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Artificial synthetic Bt antiinsect gene used for transgene antiinsect plant and its development method

A gene and insecticidal protein technology, applied in the field of Bacillus thuringiensis Btcry gene and its development, can solve problems such as unclear sequence

Inactive Publication Date: 2004-07-21
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

The sequence of these signals is not yet known, so sequence analysis can only identify possible polyadenylation signals. The following table shows some possible polyadenylation signal sequences (Dean et al., mRNA transcripts of several plantgenes are polyadenylated at multiple sites in Vivo, Nuc.A.R., 1986, 14:2229-2240)

Method used

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  • Artificial synthetic Bt antiinsect gene used for transgene antiinsect plant and its development method
  • Artificial synthetic Bt antiinsect gene used for transgene antiinsect plant and its development method
  • Artificial synthetic Bt antiinsect gene used for transgene antiinsect plant and its development method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Analysis of the insecticidal activity of Cry1Ie1 protein against soybean borer

[0047] Table 2 lists the toxicity analysis of Cry1Ie1 against several pests. The activity against corn borer and diamondback moth has been patented (application number: 01124163.2). The present invention finds that it also has an obvious insecticidal effect on soybean borer.

[0048] Table 2 Analysis of insecticidal activity of Cry1Ie1 protein

[0049] Insect species LC50 (μg / mL) 95% confidence limit

[0050] Corn borer Ostrinia furnacalis 2.22 1.77-2.75

[0051] Plutella xylostella 0.20 0.13-0.27

[0052] Leguminivora glycinivorella 9.02 3.50-23.24

[0053] Helicoverpa armigera >1000 No activity

[0054] Spodotera exigua >1000 inactive

[0055] Pyrrhalta aenescens >1000 inactive

Embodiment 2

[0056] Example 2 PCR amplification of cry1Ie1 genes of different lengths

[0057] According to the full-length cry1Ie1 gene sequence (SEQ ID NO 4), 7 primers were designed to amplify the gene fragments with end deletions. P001, P045, and P105 are upstream primers with NdeI restriction site added; P633, P648, P656 and P659 are downstream primers with SalI restriction site added. The number after the primer is the amino acid number of the primer in cry1Ie1. The sequence is as SEQ ID NO 1.

[0058] Using the plasmid pET-1IE containing the full-length cry1Ie1 gene as a template (Patent Application No. 01124163.2), the upstream and downstream primers were combined respectively to perform PCR amplification to obtain gene fragments of different lengths (Table 3). The reaction system is as follows: PCR 10 Buffer 10μL, 20mmol MgCl 2 12μL, 10mmol dNTP2μL, 10μmol upstream and downstream primers each 2μL, DNA template 0.1-2ng, 2U pfu enzyme 0.5μL, make up to 100μL with ultrapure water, mix an...

Embodiment 3

[0068] Example 3 Construction of expression vectors for cry1Ie1 genes of different lengths

[0069] The cry1Ie1 gene fragments of different lengths (such as Figure 1a ), insert these gene fragments into pET-21b (purchased from Novagen) to obtain recombinant plasmids with these fragments (such as Figure 1b ), and through sequencing, it was proved that the obtained sequence was identical to the cry1Ie1 gene sequence, and BL21(DE3) (purchased from Novagen) was transformed to obtain a recombinant engineering bacteria.

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Abstract

An artificially synthetic insecticiding gene Bt used for insect-resisting transgenic plant is prepared from the high-toxic Bt CryIIe protein of lepidopterous pest through insecticiding activity deficiency, amplifying the termination-deficient gene fragment, configuring expression carrier, transforming it to obtain recombinant engineered bacteria, inducing expression analyzing insecticiding activity, determining the minimal terminal active region and active region, reinforming polyadenylation signal sequence and ATTTA sequence, optimiizng plant prefaverable codon, and artificial synthesis.

Description

Technical field: [0001] The invention belongs to the technical field of biological control. Specifically, it relates to the Btcry gene of Bacillus thuringiensis for plant expression and its development method. Background technique: [0002] Bacillus thuringiensis (Bt for short) is a Gram-positive bacterium with an extremely wide distribution. While forming spores, it can produce protein-like parasporal crystals (parasporal crystal), for Lepidoptera (Lepidoptera), Diptera (Diptera), Coleoptera (Coleoptera), Hymenoptera (Hymenoptera), Homoptera (Homoptera), Orthoptera (Orthoptera), Mallophaga (Mallophaga) and other insects, as well as nematodes, mites and protozoa have specific insecticidal activity (Schnepf.E., N. etc., Microbiol.And Mocecular Biology Review, 1998, 62: 3 775-806). Insecticidal Crystal Proteins (ICPs), also known as delta-endotoxin (delta-endotoxin), are harmless to humans and animals, and do not pollute the environment. Therefore, Bt is the most widely used in the...

Claims

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Application Information

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IPC IPC(8): A01H1/00C07H21/04C12N15/63C12N15/82
CPCY02A40/146
Inventor 宋福平黄大昉张杰何康来王国英
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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