RU2005788C1 - Rna-fragment of potato x-virus intended for foreign gene translation - Google Patents

Abstract

FIELD: biotechnology. SUBSTANCE: method involves preparing of RNA-fragment of potato X-virus showing a broad spectrum action with regard to activation of gene translation for enhanced efficacy of foreign proteins synthesis in transgenic plants and animals. EFFECT: preparing of RNA-fragment indicated above. 1 dwg, 1 tbl

Description

The invention relates to biotechnology, in particular to the genetic engineering of plants, and is a fragment of the potato X-virus RNA, containing a 5 ^l -translated region of the genome and having the ability to enhance the translation of foreign genes in eukaryotic systems.

A known fragment of the genomic RNA of the tobacco mosaic virus (TMV) with a length of 70 nucleotides (5 ^l untranslated sequence, or ω sequence), which is one of the most effective amplifiers for gene translation.

 The disadvantages of the prototype include the following: the described TMV RNA fragment effectively enhances the translation of foreign genes not only in plant cells, but also in bacterial cells, which can have undesirable consequences during genetic engineering work on plant transformation, in particular, this can cause premature lysis of bacterial cells due to poisoning by toxic foreign proteins.

 The aim of the invention is to obtain a fragment of the potato X-virus RNA having a wide spectrum of activity with respect to activation of gene translation to increase the efficiency of the synthesis of foreign proteins in transgenic plants and animals.

To achieve this goal, a transcription vector based on the pTZ19 plasmid was constructed in which the neomycin phosphotransferase 1 (NFT 1) genes from the Tn903 transposon were controlled by a chemically synthesized fragment carrying the αβ leader and the 5 c proximal initiator codon of the 5 ^l proximal CVK RNA gene (αβ NTP 1 design). In the control construct KM), the NFT 1 gene was preceded by an untranslated sequence representing a modified polylinker of plasmid pTZ19. After linearization of the plasmids using RNA polymerase of the bacteriophage T7, the corresponding transcripts were obtained. Their leader sequences are shown in the drawing.

To prove the effect of the obtained RNA fragment, uncaptized transcripts of both plasmids were translated in cell-free protein synthesizing systems from rabbit reticulocytes, Krebs-2 ascites carcinoma cells according to and wheat germ. [ ³⁵ S] -labeled translation products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The gel was dried, exposed and quantified translation products by scanning autographs using a laser densitometer 2202 company LKB. The presence of the αβ sequence enhanced the translation of the NFT1 gene by 30–40 times in the rabbit reticulocyte lysate, 10–14 times in the system from Krebs-2 cells, and 6–8 times in extracts from wheat germ (table). Plasmids containing the αβ leader and marker gene are prepared as follows: 5 ng of oligodeoxyribonucleotides containing the target sequence and its complementary sequence are annealed in 10 μl of buffer, which contains 40 mM Tris-HCl, pH 7.5; 6 mM MgCl ₂ and 1 mM ATP at ⁵⁶ ° C for 2 hours. Oligodeoxyribonucleotides were synthesized on an automatic synthesizer of the firm Applied Biosystems (USA). After annealing, 200 ng of the plasmid pTZ19 containing the NFT 1 gene (drug from Pharmacia, Sweden) is added to the oligodeoxyribonucleotide mixture. 1 unit was added to the reaction mixture. Phage T4 DNA ligases. Ligation was carried out at ¹⁰ ° C for 12 hours. After ligation, preparation obtained transformed E. coli cells and isolated from the transformants, recombinant plasmid. The primary structure of leader regions of αβ -NFT1 and CM transcripts is presented below.

5 ^l GAAAACUAAACCAUACACCAC-
CAACACAACCAAACCCACCAC
α sequence
GCCCAAUUGUUACACACCCGCUUGA- GAAAGCAAGUCUAACAA AUG-NFT 1
β - sequence
5 l GGGAAAGCUUGGAUGCCUGC-
CUGCAGGUGGACUCUAGAGGA-
UCCCCC AUG-NFT1 The above plasmids containing the αβ leader and marker gene are transcribed as follows: a 20 μl transcription mixture contains 40 mM Tris-HCl, pH 7.5; 6 mM MgCl ₂ ; 2 mM spermidine; 10 mM NaCl; 10 mM dithiothreitol; 1 mM ATP, CTP, UTP and GTP; 20 units placental RNase inhibitor and 1 μg of plasmid DNA. After adding 10 units. PNK bacteriophage T7 polymerase mixture is incubated at ³⁷ ° C for 60 min. After the reaction, deposited transcripts overnight at 4 ^{° C} in the presence of 2 M LiCl. After centrifugation, the precipitate is washed with 70% ethanol, dried and dissolved in sterile water. Translation is carried out in a rabbit reticulocyte lysate. A translation mixture with a volume of 25 μl contains 10 μl of lysate: 20 mM Nerev, pH 7.6; 1 mM ATP; 200 μM GTP; 2.5 mM magnesium acetate; 100 mM potassium acetate; 2 mM dithiothreitol; 15 mM creatine phosphate; 1 μg creatine phosphokinase; 5 mM cAMP; 2 mM EGCTA; 3 μg yeast tRNA; 125 μM amino acids excluding methionine; 800 μCi / ml [ ³⁵ S] -methionine and 40 μg / ml RNA transcript. The reaction mixture was incubated 60 min at 30 ^° C ^[35 S] -labelled translation products were analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The gel is dried, exposed and quantified translation products by scanning autographs using a laser densitometer 2202 company LKB. Strengthening translation reaches 40 times (see table).

 Thus, the present invention allows for efficient amplification of the translation of a foreign (bacterial) gene in a eukaryotic protein synthesizing system, i.e., the proposed fragment of the PVA genome is a translational amplifier of a wide spectrum of action. (56) Reports of the USSR Academy of Sciences. 1988, vol. 300, No. 3, p. 711-716.

Claims (1)

POTATO X-VIRUS RNA Fragment, INTENDED TO STRENGTHEN ALIEN GENE TRANSLATION, obtained by chemical synthesis and has the following nucleotide sequence:
1 GAAAACUAAA CCAUACACCA CCAACACAAC CAAACCCACC ACGCCCAAUU
51 GUUACACACC CGCUUGAGAA AGCAAGUCUA ACAA.

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