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Method for changing feeding habits of bombyx mori and application of method

A technology of silkworm and feeding habits, applied in genome editing and biological fields

Active Publication Date: 2018-11-13
CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there are no related genes reported in silkworm

Method used

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  • Method for changing feeding habits of bombyx mori and application of method
  • Method for changing feeding habits of bombyx mori and application of method
  • Method for changing feeding habits of bombyx mori and application of method

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1, the cloning of hestia gene

[0050] The genome sequence of the target gene hestia is as follows (SEQ ID NO: 9, wherein the underlined sequence is an exon sequence, and the non-underlined sequence is an intron sequence):

[0051] ATGTCACCACCGCTAGTCCATATCAATACATTTGTTCAACCCAAGCAAAGTACACTGTAGACAAAAGTA TCAAAGTTTTTTATAATATGTAGTTTTTCTTAGGCGTTAATAGGTTGCCTATTATATCATCGAAACATGTGTATAC AATTCCTTCTATAATTTATACTTTCGTACTAATGTGTGTACTAAATTTTTTCGGGTTTGATTCTGTCTCATTATCTA TCATGAGTTTGAACCTTGTATTACATATCTTATGTTCCTTCCTTGGTATGTTTTTTTTGGAAGAGAATGCGCCTGTAT TATTCAGAGCTCTGTAAGTTTGATATTTGCATCGGATGCAGACCAATAACTGCACAAGGTTCCAGTAAACTTGTAAT TCAGACTTGCATTATAAATGTTTTGATAGCTTTGGTGTTTATTGTGCCGAATTCACTTCAGATTCTAATCAAACCAG TTATATATTTGCTTCCCATGCACGCCTTTGTGTCGTTCGAAGTGCATTATTATGGCCACCTTCTCAATTTACTTATC CCGCGTTTACATTTAATAAACTATTACATGGAATCCTCATTAACTACCACAAGCGATAAAAGGGAGTCGAGTGTACT GAAACATGTTATTTTTATTTAAATATTATAATAAGGAATCGAACTGTCAAATGAAAAAATTTATGGATCTTTATTATA TTATCGTAGAATTCTTACAGATATCTTA...

Embodiment 2

[0059] Embodiment 2, the selection of sgRNA target (TS) and the synthesis of sgRNA

[0060] The present invention uses the CRISPR / Cas9 system to knock out the hestia gene, and screens out a 23bp sgRNA recognition target (TS) located in exon 1 after extensive selection and testing of the gene sequence. Schematic diagram of the structure of the silkworm hestia gene and its target (TS) sequence as shown in figure 1 shown.

[0061] The sequence of the sgRNA recognition target is as follows:

[0062] 5'-GGATAAGTAAATTGAGAAGGTGG-3' (SEQ ID NO: 1).

[0063] sgRNA framework sequence (SEQ ID NO:11):

[0064] GNNNNNNNNNNNNNNNNNNNNN GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT

[0065] Wherein, the underlined part is the sgRNA targeting sequence.

[0066] According to the screened target (TS), the following primers sgF and sgR were synthesized:

[0067] sgF:

[0068] TAATACGACTCACTATAGGATAAGTAAATTGAGAAGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAG ...

Embodiment 3

[0073] Embodiment 3, the acquisition of Cas9 mRNA

[0074] The template plasmid used to synthesize Cas9 mRNA is pTD1-Cas9 (Yueqiang Wang, Zhiqian Li, JunXu, Baosheng Zeng, lin ling, Lang You, Yazhou Chen, Yongping Huang*, and Anjiang Tan* .The CRISPR / Cas System mediates efficient genome engineering in Bombyxmori. Cell Research 23(12):1414-6.2013). The nucleotide sequence of the plasmid is shown as SEQ ID NO: 8, the first ATG is a start codon, and the last TAG is a stop codon. A nuclear localization signal precedes the stop codon.

[0075] The pTD1 vector contains the T7 promoter, 5'-UTR and 3'-UTR sequences, and the ORF of the Cas9 gene is connected between the 5'-UTR and 3'-UTR sequences, so pTD1-Cas9 can be used as a template for in vitro transcription of Cas9 mRNA . The pTD1-Cas9 vector was linearized with Not I restriction endonuclease, and the gel was collected and purified as a template for Cas9 mRNA synthesis. Use mMESSAGEmMACHINE T7 kit to transcribe mRNA in vit...

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Abstract

The invention relates to a method for changing the feeding habits of the bombyx mori and application of the method. The hestia gene of the bombyx mori is targeted at a fixed point, a gene structure ofthe bombyx mori is damaged to affect normal feeding behavior of the bombyx mori, and thus, the feeding habits of the bombyx mori are changed into omnivory from oligophagy. By the method, a breeding mode of feeding the bombyx mori with folium mori only in sericultural production is solved, and a new through is provided for large-scale bombyx mori feeding.

Description

Technical field [0001] The invention belongs to the field of biotechnology and genome editing, and relates to methods and applications of using the CRISPR / Cas9 system to knock out the hestia gene of silkworm to change the phagocytosis of silkworm. Background technique [0002] Insects are the most diverse and numerous biological species in the animal kingdom. The diversity of insect species is closely related to the differentiation of their feeding habits. According to the food range of insects, they are divided into polyphagous, oligophagous and monophagous. According to the nature of insect food, they are divided into several categories such as herbivorous, carnivorous, detritivorous, and omnivorous. Herbivorous and carnivorous insects generally feed on live plants and animals respectively, detritivorous insects feed on the carcasses or remains of animals and plants, and omnivorous insects eat both plants and animals. During the long process of evolution, a unique relat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/90A01K67/033
CPCA01K67/0339A01K2207/05A01K2217/058A01K2227/706A01K2267/02C07K14/43586C12N15/113C12N15/902C12N2310/10C12N2310/11C12N2310/14C12N2310/141
Inventor 谭安江牛宝龙计东风黄勇平张忠杰
Owner CAS CENT FOR EXCELLENCE IN MOLECULAR PLANT SCI
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