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HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof

An inhibitor and gene technology, applied in the field of microbial genetic engineering and plant genetic engineering, can solve the problems affecting the biosynthesis of carotenoids and the obstruction of phytoene desaturase catalysis

Inactive Publication Date: 2016-05-11
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, plastoquinone is also a key cofactor of phytoene desaturase, and the reduction of plastoquinone hinders the catalytic action of phytoene desaturase, thereby affecting the biosynthesis of carotenoids

Method used

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  • HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof
  • HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof
  • HPPD inhibitor-resistant gene derived from ochrobactrum anthropi and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1 Isolation, Purification and Identification of HPPD Inhibitor-Resistant Bacterial Strain-Human Paleobacterium OchrobactrumanthropiH-2

[0063] 1. Source of soil samples

[0064] Soil samples were collected from 5 cm below the soil layer of the experimental fields sprayed with HPPD inhibitors.

[0065] 2. Isolation and purification of HPPD inhibitor-resistant strains

[0066] Enrichment of bacterial strains: Weigh 20 grams of soil samples respectively, one of which is added to 1 / 10LB liquid medium containing 0.3mM HPPD inhibitor (filling volume is 50ml / 300ml Erlenmeyer flask), the other soil sample Add to the 1780 modified liquid culture medium containing 0.3mM HPPD inhibitor (75% isoxaflutole water dispersible granule) (filling volume is 50ml / 300ml Erlenmeyer flask), set 30 o The enrichment culture was carried out in a C shaker at 200 rpm.

[0067] Acclimatization and purification of strains: Each time of enrichment culture for 7 days, the culture was centri...

Embodiment 2

[0073] Cloning of DNA fragments of embodiment 2 highly resistant to HPPD inhibitors

[0074] Partial DNA Fragments Highly Tolerant to HPPD Inhibitors Obtained by PCR Method

[0075] Designed according to the OchrobactrumanthropiHPPD inhibitor resistance gene sequence published in NCBI

[0076] Primer 1-F: 5′-ATGAAGACTTCGATAGCAACCGT-3′

[0077] Primer 1-R: 5′-TCAGAGTGCCACAGCACTTTTTG-3′

[0078] Using high-fidelity Taq enzyme KOD-FX to amplify the partial sequence of the anti-HPPD inhibitor gene from the Ochrobacttrumanthropi genome, PCR program: 98 o C10sec, 60 o C30sec, 68 o C2min, a total of 30 cycles.

[0079] The PCR product of primer 1-F (shown in SEQIDNo: 3) and primer 1-R (shown in SEQIDNo: 4) is a 1902bp DNA fragment, which is a DNA fragment for obtaining high tolerance to HPPD inhibitors.

[0080] From Ochrobacttrumanthropi genomic DNA, the complete sequence of a DNA fragment highly resistant to HPPD inhibitors was amplified by Taq enzyme KOD-FX high-fidelity enz...

Embodiment 3

[0081] The construction of embodiment 3 plant expression vector PTG2-OchHPPD

[0082] According to the Och-HPPD gene sequence, design primers 2-F and 2-R

[0083] Primer 2-F: 5′-GTCGACATGAAGACTTCGATAGCAACCGT-3′

[0084] Primer 2-R: 5′-CCCGGGTCAGAGTGCCACAGCACTTTTTG-3′

[0085] The Och-HPPD gene sequence was amplified by PCR with primers 2-F (shown in SEQIDNo: 5) and 2-R (shown in SEQIDNo: 6), and then digested with SalI and XmaI to obtain an insert fragment; it was taken from the company and kept The PTG1 vector was digested with SalI and XmaI, and then the above-mentioned insert fragment and the vector were connected with ligase, and the ligation product was transformed into E. coli DH5α strain, and the positive selection was selected after culturing and screening in 2×YT medium containing 50 mg / L kanamycin Cloning, after extracting the plasmid, the clone with the correct sequence was selected by sequencing, and the plasmid with the target gene was obtained, named PTG2-OchHP...

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Abstract

The invention discloses an HPPD inhibitor resistant gene having high resistance to an HPPD inhibitor and derived from ochrobactrum anthropi and an application thereof in transgenic plants. A method for transforming plants by the gene and high HPPD inhibitor-resisting activity expressed in the transgenic plants play an important role in research and development of plants resisting the HPPD inhibitor.

Description

technical field [0001] The invention belongs to the field of microbial genetic engineering and plant genetic engineering, and specifically relates to an anti-HPPD inhibitor gene derived from human paleobacterium and its application in transgenic plants. Background technique [0002] p-Hydroxyphenylpyruvate dioxygenase (4-HPPD, EC1.13.11.27) is an important enzyme lknderDA that catalyzes tyrosine metabolism in most organisms. Amino Acid Metabolism (2nded) [M]. Lon-don: John Wiley & Sons, 1985: 208-221. It can catalyze the conversion of tyrosine degradation product p-hydroxyphenylpyruvate into homogentisic acid, which is an important substance in plants, and it can be further deacidified, polypentadienylated and alkylated to generate Plastoquinone and tocopherol required for electron transfer in photosynthesis (Crouch N.P. et al. 1997, Tetrahedron, 53, 20, 6993-7010, Fritze et al., 2004, PlantPhysiology 134:1388-1400) Since HPPD enzymes in 20 Since it was discovered in the ...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12Q1/68A01H1/02A01H5/00G01N33/68
CPCC07K14/195A01H1/02C12N15/8271
Inventor 徐海英何峰初宇陈磊张冉
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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