Application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice

A technology of acid oxidase and antiscorbic acid, applied in the direction of oxidoreductase, application, enzyme, etc., can solve the problems of reduced oxidative stress tolerance, lack of AsA content, etc., and achieve the effect of enhancing drought resistance and improving plant drought resistance

Inactive Publication Date: 2020-11-03
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Conversely, the deficiency of AsA content in plants res

Method used

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  • Application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice
  • Application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice
  • Application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] Example 1 Acquisition of Rice RIP5 Gene Knockout Mutant rip5

[0049] (1) Gene editing of RIP5 target sites

[0050] The rice ascorbate oxidase gene RIP5 is numbered LOC_Os06g37150 in the Rice Genome Annotation Project (the nucleotide sequence is shown in SEQ ID NO.1; the amino acid sequence is shown in SEQ ID NO.2) (ShengzeYao, Zhirui Yang, Rongxin Yang, et al. Transcriptional regulation of miR528 by OsSPL9 orchestrates antiviral response in rice [J]. Mol Plant, 2019, 12:1114-1122.). The gene knockout vector backbone used in the present invention is CRISPR / Cas vector BGK03 (http: / / www.biogle.cn / index / excrispr)( figure 2 ), connect the gRNA target sequence of RIP5 to the CRISPR / Cas9 vector pBGK03 containing the Cas9 enzyme expression cassette, and obtain the pBGK037 vector containing the gRNA target sequence. For the specific construction method, refer to the instructions of the Baige CRISPR / Cas Vector Construction Kit Cat# BGK03. Among them, the 158-180 positions o...

Embodiment 2ZH11

[0053] AO activity determination of embodiment 2 ZH11 and mutant rip5

[0054] Ascorbic Oxidase (Ascorbic Oxidase, AO) activity was determined with reference to the instructions in the Ascorbic Oxidase (AAO) Activity Test Kit (Colorimetric Method) (Nanjing Jiancheng Bioengineering Institute), which is briefly described as follows:

[0055] (1) Extraction of crude enzyme solution: Weigh 0.1 g of the rice leaves of the rip5 mutant and wild-type ZH11 screened in Example 1, add 1 mL of reagent-homogenized in an ice bath, centrifuge at 16,000 g at 4°C for 10 min, and take the supernatant Tested on ice.

[0056] (2) Preparation of reaction system: 100 μL of crude enzyme solution, 850 μL of preheated reagent II and 50 μL of reagent III, with 100 μL of double-distilled water as a control, mix quickly, transfer to a 1 mL quartz cuvette, and measure at a wavelength of 265 nm absorbance value. Adjust to zero with double distilled water, measure the absorbance value A for 10s respective...

Embodiment 3

[0058] Embodiment 3 drought treatment ZH11 and mutant rip5

[0059] Appropriate amount of seeds of wild-type ZH11 and mutant rip5 were taken respectively, soaked in a petri dish with filter paper for 24 hours, then poured out the water, kept the filter paper moist, put them in an incubator at 37°C for 2 days, and then transferred them to soil. , moved to an incubator for growth, day / night were 28°C 14h / 26°C 12h, respectively. Mix the soil thoroughly, and then divide it into small plastic pots equally. When the rice grows to one leaf and one heart, transplant it into small plastic pots with soil prepared in advance. Plant 4 plants in each pot and place them in an artificial climate room at 28°C. 14h / 26℃12h, stop watering for two weeks after the tillering stage, and then rewater for one week to investigate the survival rate.

[0060] The result is as Figure 4 Shown: The results show that mutant rip5 is significantly more drought-resistant than its wild-type ZH11 counterpart. ...

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Abstract

The invention discloses an application of ascorbic acid oxidase RIP5 to regulating and controlling of drought resistance of rice. An RIP5 gene knockout mutant is constructed by targeted mutation of anRIP5 gene through a GRISPR/Cas9 system, and the inventor finds that the drought resistance of plants can be effectively improved by inhibiting the expression quantity and/or activity of the RIP5 protein, that is, the ascorbic acid oxidase gene RIP5 of rice can negatively regulate and control the drought resistance of the plants. The invention also provides a gRNA target sequence for editing the ascorbic acid oxidase gene RIP5 of the rice, and the gRNA target sequence cooperates with a CRISPR/Cas9 gene editing system to realize targeted mutation of the coding gene of the RIP5 protein, so thatthe drought resistance of the rice can be regulated and controlled. The research of the invention provides new gene targets and resources for drought resistance genetic breeding of the plants, and knockout mutant plants obtained by editing the gene have important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and particularly relates to the application of ascorbate oxidase RIP5 in regulating the drought resistance of rice. Background technique [0002] Rice is an important food crop. More than one-third of the world's population uses rice as a staple food. Therefore, promoting stable and high rice production is of great significance to ensuring food security and social stability. In recent years, with global warming, frequent natural disasters, uneven distribution of rainfall in time and space, and water shortages, the production area and yield of rice have declined severely. One of the main limiting factors. Drought stress has an important impact on plant growth and development, crop yield reduction and environmental degradation. In the face of natural environmental hazards, plants have evolved a series of adaptations in terms of morphology, physiology, biochemistry, cellular and molecular levels, such...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/82A01H5/00A01H6/46
CPCC12N9/0063C12N15/8273C12Y110/03003
Inventor 张泽民张晶晶何焱梁嘉燕
Owner SOUTH CHINA AGRI UNIV
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