Maize MS8 gene mutant as well as molecular identification method and application thereof

A corn and gene coding technology, applied in the field of plant molecular biology and genetic engineering, can solve problems such as limiting the efficiency of breeding of good varieties, large outbreaks of corn spot disease, and male sterility

Active Publication Date: 2017-05-31
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cytoplasmic male sterile lines that have been used in maize have some defects: First, because the cytoplasmic male sterile lines need specific restorer genes to restore fertility, the utilization rate of germplasm resources is very low, which limits the efficiency of breeding of excellent varieties Secondly, the fertility of some sterile lines is unstable, and the fertility can be restored under certain conditions, which affects the purity of the hybrid; finally, due to the single cytoplasmic genotype, it causes a large outbreak of corn spot disease, which directly leads to cytoplasmic male sterility technology. qiu...

Method used

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  • Maize MS8 gene mutant as well as molecular identification method and application thereof
  • Maize MS8 gene mutant as well as molecular identification method and application thereof
  • Maize MS8 gene mutant as well as molecular identification method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2M2

[0049] Example 2M 2 Substitute Planting and Character Observation

[0050] in M 2 During the period of heading and flowering, the morphology of anthers was observed in the field, and anthers with abnormalities such as light white color, small shape, and small pollen amount were selected for further microscopic examination under a microscope. Six plants with abnormal fertility were found in the family numbered 4505. The anthers of the mutant are smaller than the wild type, light yellow in color, and have no visible pollen, but there is no obvious difference from the wild type in vegetative growth, heading stage, and panicle type, so it is selected as a candidate mutant material for further research.

Embodiment 3

[0051] Example 3 Microscopic examination of pollen, selfing, outcrossing

[0052] The pollen fertility was counted by the ratio of iodine-stained pollen to non-colored pollen. Observing the male flower morphology of the 4505 mutant under a stereomicroscope, the anthers were smaller and lighter in color than the wild type (see figure 1 ). Collect florets at the flowering stage in the field, take out the anthers with tweezers, and put them in iodine-potassium iodide solution (0.6% KI, 0.3% I 2 , w / w), gently squeeze the anthers, drop them on a glass slide, cover with a cover glass, observe the pollen iodine staining under a microscope and take pictures. The wild type has more pollen and is stained blue-black, while the mutant cannot see the pollen grains ( figure 2 ).

[0053] Mutants can bear fruit normally under open pollination ( figure 1 ), indicating that the mutant is male sterile and females are not affected. The wild type of the same family was bagged and selfed, ...

Embodiment 4

[0054] Example 4 Leaf Sampling and DNA Extraction

[0055] Use the CTAB method to extract DNA from corn leaves. The specific method is as follows: Weigh about 0.1g leaves, put them into a centrifuge tube, add 600 μL CTAB extraction buffer, 5 μL RNase A, oscillate to disperse, put in a water bath at 65°C for 0.5 hr, and shake gently for 2-3 Add an equal volume of chloroform / Tris-saturated phenol (1:1, v / v), mix well, and shake gently for 10 minutes; centrifuge at 10,000 rpm at 4°C for 20 minutes; transfer the supernatant to a new tube, and add 1 / 10 volume of 3M sodium acetate (pH value 5.2), 0.6-1 times the volume of cold isopropanol; gently shake and mix until flocculent precipitate appears; centrifuge at 10,000 rpm at 4°C for 10 min; discard the supernatant, and wash the precipitate with 70% ethanol by volume 2 time; air-dry, add 50 μL 1×TE to dissolve the precipitate, and store at -20°C. The DNA concentration was detected by Nanodrop2000 and diluted to 10ng / L for use as a P...

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Abstract

The invention provides a maize Ms8 gene mutant as well as a molecular identification method and application thereof, belonging to the technical field of gene engineering. A waxy corn variety namely Jingkenuo 2000 is subjected to cobalt60 radiation induced mutation to induce a basic group T in a sixth exon of a maize beta-1,3-galactosyl transferase (Ms8) gene to mutate into a basic group C, thereby resulting in that a tyrosine is transformed into a histidine. The Ms8 gene mutant is named as ms8-4505, a nucleotide sequence thereof is shown by SEQ ID No.1, the mutant is further proved to cause maize recessive male sterility, and the mutant can be used for preparing a maize selfing line and transgenic maize with recessive male sterility, and plays an important role in genetic improvement breeding of maize germplasm resources. The invention also provides a molecular marker identification method of the mutant and application of the mutant in breeding and seed production.

Description

technical field [0001] The invention belongs to the fields of plant molecular biology and genetic engineering, and in particular relates to a corn MS8 gene mutant ms8-4505 and its molecular identification method and application. Background technique [0002] Plant male sterile mutation is a very common phenomenon in nature, at least male sterile mutants have been found in 617 species of 43 families and 162 genera. Genetically, plant male sterility is divided into three categories: nuclear male sterility, cytoplasmic male sterility and nuclear-cytoplasmic interaction male sterility: 1) Nuclear male sterility is produced by nuclear gene mutations, and there are dominant mutations and recessive mutations , There are sporophytic gene mutations and gametophytic gene mutations. Dominant mutations and gametophytic gene mutations can only be inherited through female gametes, recessive mutations can be inherited through both female gametes and male gametes, and follow Mendel's laws....

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82A01H5/00C07K14/415C12Q1/68C12N15/11
CPCC07K14/415C12N15/8289C12Q1/6895C12Q2600/156
Inventor 黄培劲李新鹏李京琳张维安保光龙湍
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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