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Early pickling transgene method for cultivated silkworm diapause breed variety

A transgenic and diapause technology, applied in the fields of bioengineering and biotechnology, can solve the problems of wasting manpower, restriction of transgenic operations, and death, saving manpower and material resources, shortening the breeding cycle, and avoiding the breeding process.

Inactive Publication Date: 2008-06-11
SOUTHWEST UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the use of polymorphic silkworm varieties as transgenic materials has obvious defects in the following two aspects: ① the economic traits of polymorphic silkworm varieties are poor, and the additional traits created by transgenes cannot reach the standard for direct promotion and application in terms of yield and quality , transgenic strains with polymorphic silkworm eggs as raw materials can only be used as raw materials for breeding, and as the original carrier of a certain related excellent trait, they can be used in the long and arduous traditional breeding
②Pluripotent silkworm varieties do not have a diapause period, and the maintenance of their subculture can only rely on continuous feeding and reproduction, which requires a lot of waste of manpower and material resources to maintain and subculture the obtained transgenic silkworm strains, and also greatly increases The risk of resource loss of obtained transgenic silkworm varieties
At present, the most effective time for silkworm transgene microinjection is 3-6 hours after laying eggs, because injection holes will be left on the eggs after the transgenic operation, and if the conventional instant pickling method is used after injection, hydrochloric acid will infiltrate In the silkworm eggs, it is fatal, and it is impossible to realize the transgenic research of silkworm diapause varieties
Therefore, the eggs of silkworm diapause species will diapause, which brings great restrictions to the transgenic operation.
[0006] So far, the transgenic technology of diapause silkworm varieties has not been reported at home and abroad, and there is no mature and reliable method to realize the transgenesis of silkworm diapause varieties. Silkworm transgenic technology and requirements for accelerating the practical application of scientific and technological achievements

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0020] Using the silkworm diapause variety "Bibo" as the raw material, the silkworm eggs obtained by normal feeding and mating according to the above-mentioned method were collected and treated, and treated in HCl with a liquid temperature of 46°C and a specific gravity of 1.075 for 2 minutes and 30 seconds 2 hours and 30 minutes after laying, and rinsed with tap water. After cleansing, use the Eppendorf microinjector to inject the red fluorescent protein-labeled piggyBac vector and auxiliary vector with nerve and eye-specific promoters together into the 1518 cells that were released from diapause by early pickling method at 3 hours after delivery. The silkworm eggs were sealed with non-toxic glue and placed in a high-humidity environment at 25°C and a relative humidity of 85% to accelerate the hatching. The hatched 70 G0-generation ant silkworms were collected and raised with artificial feed until they became moths, and 42 were obtained. A total of 23 silkworm eggs of the G1 g...

Embodiment 2

[0022] Using the silkworm diapause variety "Qiubai" as the raw material, the silkworm eggs that were normally reared and mated according to the aforementioned method were collected and treated for 3 minutes and 30 seconds in HCl with a liquid temperature of 46°C and a specific gravity of 1.075 at 2 hours and 20 minutes after laying, and tap water After rinsing, use an Eppendorf microinjector to inject the red fluorescent protein-labeled piggyBac vector and auxiliary vector with nerve and eye-specific promoters into the diapause-relieved early diapause 5 hours after delivery. 2008 silkworm eggs were sealed with non-toxic glue and placed in a high-humidity environment at 25°C and a relative humidity of 90% to accelerate the hatching. The hatched 109 G0-generation ant silkworms were collected and raised with artificial feed until they became moths, and 72 A total of 34 silkworm eggs of the first G0 generation silkworm moths were obtained by selfing or backcrossing, and the G1 gene...

Embodiment 3

[0024] Using the silkworm diapause variety "Dazao" as the raw material, the silkworm eggs obtained by normal feeding and mating according to the above method were collected and treated, and treated in HCl with a liquid temperature of 46°C and a specific gravity of 1.075 for 1 min and 30 sec at 2 h and 15 min after laying, and rinsed with tap water. After cleaning, use an Eppendorf microinjector to inject the red fluorescent protein-labeled piggyBac vector and auxiliary vector with nerve and eye-specific promoters together into the 1623 cells that were released from diapause by early pickling method 6 hours after delivery. The silkworm eggs were sealed with non-toxic glue and placed in a high-humidity environment at 25°C and 95% relative humidity to accelerate the green hatching. The hatched 212 G0-generation ant silkworms were collected and raised with artificial feed until they became moths, and 153 were obtained. A total of 71 silkworm eggs of the G1 generation were obtained ...

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Abstract

The invention relates to a silkworm diapause variety early pickling transgene method. The hydrochloric dipping treatment is performed to a silkworm diapause ovum, by utilizing a certain time before silkworm egg embryo is developed to a germ band formed, namely, before 2 hours and a half after egg casting, then the embryo microinjection is performed to the graine of diapause treatment canceling, the G0 generation silkworm obtained from the incubation breeding bred to eclosion, the G1 generation silkworms are produced through the idiogamy or the backcross of the silkworms, and labeled and sieved through fluorescence, and therefore the transgene silkworms are effectively manufactured. The method directly adopts the universal use utility silkworm diapause variety as original material in production, can avoid the long and troublesome breeding process, and directly utilizes the transgene technology to create the good new special silkworm variety with various characteristics, the breeding cycle can be greatly shortened, and massive manpower and physical resources are saved.

Description

technical field [0001] The invention relates to the fields of biotechnology and bioengineering, in particular to an early pickling transgenic method for diapause varieties of silkworm. Background technique [0002] Transgenic technology is an important technology in modern biological research, and plays an important role in functional gene research, new biological product development, biological material innovation, modern genetic breeding, etc. [0003] The silkworm is an important economic insect and a model organism of Lepidoptera. With the completion of the silkworm genome sequencing work, using efficient and stable technology to study the functions of important genes on a large scale has become an important topic in the era of silkworm functional genome. At the same time, silkworm is an ideal "insect factory", using modern genetic engineering technology to develop silk glands Bioreactors have huge market prospects. The silkworm transgenic technology is a favorable wea...

Claims

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Application Information

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IPC IPC(8): C12N15/85C12N15/89A01K67/04
Inventor 赵爱春夏庆友徐汉福鲁成向仲怀
Owner SOUTHWEST UNIVERSITY
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