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Method for inserting exogenous genes into W chromosomes of bombyx mori in fixed-point mode

A technology for exogenous genes and chromosomes is applied in the application field of the identification of male and female silkworms, which can solve problems such as difficulties, and achieve the effects of improving yield and quality, saving production costs and improving quality.

Active Publication Date: 2017-11-24
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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Problems solved by technology

[0006] Since there are a large number of autosomal repetitive sequences on the silkworm W chromosome, and there are also a large number of transposable elements, it is very difficult to insert an exogenous gene into the W chromosome sequence

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  • Method for inserting exogenous genes into W chromosomes of bombyx mori in fixed-point mode
  • Method for inserting exogenous genes into W chromosomes of bombyx mori in fixed-point mode
  • Method for inserting exogenous genes into W chromosomes of bombyx mori in fixed-point mode

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Embodiment Construction

[0037] The preferred embodiments of the present invention will be described in detail below with reference to the accompanying drawings. The experimental method that does not indicate specific condition in the embodiment, usually according to the conventional method, for example the method described in the Molecular Cloning Experiment Guideline (Third Edition, J. Sambrook et al.), or according to the method provided by the reagent company .

[0038] 1. The silkworm W chromosome site-specific insertion of exogenous gene target sequence and the selection of TALENs target,

[0039] According to the genome sequence of the silkworm W chromosome-specific molecular marker W-Rikishi RAPD marker genome sequence in the NCBI database, the forward primer was designed: the sequence is shown in SEQ ID NO.1, and the reverse primer: the sequence is shown in SEQ ID NO.2, and the silkworm Nistari Line female and male genome DNA are templates, detect and find that only the female genome DNA has...

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Abstract

The invention belongs to the field of biotechnology (genome editing), and relates to a method for inserting exogenous genes into W chromosomes of bombyx mori in a fixed-point mode through mediating of transcriptional activator-like nuclease, and application of the method to bombyx mori sex discrimination. The method for inserting exogenous genes into W chromosomes of bombyx mori in the fixed-point mode specifically comprises the steps that W chromosome molecular markers in a bombyx mori genome database are analyzed, a TALENs targeting site is screened, TALENs mRNA expression plasmid is designed and obtained by synthesis, homologous recombinant carrier plasmid for expressing exogenous (reporter) genes (such as red fluorescent protein) is constructed, the microinjection technique is used for injecting the TALENs mRNA expression plasmid and the homologous recombinant (donor) carrier plasmid into eggs of the bombyx mori Nistara strain, the exogenous genes are inserted into the W chromosomes of bombyx mori in the fixed-point mode, the exogenous genes are only expressed in female individuals, and thus the purpose of rapid and efficient sex discrimination is achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology (genome editing), and relates to a method for inserting an exogenous gene into silkworm W chromosome at a fixed point mediated by transcription activator-like effector nucleases (TALENs) and its production in male and female silkworms. applications in identification. Background technique [0002] Bombyx mori is a kind of silk insect with high economic value, and it is also a model insect of Lepidoptera, which plays an important role in my country's economic production, history and culture. The silkworm has 28 pairs of somatic chromosomes, 27 of which are autosomes and the other pair are sex chromosomes. The neutral sex chromosome in female silkworm is ZW; the neutral sex chromosome in male silkworm is ZZ. Therefore, the silkworm is a ZW-type sex determination model, and the sex of offspring individuals is determined by whether they carry W chromosomes. [0003] Silkworm eggs are divided into mother...

Claims

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Application Information

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IPC IPC(8): C12N15/90C12N15/85C12N15/11
CPCC12N9/22C12N15/85C12N15/902C12N2800/105
Inventor 牛宝龙谭安江黄勇平张忠杰计东风陈诗
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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