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Mannose-1-phosphate guanylyl transferase, coding gene and application

A phosphoguanylyl transferase and guanosyl phosphate technology, applied in transferase, application, genetic engineering and other directions, can solve the problems of ineffectiveness, increase the difficulty of sugar transfer genes, etc., and achieve reduced pathogenicity and good application. Foreground effect

Pending Publication Date: 2021-11-05
天津市农业科学院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The difficulty in solving the above problems and defects is: the number of genes predicted to encode products in the Pst genome is large, but the number of functional annotations has been completed is very small. The usual method of gene product function analysis based on phenotype after gene knockout and complementation is not effective in vivo. Difficult to work in sexual parasite Pst, increasing the difficulty of screening sugar transporter genes

Method used

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  • Mannose-1-phosphate guanylyl transferase, coding gene and application
  • Mannose-1-phosphate guanylyl transferase, coding gene and application
  • Mannose-1-phosphate guanylyl transferase, coding gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Recombinant plasmid construction

[0062] 1. Primer Design

[0063] Referring to the multiple cloning site of the pDR196 vector and the restriction site of the cds of the PsMT1 gene, BamHI was introduced at the 5' of the gene, and an XhoI restriction site was introduced at the 3'. The primer sequences are as follows:

[0064] PrimerID PrimerSequence(5'-3') PMT CGGAATTCatggcttcgaaagctgtct PMTr CCGCTCGAGtcataacaagacctcgttgg

[0065] 2. Template amplification

[0066] The guaranteed enzyme used is Toyobo KOD (KOD-101), and the reaction system configuration is as follows:

[0067] Component Volume(μL) 10×KODBuffer 5 2mMdNTPs 5 gDNA 1 216 (10μM) 1 217 (10μM) 1 KOD DNA Polymerase 1 wxya 2 o

36 Total 50

[0068] 1) The PCR reaction procedure is as follows:

[0069]

[0070] 3. PCR product recovery

[0071] 2) Add 4 times the volume (800 μL) of Buffer CP to a 1.5...

Embodiment 2

[0103] Example 2 Functional verification of PsMT1 product

[0104] 1. Recombinant plasmid transformed yeast competent

[0105] Boil 1mL salmon sperm DNA at 100°C for 5min, and quickly ice-bath to prepare single-stranded DNA.

[0106] Add 100 μL of yeast competent cells, 5-10 μg of recombinant plasmid DNA to be transformed, and 50 μg of denatured SS-DNA into a 1.5 mL centrifuge tube, and gently vortex to mix the bacteria completely and evenly.

[0107] Add 600 μL of sterile 0.1M LiCl / PEG4000 solution, and vortex for 10 seconds.

[0108] Incubate in a 230rmp water bath at 30°C for 30min.

[0109] Add 70 μL DMSO and mix by inverting gently.

[0110] Incubate at 42°C for 15 minutes and cool on ice for 1-2 minutes.

[0111] Centrifuge at 14000rmp for 5min at room temperature and discard the supernatant.

[0112] Resuspend the cells in 500 μL of sterilized 1×TE Buffer (pH 7.4), centrifuge at room temperature at 14,000 rpm for 3-5 seconds, and discard the supernatant.

[0113] ...

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Abstract

The invention belongs to the technical field of gene engineering, and discloses mannose-1-phosphate guanylyl transferase, a coding gene and application. The amino acid sequence of the mannose-1-phosphate guanylyl transferase is SEQIDNO: 1; the nucleotide sequence of the DNA for coding the mannose-1-phosphate guanylyl transferase is as shown in SEQ ID NO: 2; and the cDNA (complementary deoxyribonucleic acid) nucleotide sequence of the mannose-1-phosphate guanylyl transferase is shown as SEQ ID NO: 3. The mannose-1-phosphate guanylyl transferase PsMT1 provided by the invention is a hexose transport protein family member, has a mannose transport function, and plays an important role in a process of catalyzing conversion of fructose into mannose; and the PsMT1 gene expression is inhibited through a gene engineering technology to influence the thallus growth of wheat stripe rust (Puccinia striiformis), the pathogenicity of wheat stripe rust is reduced, and the gene has a good application prospect in the aspect of developing small molecule nucleotide pesticides for preventing and treating wheat stripe rust.

Description

technical field [0001] The present invention relates to a kind of mannose-1-phosphate guanylyl transferase, coding gene and application. Background technique [0002] At present, wheat stripe rust caused by the fungus Puccinia striiformis f.sp.tritici (Pst) is one of the most important diseases of cereal crops worldwide, causing huge economic losses worldwide every year. In China, many pandemics have been caused by the physiological race of Pst, which has seriously endangered the safety of wheat production. After long-term and unremitting efforts, China has made great progress in the control of wheat stripe rust, but Pst is highly variable, and new pathogenic types are constantly emerging. Emerging, wheat stripe rust is still a very important fungal disease in China's wheat production. [0003] As a biotrophic obligate parasite, Pst needs to obtain a variety of nutrients including sugars from the host wheat for the growth and development of the bacteria. It is now clear th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54A01N63/50A01N63/30A01P3/00
CPCC12N9/1241C12Y207/07013A01N63/50A01N63/30
Inventor 刘秀峰许静杨楼辰军杨兆顺许高平王昆邵凤武
Owner 天津市农业科学院
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