such as Gibson splicing or In-Fusion cloning, target gene fragments are seamlessly cloned and inserted between the Nib and the CP to construct a recombinant virus silence vector containing a plant target gene. Plants are inoculated with the recombinant virus silence vector by agrobacterium injection, and phenotypic changes of the plants are observed after the target gene silencing the plants is induced. The potyvirus VIGS system provides a powerful tool to study gene functions of papaya by using the PLDMV silence vector, and has great scientific significance and application prospects.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and mainly relates to a gene silencing system induced by potyvirus genus virus and its application. Background technique [0002] Virus-induced gene silencing (VIGS) was first used to describe the phenomenon of plant disease recovery after virus infection (van Kammen, 1997). Because there is a defense system in plants to resist the invasion of exogenous nucleic acids, which normally protect plants from virus infection, but this defense mechanism of plants can be activated by viral RNA, which belongs to the phenomenon of post-transcriptional gene silencing. In recent years, VIGS has been specifically referred to as a reverse genetics technology that induces plant endogenous gene silencing through a recombinant virus carrying a cDNA fragment of a host plant target gene (Burch-Smith et al., 2004). The VIGS system inserts the target gene segment into the viral vector. When the vira...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/74C12N15/40C12N15/82A01H5/00
CPCC07K14/005C12N15/74C12N15/8218C12N2770/34022
Inventor 庹德财沈文涛周鹏言普赵光远黎小瑛
Owner INST OF TROPICAL BIOSCI & BIOTECH CHINESE ACADEMY OF TROPICAL AGRI SCI
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