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Corn multi-copper oxidase coding gene ZmDEK559-2 and application thereof

A technology of oxidase coding and gene, applied in the field of plant bioengineering breeding and molecular biology, to achieve good growth, promote the formation of crop yield, and broad application prospects

Active Publication Date: 2020-12-11
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

After searching, there are no reports about the maize multi-copper oxidase gene ZmDEK559-2 and its application in improving crop yield under adversity

Method used

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  • Corn multi-copper oxidase coding gene ZmDEK559-2 and application thereof
  • Corn multi-copper oxidase coding gene ZmDEK559-2 and application thereof
  • Corn multi-copper oxidase coding gene ZmDEK559-2 and application thereof

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Experimental program
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Effect test

Embodiment 1

[0043] Embodiment 1: Cloning of maize multi-copper oxidase coding gene ZmDEK559-2

[0044] 1) According to the sequence number Zm00001d043090 of the ZmDEK559-2 gene, the maize MaizeGDB database (https: / / www.maizegdb.org / ) was searched to obtain the cDNA sequence of the gene, which was used for primer design and screening of the gene clone.

[0045] 2) According to the above sequence, use PRIMER5.0 software to design PCR amplification primers.

[0046] The upstream primer is 5'TATAAGCCGTGGCCTCCC 3';

[0047] The downstream primer is 5'CTGACCATCGCCTCTTAATTT 3'.

[0048] 3) Take the leaves of Maize Qi 319 seedlings, grind them in liquid nitrogen, and extract RNA with RNAisoPlus (Takara DaLian), a plant total RNA extraction kit. Take 500 ng of total RNA, and follow the PrimeScript RT reagent Kit with gDNA Eraser (Takara, Dalian) kit. Reverse transcription was performed to obtain its cDNA template for cloning of the ZmDEK559-2 gene.

[0049] The reaction system is as follows:

...

Embodiment 2

[0053] Example 2: Construction of recombinant vector for ZmDEK559-2 expression driven by maize Ubiquitin1 promoter and transformation of Escherichia coli and Agrobacterium

[0054] 1) Based on the cDNA sequence of the above clone, use primer5.0 software to design upstream and downstream primers, and at the same time, introduce SacI restriction sites at both ends to obtain base sequences containing SacI restriction sites at both ends, so as to facilitate subsequent plasmid recombination .

[0055] 2) The primer sequences are as follows:

[0056] Upstream primer: 5'-GGAGCTCATGGTGTGGTCGGCTGGGAT-3'

[0057] Downstream primer: 5'-GGAGCTCCTAGACGGAGAGGTAGGACGG-3'

[0058] 3) Using the plasmid containing the ZmDEK559-2 cDNA sequence as a template, PCR amplification obtains the CDS nucleotide sequence with SacI restriction sites at both ends, its CDS sequence is shown in SEQ ID No.1, and the amino acid encoded by it is The sequence is shown as SEQ ID No.2. After the PCR product was...

Embodiment 3

[0067] Example 3: Genetic transformation of maize and acquisition of transgenic plants

[0068] 1) Using the maize inbred line Qi 319 as a material, carry out genetic transformation mediated by Agrobacterium. The seeds germinate after being sterilized, and the shoot tips are cultured in vitro to produce clustered buds, which are finally used as receptors for transformation. The culture medium is as follows:

[0069] Seed germination medium: KI 0.83mg / l, KNO 3 1900mg / l, CaCl 2 2H 2 O 440mg / l, MnSO 4 4H 2 O 22.3 mg / l, KH 2 PO 4 ·H 2 O 170mg / l, H 3 BO 3 10mg / l, CuSO 4 ·5H 2 O 0.025mg / l, FeSO 4 ·7H 2 O27.8mg / l, MgSO 4 ·7H 2 O 370mg / l, NH4NO 3 1650mg / l, ZnSO 4 ·7H 2 O 10mg / l, CoCl 2 ·6H 2 O0.025mg / l, Na 2 MoO 4 2H 2 O 0.5mg / l, pyridoxine hydrochloride 1.0mg / l, inositol 100.0mg / l, thiamine hydrochloride 10.0mg / l, glycine 2.0mg / l, casein hydrolyzate 500mg / l, niacin 1.0mg / l l, sucrose 30g / l, biotin 0.05mg / l, agar powder 7g / l, pH 5.8-6.0, used for seed germi...

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Abstract

The invention discloses a corn multi-copper oxidase coding gene ZmDEK559-2, the CDS sequence of the gene is shown as SEQ ID No.1, and the sequence of a coded protein is shown as SEQ ID No.2. The invention also discloses application of the corn multi-copper oxidase coding gene ZmDEK559-2 or a plant expression vector containing the gene in improvement of drought or high-salt adversity stress tolerance breeding of crops. Experiments prove that the drought resistance, salt resistance and other characters of the ZmDEK559-2 gene overexpression corn are obviously improved compared with those of an untransformed wild type control, and the ZmDEK559-2 gene overexpression corn can keep relatively good growth vigor when encountering adversity stress such as drought and high salt, so that the increaseof the crop yield is promoted, and it is indicated that the gene has wide application prospects in crop stress resistance genetic breeding improvement.

Description

technical field [0001] The invention belongs to the technical fields of plant bioengineering breeding and molecular biology, and in particular relates to a corn polycopper oxidase coding gene ZmDEK559-2 and its use in improving crop tolerance to drought or high-salt adversity stress breeding and under adversity applications in increasing crop yields. Background technique [0002] Corn is an important food, feed and energy crop, and occupies an extremely important position in my country's food and energy security system. Population growth, climate change, social and economic growth, development of animal husbandry and depletion of mineral energy have resulted in global food, feed and fuel shortages, especially the rigid growth in demand for corn. At present, with the advancement of urbanization, the area of ​​cultivated land in our country is constantly shrinking, natural disasters occur frequently, and the constraints on food production are becoming increasingly prominent. ...

Claims

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Application Information

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IPC IPC(8): C12N9/02C12N15/53C12N15/82A01H5/00A01H5/10A01H6/46
CPCC12N9/0063C12N9/0061C12N9/0091C12N15/8273C12Y110/03003C12Y110/03002C12Y116/03001
Inventor 李坤朋张珂刘柏妤李文迪张可炜
Owner SHANDONG UNIV
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