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Function and application of arabidopsis thaliana POL2A gene in reduction division recombination

A meiosis, Arabidopsis technology, applied in the field of genetic engineering

Inactive Publication Date: 2015-09-23
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the DNA polymerase epsilon is a research hotspot in homologous recombination and epigenetic regulation, and its specific regulatory mechanism and conservation among different species need further study

Method used

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  • Function and application of arabidopsis thaliana POL2A gene in reduction division recombination
  • Function and application of arabidopsis thaliana POL2A gene in reduction division recombination
  • Function and application of arabidopsis thaliana POL2A gene in reduction division recombination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Planting, hybridization, transformation and genotype identification of Arabidopsis thaliana

[0062] 1. Planting of Arabidopsis

[0063] After surface disinfection of Arabidopsis seeds (75% ethanol, 30s; wash three times with sterile water, 60s / time); sow in solid 1 / 2 MS medium under sterile conditions. Cultivate in a light incubator (22°C, 16h light / 8h dark); about 10 days later (two true leaves grow) transplant the greenhouse ( figure 2 ).

[0064] 2. Hybridization of Arabidopsis

[0065] First remove the dew-white flowers of the plants identified by the genotype, and emascate the three outermost flowers. After one to two days, observe the stigma of the emasculated flower. If the stigma presents white fluff and a shiny liquid substance appears, it proves that the stigma is suitable for pollination at this time. Smear the pollen of the male parent on the stigma, and you can see the yellow powdery substance on the stigma. To be on the safe side, you can ...

Embodiment 2

[0074] Example 2 Alexander red staining of mature pollen

[0075] Inflorescences of Arabidopsis thaliana were fixed overnight in Carnot's fixative at room temperature. Peel off the flowers that have bloomed, take the flowers that have yellow anthers and have not yet cracked, and immerse them in a small PCR tube filled with Alexander red staining solution. The staining time depends on the room temperature (RT>30°C, T<30min; 15°C<RT<30 ℃, 2h<T<4h; RT<10℃, T<12h). After the dyeing is completed, peel off the anthers, remove impurities, add a small amount of dye solution, cover the slides, press gently, and after microscopic examination, seal the slides with nail polish and store at -20°C.

[0076] Recipe of Alexander red staining solution: 95% ethanol 10mL, malachite green (1% ethanol solution) 1mL, double distilled water 50mL, glycerin 25mL, phenol 5mL, chloral hydrate 0.5g, 1% acid fuchsin 5mL , 0.5 mL of 1% Orange G, 1 mL of glacial acetic acid (ALEXANDE., 1969). The total...

Embodiment 3

[0078] Example 3 Tetrad analysis

[0079] Take the inflorescence of Arabidopsis thaliana and fix it overnight at room temperature, rinse it with tap water 2-3 times, peel off the flowers with yellow anthers, take the outermost 1-2 flowers of the inflorescence on a glass slide, add a small drop of water, and peel off the flowers. After the anthers, remove the dregs, then add a drop of water, then gently pinch the anthers with tweezers, or squeeze the pollen mother cells out of the chamber with a needle tip, and spread them on a glass slide, let them air dry naturally, add a drop of alkaline After staining with magenta for 2-3 minutes, add a cover slip and examine under the microscope.

[0080] Unlike normal tetrads in the wild type, a large number of polytids were seen in the mutant ( image 3 B), where pol2a-1 and pol2a-2 The proportions of producing polymorphs were: 36% and 49.6% ( image 3 C). right pol2a-1 According to the classification of abnormal tetrads produ...

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Abstract

The invention belongs to the technical field of genetic engineering and particularly relates to a function and application of an arabidopsis thaliana POL2A gene in reduction division recombination. The application comprises the following steps of using sequence characteristics of coding POL2A protein and the authentication of the new function of the gene in reduction division, i.e. using a fluorescent label site system to analyze the influence of POL2A to reduction division recombination frequency and distribution, and providing a fluorescent label-containing genetic material; providing the function of the gene of analyzing how complete mutation of the gene causes embryo death in reduction division, and providing the technology of a series of cell biology and the like. The important function of POL2A in reduction division recombination is proved in an eukaryote for the first time; the function is used, the modern biotechnology method is used, and the recombination frequency of a generic material in a reduction division process is controlled, so that aggregation of excellent characters is quickened, separation of the excellent characters in a hybrid offspring is delayed, and the aim of propelling and improving breeding study level is fulfilled.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, in particular to plant POL2A The function of the gene in meiotic recombination, and the application of the function of the gene in changing the process of crop breeding, especially changing the interval where exchange is difficult to occur under natural conditions, can more effectively accelerate the breeding efficiency. Background technique [0002] Meiosis is a life process necessary for the sexual reproduction of eukaryotes. In the process of meiosis, DNA is replicated once, and cells divide twice, so that a pair of homologous chromosomes from parents are separated; non-homologous chromosomes are Free combination, resulting in gametes with multiple genetic combinations and halved chromosome number, the zygote formed after gamete fusion maintains the number of chromosomes in the parents, thus ensuring the stability of genetic material and enabling species to reproduce. Homologous c...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12Q1/68C12N15/82A01H5/00
Inventor 王应祥马红黄霁月
Owner FUDAN UNIV
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