Transgenic method for improving content of silk fibroin in silkworm cocoon and silkworm variety thereof

A silk fibroin and genetically modified technology, applied in the field of bioengineering, achieves good application prospects, increased silk fibroin synthesis, and increased silk production

Active Publication Date: 2022-05-27
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, tissue-specific knockout has not yet been achieved in the silkworm

Method used

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  • Transgenic method for improving content of silk fibroin in silkworm cocoon and silkworm variety thereof
  • Transgenic method for improving content of silk fibroin in silkworm cocoon and silkworm variety thereof
  • Transgenic method for improving content of silk fibroin in silkworm cocoon and silkworm variety thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Embodiment 1, it is mainly the construction of silkworm posterior silk gland specific GAL4 / UAS expression vector, it combines above-mentioned three methods, and it comprises the following steps:

[0076] The construction of step S1 GAL4 expression vector, namely the construction of rear silk gland-specific GAL4 expression vector, wherein the silkworm fibH gene promoter (SEQ ID NO.5) sequence and GAL4BD (SEQ ID NO.6) gene sequence, enhance The sub-VP16 sequence (SEQ ID NO.7) and the termination signal Ser1-poly A (SEQ ID NO.8) are concatenated to form the target gene expression cassette. By using AscI, the backbone vector pBac[3×P3-DsRed] and the target gene expression cassette are combined Cut, the backbone vector is completed by the following steps: First, assemble the 3×P3-DsRed sequence (SEQ ID NO. 9), which is driven by the 3-fold repeated P3 promoter (eye and nerve-specific promoter) to express The red fluorescent protein (DsRed) sequence composition; then the pigg...

Embodiment 2

[0083] Embodiment 2, it is mainly the making of GAL4 / UAS transgenic silkworm, it combines above-mentioned three methods, and it comprises the following steps:

[0084] Step S3 Transgene Injection and Fluorescence Screening

[0085] After obtaining the above-mentioned GAL4 / UAS transgenic expression vector, it was mixed with the helper plasmid (A4Helper) at a concentration of 450ng / μL (ng / μL) in equal proportions, and injected by an Eppendorf microinjector. (Multiple batches of silkworm seed material can be raised within a year) as the injection recipient. Before injection, the silkworm moths need to be mated for 6 hours, placed at 4°C for a day, and then taken out to lay eggs at room temperature. The paste was adhered to the glass slide, injected using an Eppendorf microinjector, sealed with non-toxic glue, sterilized by 35% formaldehyde steam for 5 minutes, and incubated at 25°C and a relative humidity of 85%. The G0 generation (first generation after injection) ant silkworms...

Embodiment 3

[0092] Embodiment 3, which is the phenotype observation of the GAL4 / UAS transgenic Bombyx mori silk glands that specifically overexpress BmDimm (SEQ ID NO. 2) in the posterior silk gland, which further comprises the steps:

[0093] Step S4-1, feeding wild-type silkworm Nistari and GAL4 / UAS transgenic silkworm whose PSG (posterior silk gland) overexpresses BmDimm (SEQ ID NO. 2) specifically expresses GAL4 / UAS that emits both blue-green fluorescence and red fluorescence Transgenic silkworms reach fifth instar by dissecting in 1×PBS (phosphate buffered saline) buffer and observing wild-type silkworm Nistari and GAL4 / UAS transgenic silkworm overexpressing BmDimm (SEQ ID NO. 2) on sixth day of fifth instar (5L6D) silk gland, and photographed, the results are attached figure 2 As shown, the posterior silk glands of the transgenic silkworms overexpressing BmDimm (SEQ ID NO. 2) became enlarged compared with those of the wild-type silkworms.

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Abstract

The invention provides a transgenic method for increasing the content of silk fibroin in silkworm cocoons and a silkworm variety thereof, the transgenic method for increasing the content of silk fibroin in silkworm cocoons respectively constructs specific overexpression BmDimm and specific knockout BmYki and BmSd in posterior silk glands of silkworms through a GAL4 / UAS system, and transgenic silkworms with enlarged posterior silk glands are obtained; the silk fibroin content of the bombyx mori can be increased by specifically knocking out BmYki in the posterior silkgland of the bombyx mori, or the silk fibroin content of the bombyx mori can be increased by specifically knocking out BmSd in the posterior silkgland of the bombyx mori, or the silk fibroin content of the bombyx mori can be increased by specifically overexpressing BmDimm in the posterior silkgland of the bombyx mori. According to the sequence codon preference of silkworm silk gland expression endogenous genes in a silkworm genome sequence database, the gene sequence of the Cas9 protein is optimally designed, so that the Cas9 gene is more beneficial to expression in silkworm silk glands; through a GAL4 / UAS system and a CRISPR / Cas9 gene editing technology, the bombyx mori silk gland is subjected to tissue-specific knockout and overexpression.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a transgenic method for increasing the content of silk fibroin in silkworm cocoons and a silkworm variety thereof. Background technique [0002] Bombyx mori is an important economic insect and model insect of Lepidoptera. In the process of feeding and domestication for more than 5,000 years, the improvement of silkworm silk production was mainly achieved by traditional cross-breeding methods. There are technical challenges such as long breeding cycle, complicated operation, and difficulty in obtaining excellent traits. With the completion of the whole genome sequencing of the silkworm, it marks that the research and understanding of the silkworm has entered the era of genetic modification. It is a simple and fast technical means to use efficient and stable transgenic technology to genetically improve the economic traits of the silkworm from the genome level. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/12C12N15/85C12N15/55A01K67/033
CPCC07K14/43586C12N15/8509C12N9/22A01K67/0334A01K2227/706A01K2267/02
Inventor 徐汉福曾文慧欧瑶罗琴刘荣鹏马艳马静文唐艺芸胡杰陈玉琴向仲怀
Owner SOUTHWEST UNIV
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