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Target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and locus and application thereof

A chromosome and transgene technology, applied in the field of nucleotide sequences, can solve the problems of W chromosome sequence difficulties, etc., and achieve the effects of ensuring hybridization rate, improving comprehensive income, and clear fluorescent markers

Active Publication Date: 2014-04-16
SOUTHWEST UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the silkworm W chromosome contains a large number of repetitive sequences, and the genome sequencing work has not yet been completed, and a large number of transposable elements on the W chromosome will inhibit the expression of inserted genes
Therefore, it is very difficult to find a W chromosome sequence suitable for transgene insertion

Method used

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  • Target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and locus and application thereof
  • Target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and locus and application thereof
  • Target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and locus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, transgenic microinjection and screening of female-specific transgenic silkworm W-T

[0024] Silkworm eggs of silkworm "Dazao" and "P50" were soaked in acid for 5 minutes at a temperature of 46°C to remove diapause. The greening is accelerated in the environment for about 30 days until hatching. After hatching, the ant silkworms are collected and raised under standard conditions with a temperature of 25°C and a humidity of 80%. Silkworm eggs were non-diapause silkworm eggs, which were used for microinjection in the next step.

[0025] Arrange the laid silkworm eggs neatly on a clean glass slide, and inject the piggyBac [3×p3 EGFP afm] transgene vector and the auxiliary plasmid A3H into 5300 silkworm eggs with an Eppendorf microinjector 2 hours after the silkworm eggs were laid In the process, after sealing with non-toxic glue, put it in an environment of 25°C and 80% relative humidity to accelerate the greening for about 10 days. The hatched G0 generation ...

Embodiment 2

[0027] Example 2, copy number detection of transgenic system W-T

[0028] Primers were designed for the GFP mutant line EGFP and the silkworm internal reference gene GAPDH (BmGAPDH). The forward primer of EGFP was 5'-gtgagcaagggcgaggagct-3' (SEQ ID NO.1), and the reverse primer was 5'-cttgtacagctcgtccatgcc-3' (SEQ ID NO.2); the forward primer of BmGAPDH is 5'-cattccgcgtccctgttgctaat-3' (SEQ ID NO.3), and the reverse primer is 5'-gctgcctccttgaccttttgc-3' (SEQ ID NO.4). The genomes of W-T female silkworm (W-T♀), W-T male silkworm (W-T♂), non-transgenic female silkworm (NT♀) and non-transgenic male silkworm (NT♂) were extracted and used as templates for PCR amplification. The PCR reaction conditions for amplifying the BmGAPDH gene are: 94°C pre-denaturation for 4 minutes, followed by 94°C denaturation for 40 seconds, 58°C annealing for 40 seconds, 72°C extension for 10 seconds, a total of 25 cycles, and finally 72°C extension for 10 minutes; The PCR reaction conditions of EGFP w...

Embodiment 3

[0030] Example 3 Insertion site detection of transgenic system W-T

[0031] The genome of W-T♀ was thoroughly digested with Hae III and self-ligated, and then amplified by inverse PCR using transposon-specific primers pBacL and pBacR. pBacR is used to amplify the partial sequence of the right arm (PR) of piggyBac[3×p3EGFP afm] and the adjacent genomic sequence (SG-R). The pBacR primer sequence is as follows, pBacR F; NO.5); pBacR R: 5'-gtactgtcatctgatgtaccagg-3' (SEQ ID NO.6), pBacL was used to amplify the partial sequence of the left arm (PL) of piggyBac[3×p3EGFP afm] and the adjacent genome sequence (SG -L), the pBacL primer sequence is as follows, pBacL F: 5'-atcagtgacacttaccgcattgaca-3' (SEQ ID NO.7), pBacL R: 5'-tgacgagcttgttggtgaggattct-3' (SEQ ID NO.8), the position is as follows image 3 As shown in A. The PCR product was identified and recovered by agarose gel electrophoresis, and then ligated with pMD19-T vector. The ligation reaction was performed under the action...

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Abstract

The invention discloses a target sequence suitable for transgene fixed point insertion of silkworm W-chromosome and a locus and application thereof. The nucleotide sequence of the target sequence is shown in SEQ ID NO.19 and is a specific sequence in silkworm W-chromosome, and Z-chromosome and autosome do not comprise the sequence; the target sequence inserted by a foreign gene can be expressed normally; therefore, the target sequence can be used as the fixed point insertion locus of the foreign gene in the silkworm W-chromosome so as to prepare W-chromosome chain transgenic silkworms, and has significance to identification of male and female silkworms.

Description

technical field [0001] The present invention relates to a nucleotide sequence, in particular to a target sequence and its site suitable for site-directed transgene insertion on silkworm W chromosome, and also relates to the application of the target sequence and site in a reporter gene expression cassette for site-directed transgene insertion. Background technique [0002] The silkworm is a Lepidoptera insect with important economic value. The silk industry is one of the main sources of income for farmers in many areas. The silk industry has made important contributions to the development of the world economy, culture and society. Breeding of fine silkworm varieties has always been the focus of scientific research on sericulture and the basis for the development of sericulture. The main goal of traditional breeding of fine silkworms is high yield and high quality. [0003] The silkworm has 28 pairs of somatic chromosomes, 27 of which are autosomes and the other pair are sex ...

Claims

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Application Information

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IPC IPC(8): C12N15/11C12N15/85
Inventor 夏庆友蒋亮孙强刘纬强郭慧珍彭正文党颖慧
Owner SOUTHWEST UNIVERSITY
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