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HPPD protein, gene, vector, cell, composition, application of HPPD protein, gene, vector, cell and composition, and method for improving herbicide resistance of crops

A gene and protein technology applied in the field of improving crop herbicide resistance

Pending Publication Date: 2022-07-22
HUAZHONG NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, taking the HPPD protein of economic crops as the research object, without changing its normal catalytic function, there are few reports on how to enhance its resistance to herbicides by mutating individual amino acid residues

Method used

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  • HPPD protein, gene, vector, cell, composition, application of HPPD protein, gene, vector, cell and composition, and method for improving herbicide resistance of crops
  • HPPD protein, gene, vector, cell, composition, application of HPPD protein, gene, vector, cell and composition, and method for improving herbicide resistance of crops
  • HPPD protein, gene, vector, cell, composition, application of HPPD protein, gene, vector, cell and composition, and method for improving herbicide resistance of crops

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] Synthesis of coding genes and nucleotide sequences

[0126] The following coding genes and nucleotide sequences were synthesized by Wuhan Jinkarui Bioengineering Co., Ltd. according to the nucleotide sequences provided by the present invention.

[0127] (1) SEQ ID NO: 11

[0128] The gene encoding the protein shown in Table 2 and the nucleotide sequence shown in SEQ ID NO: 11 were synthesized according to the nucleotide sequence shown in SEQ ID NO: 11.

[0129] Table 2

[0130] protein coding gene Mutation site codon SEQ ID NO: 1 WT (wild type) / K418D K418D GAT E423M E423M ATG E423Q E423Q CAG E432M E432M ATG E432I E432I ATT

[0131] (2) SEQ ID NO: 21

[0132] The gene encoding the protein shown in Table 3 and the nucleotide sequence shown in SEQ ID NO: 21 were synthesized according to the nucleotide sequence shown in SEQ ID NO: 21.

[0133] table 3

[0134] protein coding gene Mutatio...

Embodiment 2

[0148] Construction of expression vector

[0149] (1) Amplification of the target gene

[0150] Using the nucleotide sequences of SEQ ID NO: 11, SEQ ID NO: 21, SEQ ID NO: 31, SEQ ID NO: 41, and SEQ ID NO: 51 as templates, respectively, obtained by PCR reaction as shown in Tables 2, 3, and 4 The wild-type and mutant gene sequences of HPPD shown in , 5 and 6 were linked into the corresponding vectors.

[0151] The PCR reaction system and PCR program settings are as follows:

[0152] Table 7

[0153] PCR reaction system composition Addition amount (volume) 10x PCR buffer 5μL 5×dNTPs (10mM) 1μL stencil 1μL forward primer 1.0μL Orientation primer 1.0μL PfuDNA polymerase 0.5μL ddH2O to a total volume of 50 μL

[0154] Table 8

[0155] step temperature(℃) time(s) 1 95 240 2 95 45 3 55 45 4 72 400 5 72 600 6 4 save

[0156] The extension time at 72°C depends on ...

Embodiment 3

[0187] Expression and purification of target proteins

[0188] (1) Expression

[0189] The protein is firstly extracted, and the optimal expression conditions and purification strategies are explored. After the conditions are determined, a large amount of extraction is carried out. Protein expression was carried out in Escherichia coli BL21(DE3) cells (purchased from Kangti Life Technology Co., Ltd., product number KTSM109), and the process is as follows:

[0190] a. Pick a single clone on a plate that has been cultured for 16 hours, add it to 100 mL of LB medium containing the desired antibiotic resistance, and culture at 37 °C and 220 rpm for 5 hours, that is, obvious turbidity is observed;

[0191] b. Expand the small bottle of bacteria in step a into a large bottle of LB medium (containing antibiotics) at a volume ratio of 1:100, and cultivate at 37°C and 220rpm for 3h; wait for OD 600 When the temperature reached 0.7h, the temperature was lowered to 20°C and then 0.2mM ...

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Abstract

The invention relates to the field of gene engineering, and discloses an HPPD protein, a gene, a vector, a cell, a composition, application of the HPPD protein, the gene, the vector, the cell and the composition and a method for improving herbicide resistance of crops. The invention discloses a p-hydroxyphenylpyruvate dioxygenase protein, a gene for coding the p-hydroxyphenylpyruvate dioxygenase protein, a recombinant vector, a transgenic cell, a composition and application of the p-hydroxyphenylpyruvate dioxygenase protein, the gene, the recombinant vector, the transgenic cell and the composition to improvement of herbicide resistance of crops, and further discloses a method for improving the herbicide resistance of the crops. The HPPD protein and the coding gene thereof provided by the invention can be used for improving the herbicide resistance of crops on the premise that the catalytic activity of enzyme is basically not influenced.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a HPPD protein, gene, vector, cell, composition and application thereof, and a method for improving crop herbicide resistance. Background technique [0002] HPPD is a member of the non-heme iron, α-ketoacid-dependent oxygenase family, and is a key enzyme in the tyrosine metabolic pathway, which can further catalyze the catalytic product of tyrosine aminotransferase, p-hydroxyphenylpyruvate produce black acid. [0003] In plants, homogentisate is a precursor for the biosynthesis of plastoquinone and tocopherol. Plastoquinone is not only a key cofactor for phytoene desaturase, but also a carrier of electron transfer, directly involved in photosynthesis. Tocopherol is one of the structural components of cell membranes and is an antioxidant of cell membranes. Once the activity of HPPD is inhibited, the synthesis of plastoquinone will be reduced, and the catalytic process...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/82C12N1/21A01H5/00A01H6/00
CPCC12N9/0069C12N15/8274C12N15/8218C12Y113/11027
Inventor 杨光富林红艳杨文超魏雪芳肖寒董进霍慧玲
Owner HUAZHONG NORMAL UNIV
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