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Gene OsWNK9 for reducing cadmium content of brown rice as well as encoding protein and application of gene OsWNK9

A gene and rice technology, applied in the field of genetic engineering, can solve problems such as the application of rice breeding with low accumulation of cadmium in difficult grains

Active Publication Date: 2022-04-29
SICHUAN AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many genes or proteins that regulate different physiological transport processes have been identified, but most of the functions of the existing genes are to increase the cadmium content of rice brown rice, and it is difficult to be directly used in the breeding application of low-grain cadmium accumulation rice

Method used

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  • Gene OsWNK9 for reducing cadmium content of brown rice as well as encoding protein and application of gene OsWNK9
  • Gene OsWNK9 for reducing cadmium content of brown rice as well as encoding protein and application of gene OsWNK9
  • Gene OsWNK9 for reducing cadmium content of brown rice as well as encoding protein and application of gene OsWNK9

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Example 1 OsWNK9 gene yeast functional complementation test

[0029] 1. Test material

[0030] The Escherichia coli DH5α used in this experiment was purchased from Sangon Bioengineering (Shanghai) Co., Ltd. The yeast strains used (including wild-type BY4741, cadmium-sensitive mutant yeast △ycf1, manganese-sensitive mutant yeast △pmr1 and zinc-sensitive mutant yeast The mutant yeast Δzrc1) was purchased from EUROSCARF (http: / / www.euroscarf.de).

[0031] 2. Test method

[0032] (1) Amplification of OsWNK9 gene

[0033] Using the cDNA of OsWNK9 in the rice Yahui 2816 with low grain Cd accumulation (the same sequence as OsWNK9 in the wild-type Zhonghua 11) as a template, PCR was performed according to the method provided by the high-fidelity DNA amplification kit (NEW ENGLAND BioLabs), and the amplified The coding region fragment of the gene was recovered and purified by agarose gel electrophoresis.

[0034] (2) pFL61-OsWNK9 vector plasmid preparation and recombination ...

Embodiment 2

[0052] Example 2 Construction and Screening of OsWNK9 Mutants

[0053] 1. Test materials.

[0054] The test plant was wild-type rice Zhonghua 11, the test vector was pYLCRISPR / Cas9 multi-target vector, and the Escherichia coli DH5α used was purchased from Sangon Bioengineering (Shanghai) Co., Ltd.

[0055] 2. Candidate gene CRISPR target site design and vector construction.

[0056] Two CRISPR target sites (20KN120T1: 5'-CCATGACAGAGTCATCGATC-3'; 20KN120T2: 5'-GGCATACTGTGTCAAG CTCC-3') were designed according to the OsWNK9 coding region sequence. At the same time, design target site adapter primers according to the target site (20KN120T1-F: 5'-GCCGCCATGACAGAGTCATCGATC-3'; 20KN120T1-R: 5'-AAACGATC GATGACTCTGTCATGG-3'; 20KN120T2-F: 5'-GTTGGCATACTGTGTCAA GCTCC-3' ; 20KN120T2-R: 5'-AAACGGAGCTTGACACAGTATGC-3').

[0057] The target site forward and reverse primers are annealed (both ends form sticky ends that can match the enzyme-cut vector), and at the same time, the BsaI-digeste...

Embodiment 3

[0060] Embodiment 3 Identification of mutant seedling stage growth phenotype and mature stage yield phenotype

[0061] 1. Test materials.

[0062] The tested plants were wild-type rice Zhonghua 11 and the OsWNK9 mutant; the tested nutrient solution was Kimura B complete nutrient solution.

[0063] 2. Transplant the wild-type and OsWNK9 mutants at the three-leaf stage into Kimura B complete nutrient solution with cadmium concentrations of 0 and 50 μM in the experimental greenhouse of Wenjiang Campus, Sichuan Agricultural University, and collect samples 10 days after cadmium treatment. Phenotype identification at the seedling stage, such as Figure 4~6 shown.

[0064] Figure 4 Middle WT means wild type Zhonghua 11, oswnk9-1, oswnk9-2, oswnk9-3 all mean oswnk9 mutant; "*" means significant difference between mutant and wild type (p<0.05). The control means the normal treatment condition, and the cadmium treatment means the 50 μM cadmium stress condition. The seedling height...

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Abstract

The invention discloses a gene OsWNK9 for reducing the cadmium content of brown rice as well as an encoding protein and application thereof. The nucleotide sequence of the gene OsWNK9 is as shown in SEQ ID NO. 1, and the amino acid sequence of the encoded protein of the gene OsWNK9 is as shown in SEQ ID NO. 2. A novel functional gene OsWNK9 for controlling transfer of cadmium in the rice body is provided for breeding with high rice stem cadmium enrichment and low brown rice cadmium accumulation, and accumulation of cadmium in brown rice is reduced by regulating enrichment of cadmium in rice stems and lower nodes, so that the cadmium concentration of the brown rice is reduced; the OsWNK9 also influences the absorption and accumulation of iron by the rice and the iron concentration in the brown rice at the same time, and is helpful for enhancing trace elements of edible parts of the rice.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene OsWNK9 for reducing the cadmium content of brown rice, its encoded protein and its application. Background technique [0002] Reducing the cadmium content of brown rice and cultivating crops with safe heavy metal content in edible parts are important strategies to solve the problem of safe utilization of polluted farmland and ensure food security. The cadmium content in rice brown rice is a complex quantitative trait controlled by multiple genes, and it is also affected by the regulation of multiple physiological processes. Rice has a strong ability to absorb cadmium, so its stems have a strong ability to accumulate cadmium. Breeding rice varieties with high cadmium accumulation in stems and low cadmium content in edible parts is an effective way to achieve "production while repairing". [0003] Identifying the genes and encoded proteins that regu...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/04A01H5/10A01H6/46
CPCC07K14/415C12N15/8271
Inventor 余海英郭静怡李廷轩黄化刚张锡洲叶代桦郑子成王永东唐毓
Owner SICHUAN AGRI UNIV
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