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Strawberry vein banding virus vector, construction method thereof and application of strawberry vein banding virus vector in foreign protein expression

A technology of strawberry vein-inlaid virus and construction method, which is applied in the field of plant genetic engineering and can solve problems such as difficulty in meeting forest strawberry gene research and the like

Active Publication Date: 2021-10-19
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Strawberry vein banding virus vector, construction method thereof and application of strawberry vein banding virus vector in foreign protein expression
  • Strawberry vein banding virus vector, construction method thereof and application of strawberry vein banding virus vector in foreign protein expression
  • Strawberry vein banding virus vector, construction method thereof and application of strawberry vein banding virus vector in foreign protein expression

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Embodiment 1

[0092]Plant virus vectors are constructed from plant virus infectious clones. Therefore, the quality of plant virus infectious clones directly affects the quality of plant virus vectors, the most important of which is the incidence of viruses. The incidence of plant virus vectors constructed from plant virus infectious clones is generally higher. At the same time, the plant virus vector should be convenient for subsequent gene construction, especially the construction steps such as plasmid digestion, strain transformation, recombination connection, etc., so the present invention optimizes the construction of the Strawberry Vein Virus infectious clone.

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

[0094] Prepare the pCB301 plasmid (as shown in SEQ ID NO.21), use the restriction endonuclease Sal I / Sma I to double digest the above-mentioned pCB301 plasmid, and extract the full-length genome sequence of strawberry vein virus (as shown in SEQ ID NO....

Embodiment 2

[0107] Optimal Construction of Multiple Cloning Site Fragments

[0108] The multiple cloning site MCS is inserted into different positions of the virus-infectious clone, which has an impact on the infection and replication of the virus. There are three common ways for MCS to be inserted into the virus-infectious clone. The first is to replace the specific gene encoded by the virus with MCS The second is to insert the MCS into the downstream of the viral motor protein gene (Movement protein, MP), and among the present invention, the P1 gene encodes the motor protein of SVBV; 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, the viral vectors of these three types of MCS insertion methods were respectively 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...

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 virus 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 expression of GFP carried by the two SVBV expression vectors constructed in this study is higher in the leaves of forest strawberry.

[0146] The specific construction method is as follows:

[0147] The strawberry 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 into the linearized strawberry vein by Pml I / BamH I double enzyme digestion by the method of homologous recombination Viral vector pSVBV SY -P1-MCS, obtain strawberry vein virus transient overexpression vector pSVBV SY -P1-GFP, then transformed into A...

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Abstract

The invention discloses a strawberry vein banding virus vector as well as a construction method and application thereof in foreign protein expression, and belongs to the technical field of plant genetic engineering. The strawberry vein banding virus vector is pSVBVSY-P1-MCS or pSVBVSY-P4-MCS, and the strawberry vein banding virus vector is pSVBVSY-P1-MCS; the sequence of the pSVBVSY-P1-MCS is as shown in SEQ ID NO. 1, and the sequence of the pSVBVSY-P4-MCS is as shown in SEQ ID NO. 2. The construction method comprises the following steps: (1) constructing strawberry vein banding virus infectious clone; (2) constructing a multi-cloning-site fragment; (3) constructing a strawberry vein banding virus vector, in particular, inserting a gene sequence with Pml I and BamH I restriction enzyme recognition sites between P1 and P2 or between P4 and P5 of strawberry vein banding virus infectious clone in a homologous recombination mode. The vector constructed by the invention is subjected to vacuum filtration inoculation through an agrobacterium infiltration method and stably and efficiently infects forest strawberries, and meanwhile, foreign proteins can be efficiently expressed on the forest strawberries.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and in particular relates to a strawberry vein virus vector, its construction method and its application in exogenous protein expression. Background technique [0002] Strawberry vein virus (SVBV) is a circular double-stranded DNA (ds-DNA) virus that belongs to the family Cauli-moviridae (Cauli-moviridae) and the genus Caulimovirus. The complete genome sequence of SVBV was first reported in the United States. The full length is about 7.8kb, including 7 ORFs. SVBV can be transmitted through 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. After being infected by the virus, it shows the typical symptoms of leaf vein yellowing and discontinuous yellow border. SVBV alone infects cultivated strawberry (F. ananassa) without obvious symptoms, but when co-infe...

Claims

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Application Information

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Patent Type & Authority Applications(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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