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Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488

A technology of FSML-1488, chromosome localization, applied in the field of genetic engineering

Active Publication Date: 2012-09-12
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, FISH technology has not been used so far to locate the target DNA sequence on the algae chromosome

Method used

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  • Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488
  • Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488
  • Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Extraction and Identification of Specific Molecular Marker FSML-1488

[0026] 1) Take Laminaria gametophytic clones with a fresh weight of 0.1 g.

[0027] 2) After gently grinding the gametocyte clones into powder in liquid nitrogen, immediately transfer into pre-warmed 0.6 mL CTAB extraction buffer containing 3% CTAB (hexadecyl trimethylammonium bromide), 1.4 M NaCl, 20 mM EDTA, 10 mM Tris-HCl with a pH of 7.8 and 2% mercaptoethanol by weight to volume, and mixed for 1 min on a vortex mixer.

[0028] 3) Incubate at 65°C for 30-60 minutes, and shake upside down every 10 minutes.

[0029] 4) Add an equal volume of chloroform:isoamyl alcohol mixture with a volume ratio of 24:1, and gently invert and mix for 3-5 minutes until an emulsified layer appears.

[0030] 5) Centrifuge at 12 000 rpm (rev / min) for 10 min at room temperature, and transfer the supernatant to another sterilized centrifuge tube.

[0031] 6) Add 2 / 3 volume of isopropanol pre-cooled at -20°...

Embodiment 2

[0093] Example 2 Chromosomal location of specific molecular marker FSML-1488

[0094] The steps of chromosomal localization of Laminaria female gametophyte-specific molecular marker FSML-1488 include:

[0095] (1) Preparation of plasmid DNA with target fragment (SEQ ID NO.5)

[0096] After extracting Laminaria gametophyte DNA by CTAB method, PCR technology was used to amplify the 1488 bp female gametophyte-specific fragment of FSML-1488, then TA cloned and transformed into Escherichia coli, and finally the plasmid was extracted to obtain the target fragment (SEQ ID NO .5) Plasmid DNA.

[0097] (2) Preparation of Laminaria gametophytic chromosomes

[0098] Using cellulase, pectinase, isolate enzyme and abalone enzyme and optimize the combination to treat Laminaria gametophyte cells, stain with DAPI, and obtain high-quality chromosomes with better division ratio.

[0099] (3) Preparation of probes

[0100] The plasmid DNA of the target fragment (SEQ ID NO.5) was obtained f...

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Abstract

The invention relates to a chromosomal localization method for FSML (female-specific marker of Laminaria japonica Aresch)-1488. The method comprises the steps of preparation of DNA (deoxyribonucleic acid) with target fragment FSML-1488 plasmids, preparation of gametophyte chromosomes of the Laminaria japonica Aresch, preparation of probes, and fluorescence in situ hybridization (FISH). The method has the advantages of preparing the high-quality chromosomes on the basis of obtaining protoplast of antiphyte and gametophyte of the Laminaria japonica Aresch by the optimal coordination of various tool enzymes such as macerozyme, abalone enzyme, cellulose and pectinase, breaking through the technology bottleneck of the FISH technology in the research of algology, and successfully locating the FSML-1488 with the length being 1488bp on the chromosomes.

Description

Technical field [0001] The invention involves the field of genetic engineering. Specifically, it is a specific FSML-1488 (FEMALE-SPECICIC Marker ofer of Laminaria japonica AreSch) molecules are marked on chromosomes. Background technique [0002] kelp( Sacchaarinajaponica Or Laminaria japonica ARESCH.) It belongs to Phaeophyta and Laminariales. It is one of the important economic seaweed in my country's sea water breeding industry.History of kelp life has obvious alien generation alternation.When the spores (that is, the main part of people usually eat) mature, the spore parent cell with a single -chamber spore sac on the tablet has been reduced by merit and multiple splitting, producing 32 bangs of the same type of spores of bangs.They are attached to the matrix shortly after the release. When the growth environment is suitable, they develop them into female and male supporting, and the gender ratio is 1: 1 (Schreiber, 1930).This gender ratio indicates that the kelp may have a g...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 周志刚刘宇谷俊刚毕燕会
Owner SHANGHAI OCEAN UNIV
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