An Arabidopsis thaliana seed iron accumulation regulatory gene ino and its encoded protein and application

A technology that regulates genes and encodes proteins, and is used in applications, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2022-04-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how transcription factors regulate transporters to load iron from the mother into the seed during plant seed formation has not been reported.
However, there is no report on the involvement of the transcription factor INO in the iron transport process of plant embryos.

Method used

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  • An Arabidopsis thaliana seed iron accumulation regulatory gene ino and its encoded protein and application
  • An Arabidopsis thaliana seed iron accumulation regulatory gene ino and its encoded protein and application
  • An Arabidopsis thaliana seed iron accumulation regulatory gene ino and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Cloning method of transcription factor INO gene involved in regulation of seed iron

[0056] Sow the surface-sterilized Arabidopsis seeds on 1 / 2MS solid medium, vernalize in the dark at 4°C for 2-3 days, then move to light and culture for 7 days, then move the soil and culture until the bolting Arabidopsis blooms , use scissors to cut young green pods 4-6 days after flowering for RNA extraction, and use the kit to reverse transcribe and synthesize cDNA as a template for subsequent gene cloning.

[0057] According to the published Arabidopsis whole genome sequencing results, design upstream primers and downstream primers respectively:

[0058] Upstream primer: 5'-ATGACAAAGCTCCCCAACATGAC-3' (SEQ ID No: 5);

[0059] Downstream primer: 5'-TCCCAAATTGTTATTACTCAAATGGAG-3' (SEQ ID No: 6).

[0060] Toyobo KOD FX enzyme was used for PCR amplification. The reaction program of PCR amplification was: pre-denaturation: 94°C, 2 minutes; denaturation: 98°C, 10 seconds; annealing: 57°...

Embodiment 2

[0063] Construction method of constitutive overexpression transgene vector

[0064] A cauliflower mosaic virus constitutive promoter CaMV35S was forward inserted into the multiple cloning site by double enzyme digestion and ligation of DNA fragments using the restriction sites SacI and KpnI, so that the promoter CaMV35S was successfully connected to the vector, Transformation obtains the vector 35s-pCAMBIA1301 (see figure 1 ).

[0065] Using primers: INO-F: 5'-TGGTACCTACACACACACTCTCTATGACAAAG-3' (SEQ ID No: 2) and INO-R: 5'-CGGATCCTCCCAAATTGTTATTACTCAAATGG-3' (SEQ ID No: 3), the cDNA obtained in Example 1 above Using the sequence as a template, referring to the PCR amplification reaction procedure in Example 1 above, amplify and obtain the sequence of the Arabidopsis transcription factor gene AtINO coding region containing restriction sites at both ends. According to the instructions of the pMD19T vector produced by Takara Company, the sequence of the Arabidopsis transcription...

Embodiment 3

[0067] Arabidopsis transformation method

[0068] Add 0.5 μg of the binary transgenic vector pOEINO plasmid prepared in Example 2 into the competent cells of Agrobacterium tumefaciens strain GV3101, and then bathe in ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes. , adding no anti-LB and activating in a shaker at 28°C for 1 h to obtain an Agrobacterium strain containing a binary plasmid vector. The prepared GV3101 strain containing the binary plasmid vector is used to transform Arabidopsis thaliana, and the specific steps are as follows:

[0069] The Agrobacterium containing the binary plasmid vector was cultured in LB medium containing 50mg / L kanamycin (Kan) and 50mg / L rifampicin (Rif) at 28°C with shaking overnight until the absorbance value at OD 600 When the value is 1.0, the bacterial cells were collected by centrifugation at 4000rpm for 15min, and resuspended with 1 / 2MS medium containing 50g / L sucrose. And se...

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Abstract

The invention provides an Arabidopsis thaliana seed iron accumulation regulating gene INO and its encoded protein and application, belonging to the technical field of plant genetic engineering. The nucleotide sequence of Arabidopsis thaliana seed iron accumulation regulatory gene INO is shown in SEQ ID NO: 1 in the sequence listing. Arabidopsis seed iron accumulation regulatory gene INO encodes a 231-amino acid protein. Experiments of the present invention prove that the gene INO negatively regulates the loading of iron in plant seeds, so the application of the seed iron accumulation regulation gene INO or its encoded protein in the regulation of iron accumulation in plant seeds; Accumulation in seeds also improves the resistance of plants to iron-deficiency environments at the seedling stage. Therefore, the present invention also provides the application of the seed iron accumulation regulation gene INO or its coded protein in biofortifying iron or improving plant resistance to iron deficiency.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to an Arabidopsis thaliana seed iron accumulation regulating gene INO and its encoded protein and application. Background technique [0002] Iron is an indispensable trace element for organisms to maintain life and growth, but due to the availability of iron in the soil and the low iron content in grains, it is easy to cause iron deficiency in organisms [1] . Therefore, iron deficiency is the most common nutritional deficiency disease, and the resulting iron deficiency anemia remains a worldwide concern today [2] . According to the "Recommended Intake of Dietary Nutrients for Chinese Residents" [3] It shows that the daily recommended iron intake for adult men is 12mg, while for adult women it is more than 20mg / d. Seeds, especially those of cereal crops, are a staple food for humans. However, as a heterotrophic organ, the iron storage of seeds depend...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/11C07K14/415C12N15/82A01H5/10A01H6/20
CPCC07K14/415C12N15/8243C12N15/8271
Inventor 郑绍建孙鹂李桂新丁忠杰
Owner ZHEJIANG UNIV
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