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Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application

A moth arginine kinase and plasmid technology, applied in the field of genetic engineering, can solve the problems of affecting Bt, short residual period, narrow insecticidal spectrum, etc., and achieve the effect of improving insecticidal effect and reducing cost

Inactive Publication Date: 2013-07-24
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although Bt insecticides have the advantages of being safe and harmless to humans and animals, not polluting the environment, and having certain specificity and selective insecticidal effects, they also have problems such as poor stability, short residual period, and narrow insecticidal spectrum, which seriously affect Practical application of Bt

Method used

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  • Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application
  • Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application
  • Plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, construction of plasmid pHT305AKR

[0030] 1. Bacillus thuringiensis cryⅢA Preparation of the promoter fragment of the gene

[0031] 1.1 Extraction of plasmid pHT305a

[0032] Take a single colony containing the plasmid pHT305a strain and culture it overnight in LB liquid medium (containing 50ng / ml Amp and 50ng / ml Erm), and use the high-purity plasmid miniprep kit (product catalog: DP1002) to extract the plasmid pHT305a.

[0033] 1.2 PCR amplification of Bacillus thuringiensis cryⅢA pro3α(+) promoter fragment of the gene

[0034] Using the plasmid pHT305a as a template, a pair of primers were designed (the 5' ends of the two primers were introduced into Bam H I and Sph I digestion recognition sequence and protection base).

[0035] Upstream primer 01F: 5'-GTCT GGATCC GAAATTAGTTATACAAGCATT-3' (underlined as Bam H I recognition sequence);

[0036] Downstream primer 01R: 5'- ACAT GCATGC TTTTCTTCCTCCCTTTCTT-3' (underlined as Sph I reco...

Embodiment 2

[0090] Embodiment 2, construction and application of Bacillus thuringiensis engineering bacteria

[0091] 1. Plasmid extraction

[0092] Take a single colony containing the plasmid pHT305AKR and culture it overnight in LB liquid medium (containing 50ng / ml Amp and 50ng / ml Erm), and extract the plasmid pHT305AKR with a high-purity plasmid miniprep kit (product catalog: DP1002).

[0093] 2. Competent preparation of Bacillus thuringiensis BMB171

[0094] 2.1 Cultivate BMB171 overnight at 30°C on a shaker at 200r / min.

[0095] 2.2 Transfer to LB medium according to 1% inoculum amount, and cultivate to OD at 30°C and 220r / min 600 1.2-1.5 so far.

[0096] 2.3 Place the culture medium in step 2.2 on ice for 10 minutes, transfer to a large ice pre-cooled centrifuge tube, and centrifuge at 6000 r / min, 4°C for 10 minutes.

[0097] 2.4 Discard the supernatant, add 10ml of ice-cold SG buffer, centrifuge at 6000r / min, 4°C for 10min, and remove the supernatant. Repeat 3 times.

[0098] 2...

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Abstract

The invention relates to a plasmid for expressing plutella xylostella arginine kinase genes dsRNA (double-stranded ribonucleic acid) and an application, belonging to the field of gene engineering. The plasmid pHT305AKR is a recombinant plasmid obtained by inserting deoxyribonucleic acid (DNA) shown by SEQ ID NO.1 between BamHI and PstI enzyme cutting sites of the plasmid pHT305a. The plasmid pHT305AKR not only realizes the research of expressing dsRNA in bacillus thuringiensis, but also provides a powerful means for applying RNAi (RNA interference) technique to identifying the function of bacillus thuringiensis genes and researching a functional genome of the bacillus thuringiensis, and can also be used for synthesizing dsRNA by utilizing host bacterium, so that the cost for obtaining the dsRNA is reduced. The invention also provides a direction for exogenous transformation of the bacillus thuringiensis and research of bacillus thuringiensis biopesticide, so that the plasmid has a promising application prospect. Meanwhile, a novel way is provided for controlling the plutella xylostella, and a new field is created for controlling the pests.

Description

technical field [0001] The invention relates to a plasmid expressing dsRNA of diamondback moth arginine kinase gene and its application, belonging to the field of genetic engineering. Background technique [0002] Plutella xylostella belongs to Lepidoptera Plutellaidae, English name: Diamondback moth; scientific name: Plutella xylostella (L.), is a worldwide migratory pest, mainly damaging brassicaceous plants such as cabbage, broccoli, Chinese cabbage, and rapeseed. Since the first report of diamondback moth resistance to DTT in 1953, so far, diamondback moth has developed resistance to more than 50 insecticides to varying degrees, involving almost all control drugs. [0003] Arginine Kinase (Arginine Kinase, AK) (ATP: Argine N-phosphotransferase EC 2.7.3.3) belongs to the phosphogenase family and is widely found in invertebrates and molluscs, similar to creatine kinase (Creatine Kinase) in vertebrates Kinase, CK), is an important kinase directly related to intracellular...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/75C12N1/21A01N63/02A01P7/04C12R1/07
Inventor 杨广尤民生陈金芝徐秀凤汪淑燕
Owner FUJIAN AGRI & FORESTRY UNIV
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