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Anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana

An anther and amino acid technology, applied in the field of plant biology, can solve the problems of loss of self-pollination function, pollen abortion, zero seed setting rate of 100 plants, etc.

Active Publication Date: 2011-08-03
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Many single-gene mutations in AHL families have no obvious physiological defects, but the single-gene deletion mutants of the AHL16 gene can cause complete abortion of pollen, and the microspores in the mutants are gradually broken and degraded after being released from the tetrad, making self-pollination The function is completely lost, and the seed setting rate of 100 plants is zero

Method used

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  • Anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana
  • Anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana
  • Anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana

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Experimental program
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Effect test

Embodiment 1

[0062] 1. Arabidopsis thaliana mutant ahl16, the original wild type is Columbia (Col) ecotype (preserved by the Laboratory of Plant Molecular Biology, Shanghai Normal University).

[0063] 2. Arabidopsis material cultivation conditions: Arabidopsis was cultured in black soil; vermiculite; perlite (1:6:0.25) mixed soil after vernalization in 0.1% agarose at 4° C. for 2-4 days. Cover the upper layer with a plastic film to maintain humidity during cultivation, and remove the plastic layer when Arabidopsis germinates cotyledons. The light time is 16 hours of light, 8 hours of darkness, the humidity is kept at 60-70%, the temperature is controlled at 22-25°C, and the light intensity is 50μEm -2 the s -1 , the nutrient solution is PNS (Table 1).

[0064] Table 1. PNS stock composition and 1 L 1×PNS recipe

[0065]

[0066] a: Dissolve 7.45g of Na2-EDTA and 5.57g of FeSO4 in 400mL of water and heat to boil, then mix and continue to boil for 30min, then cool and set the volume t...

Embodiment 2

[0077] Example 2 Cytological observation

[0078] 1. Light microscopy resin sections of plant anthers: get the fresh material of ahl16 mutant and wild type and put it into a penicillin vial equipped with FAA fixative (50% alcohol, 5.0% glacial acetic acid, and 3.7% formaldehyde), pump air and remove it. Seal and store overnight at 4°C. It was subsequently dehydrated through gradient alcohols (50%×2, 60%, 70%, 80%, 90%, 95%, and 100%×2), transferred into xylene, and finally embedded in resin. Slices were then stained with toluidine blue and observed under a microscope. For callose staining, the sections were stained with 0.067M phosphate buffer solution containing 0.05% (w / v) aniline blue, and placed under a UV microscope for observation.

[0079] 2. Production of plant anther materials for electron microscopy: production of scanning electron microscope materials: take fresh anthers and pollen of ahl16 mutant and wild type and fix them on a copper platform with conductive glu...

Embodiment 3

[0081] Example 3 Detection of AHL16 gene expression in anthers by in situ hybridization

[0082] Sampling and embedding: The wild-type and mutant inflorescences were taken with scissors and fixed in FAA. Apply vacuum to infiltrate the fixative into the tissue, and replace the fixative once at 4°C overnight. The fixed material was dehydrated in 50%, 60% and 70% ethanol for one hour each. All the above steps were carried out on ice with gentle shaking from time to time. On the third day, dehydrate in 85% and 95% ethanol at 4°C for 1 hour, then turn to room temperature, dehydrate in 100% ethanol for 2 hours, and then dehydrate in 25% xylene and 75% ethanol, 50% xylene and 50% ethanol. % ethanol mixture, 75% xylene and 25% ethanol mixture (stained with safranin) were placed for half an hour each. Finally the material was placed overnight in 100% xylene containing 1 / 4 volume paraffin wax. On the fourth day the material was placed at 42°C until the wax block was completely melte...

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Abstract

The invention relates to an anther development control gene and use thereof in realizing male sterility in Arabidopsis thaliana. An Arabidopsis thaliana male sterile mutant ahl16 is screened out from T-DNA-inserted mutant population, a gene AHL16 controlling the fertility of Arabidopsis thaliana is cloned and identified, the nucleotide sequence of the gene AHL16 is represented by SEQ ID N0.1, and genetic complement experiments prove that the AHL16 gene in a wild type can restore the male sterile phenotype. Amino acid sequence analysis and subcellular localization experiments indicate that the AHL16 gene codes proteins of an AT-hookmotif nuclear localized protein family and is located in the nucleus. In Arabidopsis thaliana, the mutation of the gene leads to complete male sterility. The gene has a very important application value in aspects of explanation of influences of the growth of the inner layers of the outer walls of microspores and the structures of the inner walls on the fertility of plants and improvement on yield by hybrid seed production.

Description

technical field [0001] The invention belongs to the field of plant biotechnology, and in particular relates to a gene for regulating the development of plant anthers to regulate fertility, encoded protein and application thereof. Background technique [0002] The sexual reproduction of plants is not only the main way of plant reproduction, but also one of the foundations of plant evolution and adaptation to the environment. Anthers are the male reproductive organs of plants, where pollen develops. The development of anther and pollen is an important research direction in the study of plant gene function. Abnormal development of anthers and pollen will cause anthers to fail to produce normal functioning male gametes, resulting in male sterility. Male sterility is the basis of hybrid seed production in production. Therefore, the in-depth study on anther and pollen development has both theoretical research significance and practical application value. [0003] The tapetum is...

Claims

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Application Information

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IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12P21/02A01H5/00
Inventor 杨仲南徐晓峰朱骏
Owner SHANGHAI NORMAL UNIVERSITY
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