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Transgenic method for improving silk yield

A transgenic, silk technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve problems such as lack

Inactive Publication Date: 2011-09-21
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Although it is theoretically possible to transform silkworms to obtain improved varieties through transgenic technology, there is still a lack of technology that can effectively obtain high-yielding silkworms through transgenic technology.

Method used

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  • Transgenic method for improving silk yield
  • Transgenic method for improving silk yield
  • Transgenic method for improving silk yield

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Embodiment 1, construction transgenic vector

[0114] See Fil-GAL4 / 3XP3-DsRed figure 1 a. UAS-Ras1 V12 See / 3XP3-EGFP figure 1 b. Both of the above two vectors use the Piggybac transposon as the basic skeleton of the transgene, and the GAL4 line uses the fibroin light chain specifically expressed in the posterior silk gland as the promoter, and the artificial promoter specifically expressed in the eye and ganglion is selected as the screening marker. 3XP3-driven red fluorescent DsRed2. UAS lines carry a persistently active form of Ras1 v12 , the screening marker was changed to green fluorescent EGFP.

[0115] Fil-GAL4 / 3XP3-DsRed vector construction process, wherein the original vector is pBacA3-GAL4 / 3XP3-DsRed (obtained from the National Institute of Agricultural Sciences, Japan, reference AnjangTan, Hiromasa Tanaka, Toshiki Tamura, Takahiro Shiotsuki 2005.Precocious metamorphosis in transgenic silkworms overexpressing juvenile hormone esterase.Proc Natl Acad Sci...

Embodiment 2

[0117] Embodiment 2, the experimental analysis of transgenic silkworm acquisition and hybrid progeny

[0118] Transgenic silkworms of the above two plasmids were respectively obtained from a dimorphic variety Dazao (obtained from Southwest University) by microinjection. Specific method: After the silkworm chrysalis emerges, collect the male and female silkworm moths that have turned into moths at the same time, and disassemble them after mating under weak light at 25°C for 4 hours. Silkworm eggs were collected every hour, and the collected silkworm eggs were placed in an environment of 25°C for protection. And use the Eppendorf microinjector within 3 hours of spawning, inject 10-15nl of Fil-GAL4 / 3XP3-DsRed or UAS-Ras1 with a total concentration of 400ng / ul V12 / 3XP3-EGFP carrying an auxiliary carrier (pigA4, obtained from Southwest University), injected into silkworm eggs, sealed the injection port with non-toxic glue, sterilized with 35% formaldehyde steam for 5 minutes, and...

Embodiment 3

[0143] Embodiment 3, the expression level detection of Ras1-GTP

[0144] Ras1 activity detection kit (purchased from Millipore Company, catalog number 17-218) was used to detect the expression level of Ras in the transgenic silkworm hybrid progeny, that is, the expression level of Ras-GTP. Weighed 50 mg of the four genotype silk glands respectively , use the MLB lysate provided in the kit to centrifuge and absorb the supernatant, add 10ul of active form Ras1 binding substrate Raf-1 RBD agarose per 0.5ml cell extract. After incubating at 4°C for 45 minutes, centrifuge briefly, suck off the supernatant, wash three times with MLB washing solution, resuspend the agarose in 40ul 2×Laemmli reduced sample buffer, boil in water for 5 minutes, and centrifuge briefly to take 20ul of the supernatant was subjected to SDS protein electrophoresis, the protein was transferred to a nitrocellulose membrane, blocked with TBST containing 5% skimmed milk powder, and Ras1 primary antibody ratio 1:...

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Abstract

The invention relates to a transgenic method for improving silk yield. Genes promoting cell growth are specifically expressed in posterior silk glands of silkworms, silkworms with enlarged posterior silk glands are obtained, and the yield of silk is high. The invention provides genes promoting the growth of posterior silk glands and an expression system suitable for expressing the genes promotingthe growth of posterior silk glands at high specificity in the posterior silk glands of the silkworms. The invention provides novel means and a method for improving silk producing characters of the silkworm variety.

Description

technical field [0001] The invention relates to the field of biotechnology, more specifically, the invention relates to a transgenic method for increasing silk yield. Background technique [0002] The silkworm is an important economic insect and a model organism of Lepidoptera. In the past thousands of years, silkworm silk production has been improved mainly through traditional breeding methods, which have problems such as long breeding cycle, complicated operation, unstable traits, and difficulty in integrating good traits. With the completion of the silkworm genome framework map (2004) and fine map (2008), it marks that human research and understanding of silkworms has entered the post-genome era. Improvements are possible. [0003] In modern biological research, transgenic technology is playing an increasingly important role. It effectively introduces artificially isolated or modified genes into the genome of organisms to achieve overexpression or inactivation of homolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01K67/04C12N15/85C12N15/12
Inventor 李胜马俐夏庆友徐汉福
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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