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Protein with phosphite dehydrogenase catalytic activity, coding gene and application thereof

A phosphite dehydrogenase and phosphite technology, applied in the application, genetic engineering, plant genetic improvement and other directions, can solve the problem of not giving plants phosphite dehydrogenase phosphite utilization ability, etc., to improve phosphorus utilization efficiency Effect

Inactive Publication Date: 2019-01-11
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the inventor found that the PTDH-Q gene could not be endowed with the activity of phosphite dehydrogenase and the utilization ability of phosphite after constructing the vector and transferring it into the plant.

Method used

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  • Protein with phosphite dehydrogenase catalytic activity, coding gene and application thereof
  • Protein with phosphite dehydrogenase catalytic activity, coding gene and application thereof
  • Protein with phosphite dehydrogenase catalytic activity, coding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1, the synthesis of phosphorous acid dehydrogenase gene (PtxQ) and the construction of overexpression vector

[0036] (1) Codon optimization and synthesis of phosphite dehydrogenase gene (PtxQ)

[0037] Using the Codon OptimWiz software of Jinweizhi Company, input the protein sequence of phosphite dehydrogenase PtxQ, select the codon preference of rice and corn for codon optimization, design and modify the PtxQ gene sequence, and the nucleotide sequence of the PtxQ gene output by the software As shown in SEQ ID NO:1. According to the sequence optimized by the software (see SEQ ID NO: 1), the nucleotide sequence was artificially synthesized de novo by Jinweizhi Company.

[0038] (2) Construction of overexpression vector Ubi-PtxD by using phosphite dehydrogenase gene PtxQ

[0039] According to the PtxQ gene nucleotide sequence (see SEQ ID NO: 1) design and synthesis of PCR amplification primers, the sequence is as follows:

[0040] PtxD-F: 5'tgcaggtcgactctag...

Embodiment 2

[0046] Embodiment 2, rice transgenic

[0047] (1) Test materials The wild-type (ie non-transgenic) rice variety Zhonghua 11 (also known as ZH11, from the Institute of Crop Science, Chinese Academy of Agricultural Sciences).

[0048] (2) Rice transgenic method

[0049] 1) Preparation of rice mature embryo callus

[0050] After the mature seeds of the rice variety Zhonghua 11 were shelled, the plump, smooth seeds without plaque were selected and put into a beaker, and sterilized with 70% ethanol for 1 min. Pour off the ethanol, add 20% (v / v) NaClO solution (available chlorine is about 1-1.2%) to disinfect for 90 minutes. Pour off the NaClO solution, wash 5 times with sterile water, and soak in sterile water for 30 minutes for the last time. The sterile water was poured off, and the seeds were blotted dry on sterile filter paper. Transfer the seeds into the induction medium with tweezers, and culture in the dark at 28°C for 10-14d. Open the culture dish on the ultra-clean wo...

Embodiment 3

[0086]Transgenic materials overexpressing the PtxQ gene and wild-type (non-transgenic) rice seeds Zhonghua 11 were treated with 1% nitric acid solution at room temperature for 16 hours, rinsed off the nitric acid repeatedly with clean water, and cultivated in the dark at 37°C for two days until white . After the germination was completed, it was cultivated with rice nutrient solution (Table 13). The greenhouse light cycle is 12h light / 12h dark, the light intensity is 3000lux, the daytime culture temperature is 30°C, and the nighttime culture temperature is 22°C. Seedlings grown to 10 days were treated for two weeks under the condition of supplying phosphate and phosphite, and the enzyme activity and growth phenotype of all plants were determined. The formula of rice nutrient solution is shown in Table 13.

[0087] Table 13 Formula of rice nutrient solution under hydroponic conditions

[0088]

[0089] Note in Table 13: *The nutrient solution was adjusted to pH 5.5 with 2...

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Abstract

The invention belongs to the technical field of plant genetic engineering and in particular relates to a protein with phosphite dehydrogenase catalytic activity, a coding gene and an application thereof. The cloned phosphite dehydrogenase PtxQ protein gene in the invention has a nucleotide sequence shown as SEQ ID NO:1, and the sequence of the protein coded by the gene is shown as SEQ ID NO:2. Genetic transformation validation proves that the transgenic plant with the phosphite dehydrogenase PtxQ protein genes has the advantages that the activity on the phosphite dehydrogenase is obviously improved, and the transgenic plant gains the ability of utilizing phosphate. The gene disclosed by the invention can be applied to regulating the ability of crops for utilizing the phosphate.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. It specifically relates to a protein with catalytic activity of phosphorous acid dehydrogenase, a coding gene and its application. The present invention obtains phosphorous acid dehydrogenase with high catalytic activity through mutation screening, and then utilizes artificial design and synthesis of PtxQ gene suitable for plant expression to obtain Artificially synthesized genes are transferred into host cells, and transgenic plant materials are obtained using transgenic technology. The invention also relates to utilizing the gene to enable plants to acquire phosphorous acid dehydrogenase activity and the ability to use phosphorous acid, and to achieve the fertilization effect on target plants under the condition of phosphorous acid application. Background technique [0002] The per capita arable land area in my country is far lower than that in developed countries, and nation...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N9/02C12N15/82A01H5/00A01H6/46
CPCC12N9/0004C12N15/8243C12N2800/22C12Y120/01001
Inventor 王创吴高兵袁丽丽刘同同石磊徐芳森
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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