A gene zmsmk3 encoding maize mterf protein and its cloning method and application

A cloning method, corn technology, applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems that the function of mTERF protein has not yet been resolved

Active Publication Date: 2019-11-05
HUAZHONG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Most of the mTERF genes reported in plants are concentrated in Arabidopsis. So far, the functions of most mTERF proteins have not been resolved

Method used

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  • A gene zmsmk3 encoding maize mterf protein and its cloning method and application
  • A gene zmsmk3 encoding maize mterf protein and its cloning method and application
  • A gene zmsmk3 encoding maize mterf protein and its cloning method and application

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Experimental program
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Effect test

Embodiment 1

[0065] Cloning of embodiment 1 ZmSmk3 mutant gene

[0066] There were 1 / 4 small-grain mutations in ZmSmk3 heterozygous selfed ears, and the wild type:mutant match was 3:1 (WT:Zmsmk3=1651:526, p=0.37), which proved that the mutant trait was controlled by a recessive single gene. The leaf DNA of 10 homozygous mutants (number: RS1-RS10) and 5 homozygous wild-type plants (number: RW1-5) were extracted by CTAB method. After the concentration was determined, all of them were diluted to 50ng / ul. 5. RS4-8 and RS6-10 were mixed in equal amounts to construct 3 homozygous mutant DNA pools; RW1-5 were mixed in equal amounts to construct a wild-type DNA pool, and the four mixed pools were respectively used as templates for subsequent PCR reactions.

[0067] Use thermal asymmetric staggered PCR, that is, use the specific primers TIR9-1, TIR9-2 and random primers for the mu transposon end sequence in Table 5 to amplify. For specific amplification methods, refer to: Liu Wenting, 2006; Settles...

Embodiment 2

[0070] Example 2 Detection of mutant gene Zmsmk3 in individual maize plants

[0071] The sequence of the mutant gene Zmsmk3 was compared with the maize genome to find out the genome position corresponding to the specific fragment. Centering on the insertion site of the matching position, primers were designed in combination with TIR6 to detect the genotype of a single plant. If the genotype and phenotype are co-segregated, the gene inserted at this site is the candidate gene, further expanding the population verification. The primer used in this paper to identify co-segregation is S21F / R. Its nucleotide sequence:

[0072] S21F: GGAGGAGCTGGTGTAGATCG

[0073] S21R:TACGTGCCAGATTTGATGCT

[0074] TIR6AGAGAAGCCAACGCCAWCGCCTCYATTTCGTC

[0075] Genotype detection Use S21F / R, S21F+TIR6, S21R+TIR6, three pairs of primers to detect the genotype of each individual plant. If only S21F / R has a corresponding PCR product in the amplified bands of the three pairs of primers in a certain ...

Embodiment 3

[0089] Example 3 Expression analysis and subcellular localization of ZmSmk3 gene

[0090] The expression of ZmSmk3 gene in maize tissues was analyzed by qRT-PCR. ZmSmk3 is a constitutively expressed gene. ZmSmk3 gene is expressed in all vegetative and sexual reproductive tissues, and the expression level is relatively high in the stem, ear, ovary and embryo; the expression level is low in the root , leaves, tassels, filaments and endosperm, results such as Figure 5 Shown in A: qRT-PCR detection of the expression of ZmSmk3 in roots, stems, leaves, tassels, ears, filaments, ovaries, and embryos and endosperms of 15DAP. This result indicated that the ZmSmk3 gene was constitutively expressed in various stages of plant growth and development and in various tissues. At the same time, in the seeds 15 days after pollination, it was detected that the expression level of ZmSmk3 gene decreased in mutants (the results were as follows: Figure 5 B).

[0091] The gene was connected to ...

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Abstract

The invention relates to a gene ZmSmk3 for encoding corn mTERF protein and a cloning method for the gene ZmSmk3. The cloning method comprises the following steps of: respectively extracting DNA of a plurality of homozygous mutants and homozygous wild type plant leaves, mixing the extracted DNA of the homozygous mutants and constructing a DNA pool of the homozygous mutants; mixing the extracted wild type DNA and constructing a mild type DNA pool; respectively carrying out heat-asymmetric staggering PCR (Polymerase Chain Reaction) on the constructed DNA pool of the homozygous mutants and the wild type DNA pool to obtain PCR product segments; detecting obtained PCR amplification product segments by using agarose gel electrophoresis, recovering specific bands generated in the DNA pool of the homozygous mutants, and carrying out sequencing to obtain a sequence of the mutant gene ZmSmk3. The mutation of the gene ZmSmk3 for encoding the corn mTERF protein has an influence on the seed development progress, and the gene has an important guidance significance for breeding practice.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a gene ZmSmk3 encoding maize mTERF protein and its cloning method and application. Background technique [0002] Mitochondria are important energy conversion stations, which can efficiently convert the energy stored in organic matter into ATP (adenosine triphosphate), the direct energy source of cell life activities, and provide energy sources for life activities (Mackenzie and McIntosh, 1999; Gualberto et al., 2014) . It has autonomous DNA replication, transcription and protein synthesis systems and is a semi-autonomous organelle. Among them, most mitochondrial proteins are encoded by the nucleus and are regulated by the nucleus. [0003] Mitochondrial gene expression is a complex biological process, including RNA (ribonucleic acid) transcription, RNA editing, intron splicing, RNA translation, maturation, and degradation (Hammani and Giegé, 2014). The mitoch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00C12Q1/6895
CPCC07K14/415C12N15/8261C12Q1/6895C12Q2600/13C12Q2600/158
Inventor 邱法展任雪梅潘振远谭增栋秦瑶
Owner HUAZHONG AGRI UNIV
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