Fusarium head blight virus toxin degrading gene and expression thereof

A viral toxin and gene technology, applied in the field of biomedicine, can solve problems such as the absence of Fusarium graminearum

Inactive Publication Date: 2011-08-17
HENAN ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far there is no report on Tri101 gene and its expression of Fusarium graminearium Fg0623 (Fusarium graminearium 0623)

Method used

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  • Fusarium head blight virus toxin degrading gene and expression thereof
  • Fusarium head blight virus toxin degrading gene and expression thereof
  • Fusarium head blight virus toxin degrading gene and expression thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028]Example 1 Expression of Fusarium graminearum degradative toxin gene Tri101

[0029] 1. Extraction of total RNA and synthesis of cDNA

[0030] Inoculate Fg0623 spores cultured on fresh PDA slant into liquid medium, shake at 150rpm, 28°C for 48h; draw 1mL of the cultured bacteria into a 1.5mL sterile EP tube, and centrifuge at 10000rpm for 10min; collect the bacteria, according to RNAiso TM The total RNA of the bacteria was extracted according to the method in the Plus manual. Using the extracted total RNA as a template, cDNA was synthesized. RT1 system: RNase Free dH 2 O 6.5 μL, dNTP-Mix (10 mM) 1 μL, Oligo dT (50 μM) 2 μL, Total RNA 0.5 μL were reacted on a PCR machine at 65°C for 5 min, and cooled on ice. RT2 system: 10 μL of the above denatured and annealed reaction solution, 5×PrimeScript TM Buffer 4μL, RNase Inhibitor (40U / μL) 0.5μL, PrimeScript TM RTase (200U / μL) 1μL, RNase Free dH 2 O 4.5 μL. Reaction program: 30°C for 10 minutes, 42°C for 60 minutes, 70...

Embodiment 2

[0078] Example 2 Sequence Analysis of Fusarium graminearum Degradative Toxin Gene Tri101

[0079] The positive clone samples successfully identified by enzyme digestion were sent to Sangon Company for DNA sequence determination. Using the DNAclub software and the BLAST function of the NCBI website, the determined sequences were compared and analyzed.

[0080]The result of sequence determination of Fg0623Tri101 gene showed that the Fg0623Tri101 gene contained a complete open reading frame with a total length of 1356bp. The gene has been registered in GenBank with accession number GQ907236. It is related to the nucleotide sequence of Fusarium graminearum Tri101 gene reported by Kimura (Kimura M, Shingu Y, Yoneyama K, et al. Features of Tri101, the trichothecene 3-O-acetyl-transferase gene, related to the self-defense mechanism in Fusarium graminearum[J]. Biosci Biotechnol Biochem, 1998, 62(5): 1033-1036.) has the highest homology, 99.78%, and the base sequence only changes fro...

Embodiment 3

[0081] Embodiment 3 Tri101 Gene Coded Amino Acid Sequence Analysis

[0082] The protein analysis software provided by the website http: / / www.expasy.org / tools / #translate analyzed the basic characteristics of the amino acid sequence encoded by the Tri101 gene, and the results showed that the molecular weight of the protein predicted by the gene was 49.45kDa, and the isoelectric point In the amino acid composition, there are 47 positively charged amino acids (Arg, Lys), accounting for 10.42%, 57 negatively charged amino acids (Asp, Glu), accounting for 12.64%, 217 hydrophobic amino acids, accounting for 48.12%, and hydrophilic 184 sex amino acids, accounting for 40.80%, see Table 1 and image 3 .

[0083] Table 1 Amino acid content of Tri101 protein

[0084]

[0085] Note: *hydrophobic amino acid, #hydrophilic amino acid

[0086] The protein encoded by this gene is trichothecene 3-O-acetyltransferase, and the results of Blast database analysis show that it belongs to the tr...

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Abstract

The invention discloses a fusarium head blight virus toxin degrading gene and expression thereof. The fusarium head blight virus toxin degrading gene has a nucleotide sequence represented by SEQ ID No.1 and an amino acid sequence represented by SEQ ID No.2. The invention also discloses the expression of the gene, designs two pairs of primers, constructs a recombinant plasmid Pmd19-T / Tri101 and a recombinant prokaryotic expression vector pGEX-4T2 / Tri101. The pGEX-4T2 / Tri101 carries a glutathione S-transferase (GST) tag and can be expressed to produce a GST-Tri101 fusion protein. In the invention, a Tri101 gene for coding a trichothecene 3-O-acetyl transferase is expressed in Escherichia coli with high efficiency, the nucleotide sequence and amino acid sequence of the gene are analyzed in details, and a theoretical basis is laid for further study on the biological functions of the gene and the protein thereof and plant genetic engineering.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a gene for degrading scab toxin and its expression. Background technique [0002] Fusarium head blight (FHB) caused by Fusarium is a catastrophic disease of wheat and small-grain cereal crops all over the world, causing serious economic losses, especially in high humidity and warm environments ( Parry D W, Nicholson P, McMullen M, Jones R, Gallenberg D. FHB of wheat and barley: a re-emerging disease of devastating impact[J]. Plant Disease, 1997, 81: 1340-1348; Windels C E. Economic and social impacts of Fusarium head blight: changing farms and rural communities in the Northern Great Plains[J] . Phytopathology, 2000, 90: 17-21.). Wheat head blight is caused by a variety of Fusarium species. In different regions of the world, due to different ecological and geographical environments, the dominant pathogenic bacteria that cause head blight are different. In 1984, the Nationa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/37C12N15/63C12N15/79C07H21/04C12R1/77
Inventor 杨丽荣薛保国闫海霞雷振生赵献林全鑫孙虎张振臣王恒亮吴政卿吴仁海吴坤张永超曹岳恩武超武予清刘明源马雪皎王亚南
Owner HENAN ACAD OF AGRI SCI
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