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Citrus tristeza virus based vectors for foreign gene/s expression

A virus vector, gene cassette technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problem of low stability of foreign inserts

Active Publication Date: 2016-09-14
UNIV OF FLORIDA RES FOUNDATION INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the low stability of foreign inserts is a major disadvantage of viral vectors in the commercialization of protein expression in the application field.

Method used

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  • Citrus tristeza virus based vectors for foreign gene/s expression
  • Citrus tristeza virus based vectors for foreign gene/s expression
  • Citrus tristeza virus based vectors for foreign gene/s expression

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0076] Protoplast preparation, transfection, RNA isolation and Northern Blot analysis

[0077]Tobacco benthamiana leaf protoplasts were prepared according to the method invented by Nava-Castillo et al., (1997). Disinfect the surface of Bensheng tobacco leaves for 3 weeks, use a sterile knife to lightly scratch the lower surface, and culture overnight in a dark room (16-20 hours), the medium is 0.7M MMC (0.7M mannitol, 5mM MES, 10mM CaCl 2 ) plus 1% cellulose (Yakult Honsh, Tokyo, Japan) and 0.5% polygalacturonase (Calbiochem Laboratories, La Jolla, California).

[0078] Use Sp6 RNA polymerase (Wisconsin State Epicentre Technologies Laboratory) to obtain capped in vitro RNA (Satyanarayana et al., 1999) on NotI or StuI linear plasmid DNA, use PEG (polyethylene glycol) to transfect protoplasts, this method refers to Satyanarayana et al., (1999). Four days after transfection, protoplasts were used to prepare total RNA for Northern blot analysis, and virions were isolated. Aliqu...

Embodiment 1

[0087] Example 1: System for detecting CTV-based expression vectors

[0088] CTV-based expression vectors were tested in three systems, respectively, N. benthamiana leaf pulp protoplasts, N. benthamiana whole plant, and Alemon whole plant. Vectors for infecting whole plants were constructed using a full-length CTV cDNA clone (pCTV9R) and a mostly deleted mutant of the p33 gene (pCTV9RΔp33), which has a PstI restriction site deleted, is easier to clone, and retains the ability to infect most citrus plants. Competence (Tatineni et al., 2008). Faster analysis of N. benthamiana protoplasts requires construction of a vector on the SP6 transcriptional plasmid (Satyanarayana et al., 1999). In protoplasts it is convenient to use the minireplicon pCTVΔCla333R (Gowda et al., 2001) with most 3' gene deletions. The overall goal is to obtain citrus trees infected with different CTV expression vectors, which is more difficult and time-consuming. Soil inoculation of citrus trees has been ...

Embodiment 2

[0089] Example 2: Genes added at different positions in the CTV genome

[0090] Insertion at the p13 gene locus

[0091] Some researchers (Folimonov et al., 2007) inserted other genes between the two capsid protein genes in an effective CTV vector, making the exogenous gene the sixth gene starting from the 3' end. But the CTV gene with the highest expression tends to be near the 3' end. Inserting a gene close to the 3' end will therefore result in increased expression. P13 is the third gene counted from the 3' end, and it is a relatively high expression gene, which is not required for infection of most CTV hosts (Tatineni et al., 2008; Tatineni et al., in preparation). But previous attempts to replace p13ORF with GFP ORF failed (Folimonov et al., 2007). There are several possibilities for the cause of failure. Previously the vector was designed with the assumption that translation starts at the first start codon, but the p13ORF has a second in-frame AUG. Transcription may...

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Abstract

Disclosed herein are viral vectors based on modifications of the Citrus Tristeza virus useful for transfecting citrus trees for beneficial purposes. Included in the disclosure are viral vectors including one or more gene cassettes that encode heterologous polypeptide s. The gene cassettes are positioned at desirable locations on the viral genome so as to enable expression while preserving functionality of the virus. Also disclosed are methods of transfecting plants and plants transfected with viral vector embodiments.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 USC § 119 (35 USC 119) to US Patent Application No. 61 / 537,154, filed September 21, 2011, which is hereby incorporated by reference. Background technique [0003] Early development of viral vectors aimed at inexpensive production of specialized proteins that could scale up the field. A plant viral vector was initially used, the cauliflower mosaic virus, a double-stranded DNA virus (Brisson et al., 1984; Gronenborn et al., 1981). However, this virus has too low stability to be used (Fütterer et al., 1990). The development of reverse genetics systems has enabled the use of RNA viruses, providing more options for vector development (Ahlquist et al., 1984). [0004] Viral vectors are an important element of basic research and have great potential for commercial applications. However, the low stability of foreign inserts is a major disadvantage of viral vectors in the commercializa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N15/83C12N15/11
CPCC12N15/8203
Inventor 威廉·欧·多森斯维特拉娜·弗里莫诺娃卓阿·埃尔默塔
Owner UNIV OF FLORIDA RES FOUNDATION INC
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