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Application of KRP gene to pest control

A gene and gene expression technology, applied in the application field of KRP gene in pest control, can solve problems such as unclear

Active Publication Date: 2020-12-11
SOUTH CHINA NORMAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it is unclear whether 20E can reverse the expression of Kr-h1 and the molecular mechanism of Kr-h1

Method used

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  • Application of KRP gene to pest control
  • Application of KRP gene to pest control
  • Application of KRP gene to pest control

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Bioinformatics analysis of BmKRP

[0048] The Accession number of BmKRP in NCBI's Genbank database is XM_004922171.3 (https: / / www.ncbi.nlm.nih.gov / nuccore / XM_004922171.3 / ), and there is no description or article about this gene in the NCBI database. The encoded protein is currently an unknown protein. According to the analysis of the nucleic acid sequence of BmKRP, the full length of the BmKRP gene is 2689bp, with an open reading frame of 1557bp, including 3 exons and 2 introns, and the sizes of the 3 exons are 422bp, 237bp and 899bp, The size of the first intron is 1195bp and 1889bp (such as figure 1 shown). The protein encoded by BmKRP has three typical Cys2 / His2 zinc finger domains at the N-terminus and three low-complexity regions at the C-terminus (such as figure 2 shown). It is speculated that its zinc finger domain is the binding domain of transcription factor BmKRP and DNA. BmKRP protein encodes 518 amino acids, its predicted molecular weight is ...

Embodiment 2

[0049] Example 2 Design and synthesis of sgRNA

[0050] (1) Primer design

[0051] According to the principle of target design, use the online target design program to design the sgRNA core sequence and select site1 as the target site (1015nt-1037nt of the nucleotide sequence shown in SEQ ID NO: 1 is site1), after sequencing verification, the used The P50 silkworm BmKRP target sequences are all homozygous and can be used as the target of Cas9.

[0052] (2) Primer PCR

[0053] Send the designed forward and reverse primers to the company for synthesis, and then amplify the primers for each other through the PCR program to synthesize a complete sgRNA sequence transcription template. The primer sequences used to synthesize the BmKRP target sgRNA are shown in Table 1 (SEQ ID NO: 3-4). The PCR system used is shown in Table 2. The amplification program is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 30 s, annealing at 55°C for 15 s, extension at 72°C for 20 s,...

Embodiment 3

[0074] Example 3 Construction of CRISPR / Cas9 Knockout Strain of Bombyx mori BmKRP- / -

[0075] 1. Experimental method

[0076] 1) Screening of silkworm BmKRP mutants

[0077] Test positive for injection

[0078] Injection volume: Take an appropriate amount of the prepared sgRNA and the purchased Cas9 protein, and prepare 5 μL of a mixture (the final concentration of sgRNA and Cas9 are both 600 ng / μL). . Mix 2.5 μL of Cas9 protein and 2.5 μL of site1 sgRNA evenly before injecting, the injection volume is about 2-3 nL / egg.

[0079] After the injected eggs hatched, the genome was extracted by collecting multiple dead embryos and mixing them, and the extracted genomic DNA (extracted by conventional methods) was treated with ddH 2 O diluted to 100ng / μL, 20 μL PCR system Take 1 μL dead embryo genomic DNA as template, add template / primer mixture according to Table 7, mix the mixture and centrifuge to the bottom of the tube for PCR reaction.

[0080] The PCR product was directly sen...

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Abstract

The invention relates to the technical field of gene engineering, in particular to application of a KRP gene in pest control. According to the application provided by the invention, through construction of a CRISPR / Cas9 knockout strain of silkworm BmKRP, the influence of a new transcription factor BmKRP on the reproductive process of female silkworms is preliminarily explored and studied; and through observation on the silkworm BmKRP<- / -> phenotype, the effect that the deletion of BmKRP influences the female reproduction of silkworms, resulting in ovarian reduction, oocyte volume reduction andadult egg laying number reduction is found; and the effects that the mutation deletion of BmKRP influences the combination of BmKRP with reaction elements on a BmKrh1 promoter, so that BmKr-h1 does not express or down-regulates the expression, and important significance is realized on explaining a molecular mechanism of the silkworm hormone regulation reproduction are found. Through study, a similar control mechanism existing in other insects is speculated. Clues and theoretical guidance are provided for beneficial insect utilization, and pest control new targets and new strategies, and the application significance is realized.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to the application of KRP gene in pest control. Background technique [0002] The silkworm (Bombyx mori L.) is a silk-producing insect of the order Lepidoptera, which belongs to invertebrates, and is a species of silkworm in the family Bombyxidae Arthropodae. In the field of scientific research, silkworm, as an important model insect of Lepidoptera, is widely used to study insect reproductive genetics and metamorphosis, so as to grasp and use its growth regulation mechanism, and finally achieve the effect of agricultural and forestry pest control. [0003] Bombyx mori is a holometabolous insect. One generation has to go through four completely different developmental stages: egg, larva, pupa and adult; its larvae need to go through five molts in the stage of turning into pupa. The process of molting and metamorphosis of silkworm is regulated by complex hormone endocrine...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/435C12N15/12C12N15/113C12N9/22C12N15/89C12N15/10A01N57/16A01P7/04
CPCA01N57/16C07K14/43563C12N9/22C12N15/102C12N15/113C12N15/89C12N2310/20
Inventor 朱子丹邓惠敏童春梅冯启理
Owner SOUTH CHINA NORMAL UNIVERSITY
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