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Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein

An insecticidal crystal protein and protein technology, which can be applied in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, and can solve problems such as narrow insecticidal spectrum.

Active Publication Date: 2016-07-20
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

On the one hand, its strong specificity means that it is safe to organisms other than target pests, especially humans and animals; but on the other hand, it also makes its insecticidal spectrum too narrow. For the integrated control of various pests, it is necessary to develop Effective insecticidal crystal proteins for different pests can achieve the purpose of control
[0004] However, in the prior art, only the vegetative phase insecticidal protein (Vip protein, a protein different from insecticidal crystal protein) of Bacillus thuringiensis has been found to have good activity on beet armyworm, and in the insecticidal crystal protein so far No protein with good activity against beet armyworm has been found

Method used

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  • Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein
  • Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein
  • Insecticidal crystal protein, nucleic acid, and preparation method and application of insecticidal crystal protein

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Experimental program
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Effect test

preparation example Construction

[0047] Table 2 Preparation table of SDS polyacrylamide gel

[0048]

Embodiment 1

[0050] 1.1 Cloning of cry2Aa17, cry2Ab29, cry2Ba2 and mcry2Aa17

[0051] By conventional methods in the art, the cry2Aa17 gene was cloned from the wild strain DS415 of Bacillus thuringiensis, as shown in SEQ ID No. 8, the cry2Ab29 gene was cloned from the wild strain LS5115-3, as shown in SEQ ID No. 9, and cry2Ba2 was cloned from the wild strain HD395. Gene, as shown in SEQ ID No. 10; cloned or artificially synthesized cry2Aa9 gene from a wild strain of Bacillus thuringiensis, as shown in SEQ ID No. 11; wild strain BtB-Pr-88, cloned cry2Ab4 gene, as shown in SEQ ID No. 12; The wild strain BtSC6H8 cloned the cry2Ah1 gene, as shown in SEQ ID No.13.

[0052] The cloned cry2Aa17 gene was subjected to site-directed mutation to obtain the mcry2Aa17 gene, as shown in SEQ ID No.14.

[0053] Eight kinds of cry2 genes: cry2Aa17, mcry2Aa17, cry2Ab29, cry2Ba2, cry2Aa9, cry2Ab4 and cry2Ah1 were inserted into the position of pEB vector Ecl136II. The ligation reaction was carried out according to ...

Embodiment 2

[0075] Activity analysis of insecticidal protein against Asian beet armyworm, Asian corn borer, Plutella xylostella

[0076] The artificial feed of beet armyworm was provided by the migratory pest group of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences.

[0077] Bioassay method for beet armyworm: (1) Take 30g of artificial feed and place it in a petri dish, add 3mL of sample solution to be tested, stir well, and place at room temperature. According to the degree of dryness and wetness of the feed, leave it at room temperature until the feed cannot be squeezed out. Moisture

[0078] (2) Divide all the feed into 3 sterilized 24-well cell culture plates;

[0079] (3) Use a writing brush to gently pick the larvae into a 24-well plate, each hole has a newly hatched larva that is less than or equal to 12 hours; after receiving it, cover it (with a layer of blown paper) and fix it with a rubber band;

[0080] (4) Place it in a 25℃ light incubator, with a photoperi...

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Abstract

The invention relates to the field of insecticidal crystal protein, in particular to Cry2-class insecticidal crystal protein with insecticidal activity on beet armyworm, striped rice borer, Asiatic corn borer and plutella xylostella.

Description

Technical field [0001] The present invention relates to the field of insecticidal crystal proteins, in particular to Cry2 insecticidal crystal proteins with insecticidal activity against Lepidoptera. Background technique [0002] So far, about 600 insecticidal crystal proteins have been discovered, and these insecticidal crystal proteins are mainly derived from Bacillus thuringiensis. It has insecticidal activity against more than 500 kinds of insects in 9 orders, nematodes, mites and a variety of protozoa. Due to its wide insecticidal activity, high specificity, safety, good control effect, and no environmental pollution compared with chemical pesticides, Bt has become the most studied, most widely used and most successful insect pathogenic microorganism in the world. Bt insecticides account for more than 70% of biological pesticides, and have been applied to genetically modified plants, bringing good economic and ecological benefits to humans, and their research is still expan...

Claims

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Application Information

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IPC IPC(8): C07K14/325C12N15/32C12N15/63A01N63/00A01P7/04
Inventor 束长龙张杰张风娇彭琦宋福平耿丽丽
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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