Preparation method and application of insecticidal crystal protein, nucleic acid, and insecticidal crystal protein
An insecticidal crystal protein and protein technology, which can be used in the fields of botanical equipment and methods, biochemical equipment and methods, and applications, and can solve problems such as narrow insecticidal spectrum.
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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; the wild strain The cry2Ba2 gene was cloned from HD395, as shown in SEQ ID No.10; the cry2Aa9 gene was cloned or artificially synthesized from the wild strain of Bacillus thuringiensis, as shown in SEQ ID No.11; cry2Ab4 was cloned from the wild strain Bt B-Pr-88 The gene is shown as SEQ ID No.12; the cry2Ah1 gene is cloned from the wild strain Bt SC6H8, as shown in SEQ ID No.13.
[0052] Site-directed mutation was performed on the cloned cry2Aa17 gene 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 pEB vector Ecl 136II respectively, and the ligat...
Embodiment 2
[0075] Activity analysis of insecticidal proteins against Asian beet armyworm, Asian corn borer, diamondback moth and
[0076] The artificial diet of beet armyworm was provided by the migratory pest group of 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 the sample solution to be tested, stir well, place at room temperature, and air at room temperature according to the degree of dryness and humidity of the feed, until the feed cannot be squeezed out moisture;
[0078] (2) All feeds are divided into three sterilized 24-hole cell culture plates;
[0079](3) Gently pick the larvae with a brush and place them in a 24-well plate, with one newly hatched larvae less than or equal to 12 hours old in each well; after picking up, add a cover (with a layer of blow-molded paper inside), and fix it with a rubber band;
[0080] (4) place in 25 ℃ of...
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