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Low-temperature incubation transgene method for cultivated silkworm diapause breed variety

A transgenic and diapause technology, which is applied in the field of bioengineering and biotechnology, can solve the problems of transgenic operation restrictions, waste of manpower, long time, etc., and achieve the effect of shortening the breeding cycle, saving manpower and material resources, and good economic characteristics

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 diapause of eggs of silkworm diapause breeds brings great restrictions to transgenic operations.
[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. Transgenic technology of silkworm and the urgent requirement of accelerating the practical application of scientific and technological achievements

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Using the silkworm diapause variety "Dazao" as the original material, the silkworm eggs obtained from normal rearing were placed in an environment with a constant temperature of 15 °C and a relative humidity of 75% for the whole process after conventional artificial incubation to promote greening until hatching. Raised under normal conditions, the contemporary silkworm moths were selfed as described above to obtain the next generation of non-diapause silkworm eggs. These silkworm eggs were 3h after laying, using an Eppendorf microinjector to inject 10-15nl with a total concentration of 400ng. 601 silkworm eggs were co-injected into 601 silkworm eggs, the injection port was sealed with non-toxic glue, and injected into 35% formaldehyde vapor. After sterilizing for 5 minutes, place them in a high humidity environment of 25° C. and a relative humidity of 85% to promote green incubation, and collect and raise 350 hatched G0 generation ant silkworms with artificial feed to tr...

Embodiment 2

[0021] Using the silkworm diapause variety "Dazao" as the raw material, the normally raised silkworm eggs were placed under the conditions of a constant temperature of 25 °C and a relative humidity of 85% for 5 days after conventional artificial incubation, and then placed at a constant temperature of 15 °C for 5 days. , in an environment with a relative humidity of 75% to promote green to hatch, the hatched silkworms are reared under normal conditions, and the contemporary silkworm moths are selfed as described above to obtain the next generation of non-diapause silkworm eggs, which are laid After 4 hours, using an Eppendorf microinjector, 10-15nl, a total concentration of 400ng / ul, with a red fluorescent protein-labeled piggyBac recombinant transposition vector and a helper vector with nerve- and eye-specific promoters, were co-injected into 596 capsules. In silkworm eggs, the injection port was sealed with non-toxic glue, and after sterilizing in 37% formaldehyde vapor for 5...

Embodiment 3

[0023]Using the silkworm diapause variety "Xiafang" as the raw material, the normally raised silkworm eggs were placed under the conditions of 25°C constant temperature and 85% relative humidity for 6 days after conventional artificial incubation, and then placed at 15°C for 6 days. The green silkworms are raised in an environment with a constant temperature and relative humidity of 75% to hatch, and the hatched silkworms are reared under normal conditions. 6h after the injection, use an Eppendorf microinjector to inject 10-15nl with a total concentration of 400ng / ul of the red fluorescent protein-labeled piggyBac recombinant transposition vector and the helper vector with nerve- and eye-specific promoters. In the silkworm eggs, the injection port was sealed with non-toxic glue, and after being sterilized in 36% formaldehyde vapor for 5 minutes, it was placed in a high humidity environment of 25 ° C and a relative humidity of 95% to promote green incubation, and 605 hatched G0 ...

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Abstract

The invention relates to a method for a silkworm diapause variety low temperature egg incubation transgene. Silkworm diapause variety next generation graine voltinism is changed in the special egg incubation condition, to ensure that present generation silkworm diapause variety moths obtained under the special egg incubation condition mate, to give birth to non-diapause next generation graine, finally referring to the microinjection transgene method of the non-diapause silkworm eggs embryo, the gene transferring is performed, therefore the obstruction of the silkworm diapause variety transgene technology is expertly broken, and a transgene silkworm is 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 a low-temperature green-promoting 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, and modern genetic breeding. [0003] Silkworm is an important economic insect and model organism of Lepidoptera. With the completion of the silkworm genome sequencing, the use of 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 genomes. 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 fav...

Claims

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Application Information

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