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Molecular marker of maize high lysine gene zmcytmdh4 and its application

A high-lysine, molecular marker technology, applied in the field of molecular markers of the high-lysine gene ZmcytMdh4 in maize, can solve the problems of inability to produce and process, low yield, and susceptibility to diseases and insect pests, and achieve the effect of improving efficiency

Active Publication Date: 2021-11-23
HENAN AGRICULTURAL UNIVERSITY
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  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved by the present invention is to provide a kind of corn high lysine gene ZmcytMdh4 Molecular markers and their applications aim to solve the technical problems that the existing high-lysine mutants have weak seedling growth, powdery endosperm, low yield, susceptibility to diseases and insect pests, and cannot be produced and processed

Method used

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  • Molecular marker of maize high lysine gene zmcytmdh4 and its application
  • Molecular marker of maize high lysine gene zmcytmdh4 and its application
  • Molecular marker of maize high lysine gene zmcytmdh4 and its application

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Embodiment 1

[0031] Embodiment 1: Obtaining of high lysine functional gene

[0032] (1) A segregation population containing 30,000 individuals was constructed using a high-lysine content maize mutant and an excellent inbred line Zheng 58, and a mutant with a significantly increased lysine content was obtained.

[0033] The inventor screened a mutant material with a defective grain phenotype during a large number of field breeding processes in the early stage. Then, a set of F 2 The populations were separated, and the mature grains of the wild type and the mutant were measured by near infrared spectroscopy (NIR). The results showed that the lysine content in the mutant was significantly higher than that of the wild type.

[0034] (2) The target gene was located in the physical interval of about 224 kb on the first chromosome of maize by map-based cloning, and there were 2 protein-coding genes in the candidate segment.

[0035] By chi-square test, for F 2 Statistical analysis of the grain...

Embodiment 2

[0038] Embodiment 2: Screening of high lysine functional gene

[0039] Sequence analysis and maize genetic transformation demonstrate that the enzyme encoding cytoplasmic malate dehydrogenase 4 ZmcytMdh4 gene is the gene of interest.

[0040] By querying the MaizeGDB (https: / / www.maizegdb.org / ) database, it was found that Zm00001d032695 encodes a cytoplasmic malate dehydrogenase. Knockout vectors were constructed using CRISPR / Cas9 technology and sent to China Seed Group Co., Ltd. for genetic transformation. Allelic tests were performed on the isolated homozygous positive plants with the original mutant and heterozygous individual plants. The results showed that ZmcytMdh4 for the target gene.

[0041] ZmcytMdh4 Gene structure and nucleotide polymorphism information such as figure 1 Shown:

[0042] figure 1 The figure (a) in the figure shows the positioning process of the ZmcytMdh4 gene. The upper part of the vertical line represents the molecular marker used, and the nu...

Embodiment 3

[0043] Embodiment 3: High lysine functional gene ZmcytMdh4 Analysis

[0044] (1) In mutants ZmcytMdh4 There is a 3-bp deletion in the seventh exon, and the deletion produces a frameshift mutation, which leads to decreased enzyme activity, accumulation of malic acid, and increased content of lysine.

[0045] Analysis of the sequencing results of wild-type and mutant DNA sequences and coding sequences revealed that there was a 3-bp deletion in the seventh exon of the mutant, which eventually resulted in the loss of one amino acid in the mutant. Then, the wild-type and mutant proteins were purified in vitro by the method of prokaryotic expression, and the catalytic activity of the protein was determined by adding different substrates and corresponding coenzymes. The results showed that the catalytic activity of the mutant was almost lost.

[0046] By targeting the metabolome, the changes of energy metabolism-related substances in the wild-type and mutant grains were measured 12 ...

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Abstract

The invention discloses a corn high lysine gene ZmcytMdh4 The molecular marker and its application aim to solve the technical problems that the existing high-lysine mutants have weak seedling growth, powdery endosperm, low yield, susceptibility to diseases and insect pests, and cannot be produced and processed. The present invention obtains a corn high lysine gene through screening ZmcytMdh4 , two maize high lysine genes were analyzed ZmcytMdh4 Molecular markers, and corresponding primer pairs for detecting molecular markers are provided. maize high lysine gene ZmcytMdh4 , maize high lysine gene ZmcytMdh4 The molecular markers and primer pairs are used in marker-assisted selection and aggregation breeding of maize with high lysine content. The invention can greatly improve the efficiency of molecular marker-assisted selection, lay a foundation for breeding excellent lines with high lysine content, and provide technical support for high-lysine corn breeding.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a corn high lysine gene ZmcytMdh4 Molecular markers and their applications. Background technique [0002] Corn is the largest food crop in my country, and it is also an important feed, industrial raw material and energy crop. However, most of the protein in corn nutrients is gelatin, and lacks the essential lysine required by humans and monogastric animals, which will affect human health and livestock production performance dependent on corn products. [0003] Breeding of high-quality protein maize (QPM) for the purpose of increasing lysine content has become an important way to solve this problem. opaque The discovery of similar mutants has opened a new chapter in the cultivation of high-quality protein corn. Existing typical high-lysine content maize mutants include o2 , o7 , fl2 with Zmocd1 , the location and functional analysis of such mutant genes make i...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/53C12Q1/6895C12N15/11
CPCC12N9/0006C12Q1/6895C12Q2600/13C12Q2600/156C12Y101/01037
Inventor 付志远陈永强汤继华郭战勇张雪海陈晓阳李卫华丁冬王洪秋薛亚东李浩川
Owner HENAN AGRICULTURAL UNIVERSITY
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