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A corn kernel development-related gene mir169o and its application

A technology of mir169o and corn, applied in application, genetic engineering, DNA/RNA fragments, etc., can solve problems such as increasing crop yield

Active Publication Date: 2021-06-04
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The patented technology has no guiding significance for directly increasing crop yield

Method used

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  • A corn kernel development-related gene mir169o and its application
  • A corn kernel development-related gene mir169o and its application
  • A corn kernel development-related gene mir169o and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Cloning of Example 1 Maize zma-miR169o Precursor Sequence cDNA

[0019] The cDNA library of maize B73 was used as a template for RT-PCR amplification, and the PCR primers were as follows: zma-miR169o F: 5'-GTAGCCAAGAATGACTTGCC-3' (SEQ ID NO: 3); zma-miR169o R: 5'-TGGCTAGCCAAGAAGACCTG-3 ' (SEQ ID NO: 4).

[0020] The amplification conditions are as follows:

[0021]

[0022] The amplified DNA fragment was recovered from the agarose gel and cloned into the pEASY-T1 vector. It was digested and identified by sequencing, and named pTmiR169o. The sequencing results showed that the obtained precursor was the miRNA shown in Sequence 1, which was zma-miR169o, with a full length of 138bp, and the specific sequence was shown in SEQ ID NO: 1 in the sequence table. The processed mature sequence is shown in sequence 2, with a total of 21 nts, and its sequence is shown in sequence table SEQ ID NO:2.

Embodiment 2

[0023] Example 2 Construction of pmiR169o:GUS expression vector

[0024] 1) Design a 2.0kb-long primer for amplifying the upstream of the zma-miR169o precursor sequence, and add BamHI and NcoI restriction site sequences at both ends of the primer. The primer sequences are as follows:

[0025] PmiR169oF:

[0026] 5'-GGATCCGAGAGGGAGAGGGAAGTTGC-3' (SEQ ID NO: 5);

[0027] PmiR169oR:

[0028] 5'-CCATGGCAAAAGGGGCTCTCCATCTC-3' (SEQ ID NO: 6).

[0029] Use B73 genomic DNA as a template for PCR amplification;

[0030] 2) The amplified product is recovered, and a ligation reaction is performed on the recovered product and pCAMBIA3301 double digested with BamHI and NcoI.

[0031] The reaction system is as follows:

[0032]

[0033] 3) Positive clones were identified by enzyme digestion, and the positive clones were pmiR169o::GUS vectors. figure 1 The pmiR169o::GUS expression vector map is shown, the positive clones are sequenced and verified, and the positive plasmids are obtai...

Embodiment 3

[0034] Example 3 Construction of pUBI::miR169o expression vector

[0035] 1) Design primers for amplifying the full-length precursor of Zma-miR169o, and add 20nt vector homologous sequences to both ends of the primers. The primer sequences are as follows:

[0036] Zma-miR169oF:

[0037] 5'-GCGTGGATCCGAGCTCACATGTAGCCAAGAATGACTTGCC-3' (SEQ ID NO: 7);

[0038] Zma-miR169oR:

[0039] 5'-GTCACCTGTAATTCACACTCTGGCTAGCCAAGAAGACCTG-3' (SEQ ID NO: 8).

[0040] Use the pTmiR169o plasmid as a template for PCR amplification;

[0041] 2) The amplified product is recovered, and the recovered product and the transformed pCAMBIA3301m are subjected to a recombination reaction, the reagent used is GBclonart Seamless Cloning Kit,

[0042] The reaction system is as follows:

[0043]

[0044] 3) Positive clones were identified by enzyme digestion, and the positive clones were pUBI::miR169o vectors. figure 2 The pUBI::miR169o expression vector map is shown, the positive clones are sequence...

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Abstract

The invention discloses a corn grain development-related gene miR169o and its application, which belong to the technical field of biological genes. The precursor nucleotide sequence of miR169o is shown in the sequence listing SEQ ID NO: 1; its mature coding sequence is shown in the sequence listing SEQ ID NO: 2. Under the same planting and management conditions, the single grain volume of zma-miR169o overexpressed maize lines increased by 8%-20% and the single-grain weight increased by 10%-20% compared with the control. Other agronomic traits did not change significantly, so it has important application value in improving maize yield. In addition, the zma-miR169o overexpression material obtained in the present invention can be transferred to an excellent inbred line through backcrossing to create an excellent variety for maize breeding.

Description

technical field [0001] The invention belongs to the technical field of biological genes, and in particular relates to a gene miR169o related to corn grain development and an application thereof. Background technique [0002] Grain yield is one of the most important and complex traits of crops, the ultimate embodiment of a series of physiological and biochemical processes controlled by interrelated polygenes. Corn grain yield is determined by the number of plants per unit area, the number of grains per plant and the weight of 100 grains. Under the condition of constant planting density, the grain number and grain weight per plant directly determine the grain yield. More than 80% of the total weight of corn grain is endosperm, and the development, proliferation and filling of endosperm cells determine the weight and quality of the grain. [0003] MiRNAs are a class of non-coding RNA molecules with a length of about 22 bases, which specifically post-transcriptionally regulate...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/82C12N15/11A01H5/00A01H6/46A01H6/54A01H6/20
CPCC12N15/113C12N15/8261C12N2310/141
Inventor 徐妙云王磊邹俊杰郑红艳
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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