Anther development control gene and application thereof to male sterility of arabidopsis

A technology for anthers and uses, applied in the field of plant biology, can solve problems such as unclear molecular mechanisms

Inactive Publication Date: 2013-11-27
SHANGHAI NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Although the polar transport and mechanism of action of auxin have been well understood, the signaling pathway of auxin in anther development and the molecular mechanism of regulating anther dehiscence, filament elongation, tapetum development and pollen formation are still unclear.

Method used

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  • Anther development control gene and application thereof to male sterility of arabidopsis
  • Anther development control gene and application thereof to male sterility of arabidopsis
  • Anther development control gene and application thereof to male sterility of arabidopsis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0064] 1. Arabidopsis thaliana mutant arf17, the original wild type is Columbia (Col) ecotype (preserved by the Laboratory of Plant Molecular Biology, Shanghai Normal University).

[0065] 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).

[0066] Table 1. PNS stock composition and 1 L 1×PNS recipe

[0067]

[0068] a: Na 2 - 7.45g of EDTA and 5.57g of FeSO were dissolved in 400mL of water and heated to boiling, then mixed, and continued to boil for 30min, cooled and a...

Embodiment 2

[0079] Example 2 Cytological observation

[0080] 1. Light microscope resin sections of plant anthers: get arf17 mutants and wild-type fresh materials and put them into penicillin vials equipped with FAA fixative (50% alcohol, 5.0% glacial acetic acid, and 3.7% formaldehyde), pump air and remove them. 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.

[0081] 2. Production of plant anther materials for electron microscopy: material production for scanning electron microscopy: fresh anthers and pollen of arf17 mutants and wild types were fixed on a copper platform with conductive glue, and ...

Embodiment 3

[0083] Example 3 Plant tissue extraction of RNA and RT-PCR detection of downstream genes:

[0084] The fresh material of arf17 mutant and wild type was ground thoroughly with liquid nitrogen in a mortar. Put the ground material into the EP tube that has been added with 1mL TRIZOL in advance, shake and mix well. Add chloroform after incubating at 15-30°C, after standing still, centrifuge at 12000g, then transfer the water phase to another EP tube, add isopropanol, centrifuge at 12000g at 4°C after standing still, then wash once with 75% alcohol, Dry the RNA and dissolve it in 20-40 μL DEPC-H 2in o. Then an appropriate amount of RNA was reverse-transcribed with AMV reverse transcriptase to synthesize the first strand of cDNA. The cDNA synthesized above was used as an amplification template for RT-PCR. The semi-quantitative RT-PCR program is pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 30 sec, annealing at 56°C for 30 sec, and extension at 72°C for 30 sec....

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Abstract

The invention relates to an anther development control gene and application thereof to male sterility of arabidopsis. An arabidopsis male sterile mutant arf17 is screened from a T-DNA (transfer deoxyribonucleic acid) insertion mutant pool, and a gene ARF17 (Auxin Response Factor 17) for controlling the fertility of arabidopsis is cloned and identified. The anther development control gene has a nucleotide sequence shown as SEQ ID No.1. A genetic complementary experiment proves that the ARF17 gene of a wild type can recover the male sterility phenotype of the mutant. An amino acid sequence analysis experiment indicates that ARF17 is a transcription factor of an ARF family and is positioned in a nucleus. In arabidopsis, mutation of the gene causes complete male abortion. The gene has a very important application value in the aspect of regulation and control of plant fertility by explaining and controlling callose synthesis and transcription of a key gene formed on an outer wall and the aspect of increasing in the crop yield during 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] In most plants...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/63C12N5/10C12P21/02A01H5/00
Inventor 杨仲南杨俊朱骏
Owner SHANGHAI NORMAL UNIVERSITY
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