Gene product detection method of degradative graminearum toxin

A technology of scab and toxin, which is applied in the field of biomedicine, can solve the problems that there is no detection method for Fusarium graminearum Tri101 gene expression, and achieve the effects of reducing detection cost, reducing metabolism, and improving crop yield

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

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Problems solved by technology

But so far there is no report on the detection method of Tri101 gene expression in Fusarium graminearium Fg0623 (Fusarium graminearium 0623)

Method used

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  • Gene product detection method of degradative graminearum toxin
  • Gene product detection method of degradative graminearum toxin
  • Gene product detection method of degradative graminearum toxin

Examples

Experimental program
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Embodiment 1

[0054] Example 1 Expression of Fusarium graminearum degradative toxin gene Tri101

[0055] 1. Extraction of total RNA and synthesis of cDNA

[0056] Inoculate Fg0623 spores cultivated on fresh PDA slant into liquid culture medium, shake at 150rpm, 28°C for 48h; draw 1mL of cultured spores into a 1.5mL sterile EP tube, centrifuge at 10 000rpm 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, ...

Embodiment 2

[0104] Example 2 Sequence Analysis of Fusarium graminearum Degradative Toxin Gene Tri101

[0105] 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.

[0106] The result of sequence determination of Fg0623Tri101 gene showed that the Fg0623 Tri101 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-acetyltransferase gene, related to the self-defense mechanism in Fusarium graminearum[J].Biosci Biotechnol Biochem, 1998, 62(5): 1033-1036.) has the highest homology at 99.78%. The base sequence only changes from ...

Embodiment 3

[0107] Embodiment 3 Tri101 Gene Coded Amino Acid Sequence Analysis

[0108] 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 .

[0109] Table 1 Amino acid content of Tri101 protein

[0110]

[0111]

[0112] Note: *hydrophobic amino acid, #hydrophilic amino acid

[0113] The protein encoded by this gene is trichothecene 3-O-acetyltransferase, and the results of Blast database analysis show that it belon...

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Abstract

The invention discloses a gene product detection method of degradative graminearum toxin. A rabbit anti-GST(glutathione transferase)-Tri101 antibody acting as a primary antibody, and a goat anti-rabbit IgG-HRP (horse radish peroxidase) acting as a secondary antibody and an object to be tested containing the Tri101 protein carry out Western blot analysis. The trichothecene 3-O-acetyl invertase coded by Tri101 gene in the invention is efficiently expressed in colibacillus; a purified protein immune New Zealand white rabbit obtains an antibody with relatively good specificity, and can carry out immunoblotting reaction with Tri101 protein secreted and expressed in BL21 (DE3); and a new detection method is provided for trangenosis of Tri101 gene in wheat and corns; the detection cost is reduced; and a solid basis for further researching the biological functions of Tri101 protein is provided.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to a method for detecting gene products of degraded gibberella toxin. Background technique [0002] Fusaruim head blight or scab is harmful to wheat (Triticum aestivum), barley (Hordeum vulgare), oats (Avena sativa L.), corn (Zea mays L.), rice (Oryza sativa), rye (Secale cereale L.) and other cereal crops, widely distributed in warm and humid regions of the world. Wheat head blight was first discovered in England in 1884. In recent years, with the gradual warming of the global climate and changes in cropping systems and methods such as rice-wheat, corn-wheat rotation or no-tillage, wheat scab has shown a tendency to expand in wheat producing areas around the world. The direct economic losses are also becoming more and more serious (Bai G H, Shaner G. Scab of wheat: Prospects for control [J]. Plant Disease, 1994, 78: 760-766; Parry D W, Nicholson P, Mcleod L. Fusarium ear bli...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/531G01N33/543
Inventor 薛保国杨丽荣雷振生全鑫孙虎宋玉立闫海霞鲁传涛郭松景赵献林刘红彦吴坤何文兰吴政卿王恒亮曹岳恩武超王亚南马雪皎王爱平刘明源
Owner HENAN ACAD OF AGRI SCI
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