Method for constructing transgenic Nosema bombycis
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A microsporidia and transgenic technology, applied in the field of genetic engineering, can solve the problems of lack of research platform for microsporidia gene function
Inactive Publication Date: 2012-11-21
SUZHOU UNIV
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[0004] At present, the genome sequence of N. silkworm has been nearly completed, but there is a lack of gene function research platform for N. silkworm. Gene knock-in and knock-out are the most direct and effe...
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Embodiment 1
[0032] Embodiment 1: Based on pIZT / V5-His-mediated transgene of Bombyx mori Microsporidia
[0033] (1) with 10 6 / mL of silkworm microspores smeared with mulberry leaves and fed silkworms from the 4th instar for 6 hours, and fed for 3 to 4 days.
[0034] (2) Mix 5 μL of 2 μg / μL pIZT / V5-His (Invitrogen) with 5 μL of liposomes, inject the silkworms in step (1) into the body cavity, and continue to raise them.
[0035] (3) After 2 to 4 days, silkworm hemolymph was collected and observed with a fluorescence microscope. It was observed that some blood cells showed green fluorescence ( figure 1 ), adding silkworm hemolymph to silkworm BmN cultured cells, cultured at 26°C, and observed fluorescence every day.
[0036] (4) Aspirate half of the old culture medium every 2-3 days, add corresponding fresh medium and normal silkworm BmN cultured cells, and culture at 26°C.
[0037] (5) Observing the cultured cells with a fluorescence microscope, when the microsporidia infecting the cult...
Embodiment 2
[0043] Example 2: Transgenesis of Microsporidia silkworm mediated by piggyBac transposon vector pigA3GFP
[0044] (1) with 10 6 / mL of silkworm microspores smeared with mulberry leaves and fed silkworms from the 4th instar for 6 hours, and fed for 3 to 4 days.
[0045](2) Take 2.5 μL of 4 μg / μL pigA3GFP and 2.5 μL of 4 μg / μL helper plasmid (see Tamura Toshiki. et al. Germline transformation of the silkworm Bombyx mori L. using a piggyBac transposon-derived vector. Nature Biotechnology, 2000, 18:81-84)) was mixed with 5 μL of liposomes, injected into the silkworm in step (1) into the body cavity, and continued to be reared.
[0046] (3) After 2 to 4 days, take silkworm hemolymph and observe it with a fluorescent microscope. Some blood cells were observed to show green fluorescence. Take silkworm hemolymph and add it to silkworm BmN cultured cells, culture at 26°C, and perform fluorescence observation every day.
[0047] (4) Aspirate half of the old culture medium every 2-3 da...
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Abstract
The invention discloses a method for constructing transgenic Nosema bombycis, characterized by injecting a transgenic vector wrapped by liposome in body cavity of the Bombyx mori infected with Nosema bombycis, after 2-4 days, adding silkworm hemolymph in Bombyx mori BmN culture cells to cultivate; when microsporidian presents green fluorescent light, using an insect medium to extremely dilute and adding into a cell culture plate, selecting a culture pore where only one culture cell presents green fluorescent light, adding normal culture cells to culture for 2-4 days to obtain infected culture cells; repeating for at least three times, taking the obtained infected culture cells, conducting homogenate and crushing the cells, conducting differential centrifugation, purifying microsporidian, infecting the Bombyx mori, after the Bombyx mori falls ill, purifying the infected tissues to obtain the transgenic Nosema bombycis. According to the invention, exogenous genes can be integrated into the Nosema bombycis genome, the modification of Nosema bombycis by gene engineering is realized, the adjustment of expression of the microsporidian genes is realized, and a method for microsporidian gene function research is provided.
Description
technical field [0001] The invention belongs to the field of genetic engineering, and in particular relates to a method for constructing transgenic microsporidia of silkworm. Background technique [0002] Microsporidia (microsporidia) are obligate intracellular parasitic single-celled eukaryotic organisms, from nematodes to humans, almost all animals have found microsporidia. Microsporidia are pathogens of insects, fish, rodents, lagomorphs, fur animals and primates, and are insecticides for biocontrol of locusts. So far, more than 1200 species of microsporidia have been found, distributed in 144 genera. [0003] Bombyx mori microparticle disease is an infectious disease caused by Nosema bombycis (Nb) parasitizing the silkworm body. The pathogen of the disease was first discovered in France in 1857. Countries and regions are listed as the only quarantine objects for silkworm egg production. At present, the disease still poses a major threat to the sericulture industry. ...
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