Method for cultivated silkworm transgene

A transgenic and silkworm technology, applied in the biological field, can solve the problems of unstable inheritance and low expression frequency

Inactive Publication Date: 2007-08-15
ZHEJIANG UNIV
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  • Summary
  • Abstract
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  • Claims
  • Application Information

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Problems solved by technology

[0005] In the early application of this method, more mutated individuals were also obtained, but usually these foreign DNAs only enter the cytoplasm rather than integrate into the chromosomes, so they cannot be inherited stably; at the same time, foreign DNA enters another individual, often due to the action of degrading enzymes and rapidly decomposed, the frequency of its expression is very low

Method used

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Experimental program
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Effect test

Embodiment 1

[0025] The silk glands of female adult spiders (Nephila clavata) were dissected, washed with 0.8% saline, and genomic DNA was extracted by Sambrook et al. Design primers for cross-filament gene amplification, primer 1: 5'-AATGAAAAAAGAGGGTT GGTACC TCCTGG-3' (the underline is the KpnI restriction site), primer 2: 5'-TTCGAATTTTCTTGGGT CTGCAG GTGT-3' (the underline is the PstI restriction site); the cross-filament gene was obtained by PCR amplification under the following conditions: 94°C for 1 minute, 56°C for 1 minute, 72°C for 1 minute and 30 seconds, 30 cycles. The amplified spider silk gene was cloned into pFastBacHT B (Invitrogen) and sequenced. Cut out the horizontal silk gene and clone it into the transposon vector pBac3×P3DsRed to construct a recombinant transposon vector, which contains the silkworm heavy chain promoter, red fluorescent marker gene, transposon and spider silk horizontal silk genes etc.

Embodiment 2

[0027] The silkworm eggs used for transgenic injection are the polymorphic N4 variety, and the purified recombinant transposon and Helper plasmid (providing transposase) are adjusted in a weight ratio of 1:1 with injection buffer (5mMKCl, 0.5mMPBS, pH7.0) The concentration was 0.4 μg / μl. Microinjection was carried out under a solid microscope, the egg injection age was 2-4 hours after laying eggs, and the injection volume was 15-20ng DNA. Silkworm eggs are sealed with leather glue (non-toxic) after injection. A total of 380 silkworm eggs were injected, 165 ant silkworms (GO) were hatched, 125 cocoons were formed after feeding, 48 female moths were obtained, and 45 oviposites were obtained after mating. The 45 egg circles were respectively stimulated to hatch (G1), and the single eye color of the G1 generation ant silkworm was observed under a fluorescent binocular dissecting microscope. Select area 2 of the ant silkworm whose single eye shows red, and continue to raise and p...

Embodiment 3

[0029] After the G2 generation, the same method was used to select, raise and subculture with the red mark of the monocular eye. Genomic DNA was extracted from the posterior silk glands of G1 moths positive for red fluorescence and G2 5th instar 3rd day larvae, and total RNA was extracted with the RNA extraction kit provided by ISOGENE (Japan). Methods To detect the transposition of spider silk genes. The result of implementation shows that the horizontal silk gene of spider silk has been correctly introduced into the silk fibroin gene of Bombyx mori, and its introduction site is 1-2. The silk quality of the transgenic domestic silkworm silk and the control N4 cocoon silk were tested, and the strength and elasticity were increased by 6-8% respectively.

[0030] The advantages of the scheme of the present invention are: the silkworm egg injection method is simple and convenient, the injection success rate is high, and the transgenic efficiency is greatly improved.

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Abstract

The invention discloses a method of cultivated silkworm transfer-gene, which comprises the following steps: formulating paste with forced fecula to fix silk seed; utilizing 70% alcohol to sterilize silk seed; arranging the silk seed one by one with fine writing brush; colonizing extra-source gene on the rotor seat carrier; constituting recombinant rotor seat; setting the mass ratio with Helper plasmid as 1 : 1; adjusting the density at 0.4 mu g/mu l with injection buffer liquid; carrying out microscopic injection with stereoscopic microscope; setting the injecting quantity at 15-20ng DNA; sealing with leather and gel; transferring the silk seed and slide into incubation vessel; incubating at 25 deg.c; hatching and seeding; choosing successive; checking.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for transgenic silkworm. Specifically, exogenous genes are introduced into the egg injection method, including silkworm egg laying, injection embryo stage, ovulation method, injection and sealing, and greening, which greatly improves the transgenic efficiency. Background technique [0002] The so-called transgene refers to the transfer of genes, that is, inserting genes that the organism itself does not have by various engineering methods, and changing its expression due to the exchange of chromosomal genetic material. Organisms obtained by means of genetic modification are called genetically modified organisms, that is, genetically modified plants and genetically modified animals. Furthermore, the so-called transgenic insects refer to recombinant insects in which genes from other organisms are inserted into the insect chromosomes. When a virus is used to introduce foreign g...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/89C12N15/85A01K67/04
Inventor 缪云根李广李
Owner ZHEJIANG UNIV
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