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AccCDK5 gene and AccCDK5r1 gene of Chinese honeybee and application of genes

A Chinese honeybee and gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of population continuation threat and sudden decrease in the number of Chinese honeybee colonies.

Active Publication Date: 2017-05-31
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Apis cerana has good economic benefits and important ecological functions. However, due to the introduction of western honeybees, pests and diseases, deforestation, overuse of pesticides, environmental pollution and other factors in recent years, the number of Apis cerana colonies has decreased sharply, and the continuation of the population has been restricted. extremely serious threat

Method used

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  • AccCDK5 gene and AccCDK5r1 gene of Chinese honeybee and application of genes
  • AccCDK5 gene and AccCDK5r1 gene of Chinese honeybee and application of genes
  • AccCDK5 gene and AccCDK5r1 gene of Chinese honeybee and application of genes

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Experimental program
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Effect test

Embodiment 1

[0029] Example 1: Cloning of the AccCDK5 and AccCDK5r1 genes of Apis chinensis

[0030] With Apis cerana cDNA as template, forward primer (its nucleotide sequence is shown in SEQ.ID.NO.5) and reverse primer (its nucleotide sequence is shown in SEQ.ID.NO.6) to amplify To increase the AccCDK5 fragment, the reaction procedure is as follows: pre-denaturation at 94°C for 10 min → (94°C for 40 s, 52°C for 40 s) × 35 cycles → 72°C for 1 min → 72°C for 10 min.

[0031]Using the amino acid sequences of mammalian CDK5 activators p35 and p39 as a reference sequence, design AccCDK5r1 specific primers and use the cDNA of Apis chinensis as a template, and use the forward primer (its nucleotide sequence such as SEQ.ID.NO.7 Shown) and reverse primer (its nucleotide sequence is shown in SEQ.ID.NO.8) amplification AccCDK5r1 fragment, reaction procedure is as follows:

[0032] Pre-denaturation at 94°C for 10 minutes→(94°C for 40s, 55°C for 40s)×35cycles→72°C for 1min→72°C for 10min.

[0033] A...

Embodiment 2

[0035] Embodiment 2: Recombinant vector construction

[0036] After obtaining the nucleotide sequences of AccCDK5 and AccCDK5r1 genes, the recombinant vector and AccCDK5 prokaryotic expression vector required for the yeast two-hybrid experiment were first constructed:

[0037] First, the AccCDK5-BD recombinant vector was constructed: using the above-mentioned plasmid with correct sequencing as a template, using a forward primer, the nucleotide sequence of which is shown in SEQ.ID.NO.9 (5'- Catat GATGCAAAAATATGAGAAACTCGAG-3', the underlined part is the Nde I restriction site) and the reverse primer, the nucleotide sequence of which is shown in SEQ.ID.NO.10 (5'- GGATCC CTGACAACGATCGTTTTTAATGG-3′, the underlined part is the BamH I restriction site) to amplify the AccCDK5 fragment.

[0038] After the reaction, the PCR product was detected by 1.0% agarose gel electrophoresis, the target fragment was recovered and purified, connected with the vector pEASY-T1 simple, and sequenced...

Embodiment 3

[0041] Embodiment 3: yeast two-hybrid experiment

[0042] The Yeast Two-Hybrid System of the present invention adopts Matchmaker GoldYeast Two-Hybrid System, and this experiment is divided into three parts.

[0043] First, the AccCDK5-BD recombinant vector self-activation detection was carried out. The yeasts transformed into pGBKT7 empty vector and AccCDK5-BD recombinant vector were co-spread on SD / -Trp / -His solid medium and SD / -Trp / -Ade solid medium, and the results showed that neither of them grew, proving that AccCDK5 -BD recombinant vector has no self-activation phenomenon (such as Figure 4 shown in A).

[0044] Then AccCDK5-BD recombinant vector toxicity test was carried out. The yeast competent cells transferred into pGBKT7 empty vector and AccCDK5-BD recombinant vector were co-coated on SD / -Trp solid medium, and the results showed that the growth of the two was the same, which proved that the AccCDK5-BD recombinant vector was non-toxic (such as Figure 4 shown in ...

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Abstract

The invention relates to the technical field of molecular biology and the biotechnology field, and in particular relates to effect of an AccCDK5 gene and an AccCDK5r1 gene of a Chinese honeybee in an antioxidation process and application of the genes. The invention provides a method for cloning two Chinese honeybee genes, namely the AccCDK5 gene and the AccCDK5r1 gene, and constructing a recombinant carrier and also provides an interaction evidence between an AccCDK5 protein and an AccCDK5r1 protein and an in vitro antioxidant capacity qualitative research method for an AccCDK5 recombinant protein, so that a certain theoretical basis is provided for breeding of a new high-quality variety of the Chinese honeybee, research on a honeybee nervous system is also enriched, and the theoretical basis is provided for research of development and nervous lesion of the honeybee nervous system.

Description

technical field [0001] The invention relates to the fields of molecular biology and biotechnology, in particular to the cloning of AccCDK5 gene and AccCDK5r1 gene of Apis cerana and the construction of recombinant vectors, and the first verification of the interaction relationship between AccCDK5 and its activator AccCDK5r1 in Apis cerana, providing A qualitative research method for the antioxidant capacity of AccCDK5 recombinant protein in vitro. Background technique [0002] Cyclin-dependent kinase-5 (CDK5) is a kind of serine / threonine protein kinase, which belongs to the cyclin-dependent kinase family CDKs. Studies in recent years have shown that in mammals, CDK5 does not participate in the regulation of the cell cycle, but mainly plays a role in the nervous system, participating in neuron migration, actin movement, microtubule transport and its stability, cell Adhesion, axon guidance, synaptic structural plasticity, myogenesis, and membrane trafficking are diverse cell...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/12C12N15/12C07K14/435
CPCC07K14/43572C12N9/12C12Y207/11022
Inventor 郭兴启赵光栋王琛胥保华崔学沛
Owner SHANDONG AGRICULTURAL UNIVERSITY
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