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A strawberry vein virus vector and its construction method and application in foreign protein expression

A technology of strawberry vein virus and vector, which is applied in the field of plant genetic engineering, can solve the problems that are difficult to meet in the research of forest strawberry genes

Active Publication Date: 2022-05-24
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

According to reports, the apple latent spherical virus (ALSV) vector can overexpress the exogenous AtFT gene in cultivated strawberry, and promote the early flowering of cultivated strawberry, but ALSV can only be used in the cultivation of strawberry tissue culture seedlings, and it is not effective for soil cultivation. The infection rate of cultivated strawberry seedlings is 0%, therefore, both TRV and ALSV vectors are difficult to satisfy the research on forest strawberry genes

Method used

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  • A strawberry vein virus vector and its construction method and application in foreign protein expression
  • A strawberry vein virus vector and its construction method and application in foreign protein expression
  • A strawberry vein virus vector and its construction method and application in foreign protein expression

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Embodiment 1

[0092]Plant virus vectors are constructed from plant virus infective clones. Therefore, the pros and cons of plant virus infective clones directly affect the pros and cons of plant virus vectors. The most important thing is the incidence of the virus, which is determined by the high incidence of the virus. Plant viral vectors constructed from infective clones of plant viruses also generally have a higher incidence. At the same time, the plant virus vector needs to facilitate the construction of subsequent genes, especially the construction steps such as plasmid digestion, strain transformation, recombination connection, etc. Therefore, the present invention optimizes the construction of the infective clone of strawberry mosaic virus.

[0093] Strawberry Vein Virus Infectious Cloning pSVBV SY optimized build of

[0094] Prepare pCB301 plasmid (as shown in SEQ ID NO.21), use restriction endonuclease Sal I / Sma I double enzyme to cut the above pCB301 plasmid, and make the full-le...

Embodiment 2

[0107] Optimized Construction of Multiple Cloning Site Fragments

[0108] Multi-cloning site MCS is inserted into different positions of the virus infective clone, which affects the infection and replication of the virus. There are three common ways of inserting MCS into the virus infective clone. The first is to replace the specific gene encoded by the virus with MCS. The second is to insert MCS into the downstream of the viral motion protein gene (Movement protein, MP), and the P1 gene encodes the motor protein of SVBV in the present invention; the third is to insert the MCS into the downstream of the viral coat protein gene (Coat protein, CP), In the present invention, the P4 gene encodes the coat protein of SVBV. Therefore, in this study, three types of MCS-inserted viral vectors were constructed. It should be reminded that the P1 gene sequence is shown in SEQ ID NO.24, and the P4 gene sequence is shown in SEQ ID NO.25.

[0109] The specific optimization scheme is as fol...

Embodiment 3

[0144] Application of SVBV virus vector in expressing foreign protein

[0145] Insert the GFP gene (as shown in SEQ ID NO. 23) into the recombinant viral vector pSVBV respectively SY -P1-MCS and pSVBV SY -P4-MCS, to obtain the expression vector pSVBV SY -P1-GFP and pSVBV SY -P4-GFP, compared with the TRV-GFP expression vector, the two SVBV expression vectors constructed in this study carried a higher GFP expression in forest strawberry leaves.

[0146] The specific construction method is as follows:

[0147] Described strawberry inlaid vein virus vector is pSVBV SY -P1-MCS;

[0148] Using the forward primer SEQ ID NO.12 and the reverse primer SEQ ID NO.13 to amplify the fragment P1GFP, the fragment P1GFP was connected to the strawberry inlaid vein linearized by Pml I / BamH I double digestion and linearization by the method of homologous recombination viral vector pSVBV SY -P1-MCS, obtain the transient overexpression vector pSVBV of strawberry mosaic virus SY -P1-GFP, th...

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Abstract

The invention discloses a strawberry vein virus vector, its construction method and its application in exogenous protein expression, belonging to the technical field of plant genetic engineering. Strawberry vein virus vector pSVBV SY ‑P1‑MCS or pSVBV SY ‑P4‑MCS; pSVBV SY The sequence of ‑P1‑MCS is shown in SEQ ID NO.1, pSVBV SY The sequence of ‑P4‑MCS is shown in SEQ ID NO.2. Its construction method is as follows: (1) construction of strawberry vein virus infectious clone: ​​(2) construction of multiple cloning site fragments: (3) construction of strawberry vein virus vector, specifically, will have Pml I The gene sequence of the BamH I restriction endonuclease recognition site is inserted between P1 and P2 or between P4 and P5 preparations of the Strawberry Vein Virus infective clone by means of homologous recombination. The vector constructed by the invention is vacuum-filtered and inoculated through the Agrobacterium infiltration method, and stably and efficiently infects the forest strawberry, and can also efficiently express the foreign protein on the forest strawberry.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a strawberry inlaid vein virus vector and its construction method and application in the expression of exogenous proteins. Background technique [0002] Strawberry Veined Vein Virus (SVBV) is a circular double-stranded DNA (ds-DNA) virus that belongs to the cauliflower mosaic virus family (Cauli-moviridae) in the genus Caulimovirus. The SVBV whole genome sequence was first reported in the United States. The total length is about 7.8kb and contains 7 ORFs. SVBV can be transmitted by aphid vectors and grafting, and is one of the viruses that seriously endanger strawberry production. Forest strawberry (Fragaria vesca) is a sensitive indicator plant of SVBV, which shows typical symptoms of leaf vein yellowing and discontinuous yellowing and bordering after being infected by the virus. SVBV alone infects cultivated strawberry (F.ananassa) without obviou...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/66C07K14/435
CPCC12N15/8203C12N15/66C07K14/43595
Inventor 江彤杨先初蒋磊赵青青张汉平胡亚会蒋西子
Owner ANHUI AGRICULTURAL UNIVERSITY
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