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Plant expression carrier construction for divalent insect-resistant gene and transgenic plant acquiring method thereof

A technology for expressing vectors and genes, which is applied to the construction of plant expression vectors for bivalent insect-resistant genes and the acquisition of transgenic plants. The effect of resistance

Inactive Publication Date: 2011-01-26
BEIJING CHANGLE ERSHENG GENE TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] As far as the current production and application situation is concerned, the target insects of cotton with Bt insecticidal genes are pests such as cotton bollworm and red bollworm. Adaptability or resistance, which will not only affect the application of Bt cotton, but also affect the insect control effect of Bt pesticide formulations

Method used

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  • Plant expression carrier construction for divalent insect-resistant gene and transgenic plant acquiring method thereof
  • Plant expression carrier construction for divalent insect-resistant gene and transgenic plant acquiring method thereof
  • Plant expression carrier construction for divalent insect-resistant gene and transgenic plant acquiring method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Cloning of Amaranthus retroflexus Lectin Gene, Construction of Prokaryotic Expression Vector and Experiment of the Insecticidal Effect of Prokaryotic Expression Product

[0051] In this study, according to the characteristics of high homology of genes in the same family and genus, the wild amaranth collected from the roadside dry land in the suburbs of Beijing was identified as Amaranthus retroflexus L., and the seeds of Amaranth retroflexus were isolated from the leaves after aseptic culture. The total DNA was extracted, and the two exons were respectively amplified using the total DNA as a template by using primers designed with overlapping sequences and the polymerase chain reaction (PCR) technology, and then further PCR was performed using the recovered products of the two exons as templates. Amplify, complete the in vitro splicing of two exons, obtain the complete sequence of the gene coding region, and connect it to the pUCm-T vector. Analysis of the dige...

Embodiment 2

[0139] Example 2 Artificial synthesis of Bt gene, construction of prokaryotic expression vector and experiment on insecticidal effect of prokaryotic expression product

[0140] 1. Artificial synthesis of Bt insecticidal protein gene and construction of its prokaryotic expression vector

[0141]1.1 Artificial synthesis of Bt insecticidal protein gene

[0142] The Bt insecticidal protein gene of the present invention is artificially synthesized, and this new artificially synthesized Bt gene is synthesized after referring to the nucleotide sequence design and transformation of the Bt cryI A (C) insecticidal protein gene. The synthetic Bt insecticidal protein gene of the present invention The protein gene (SEQ ID NO: 3), under the premise of not changing the amino acid sequence, partially transforms the insecticidal protein gene, selects the codons favored by plants, removes the unstable elements in the original sequence in plants, and synthesizes it through transformation The se...

Embodiment 3

[0149] Example 3 Construction of ARA and Bt Bivalent Insect-resistant Gene Plant Expression Vector

[0150] 1. Construction of expression vector driven by phloem-specific promoter BSP

[0151] 1.1 Construction of ARA gene plant expression vector pBI-ARA with Ca35S-ARA-NOS expression unit

[0152] pBI121 was single-digested with BamHI, then filled in with the large Klenow fragment, and the filled-in product was recovered; then digested with SacI, the large fragment in the digested product was recovered, and the small fragment where the GUS gene was located was discarded. Use NdeI to digest pUCm-T-ARA, then use Klenow large fragment to fill in, and recover the fill-in product; then use SacI to recover the target fragment ARA. The recovered ARA gene fragment was connected with the large fragment recovered by pBI121 enzyme digestion, and introduced into competent E.coliDH5α, and the recombinant clone was screened, and the recombinant was identified by PCR and enzyme digestion, an...

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Abstract

The invention relates to an Amaranthus retroflexus agglutinin (ARA) gene derived from Amaranthusr etroflexus L. and the gene sequence of gene sequence-Bt of Bacillus thuringiensis (Bt) parasporal crystal protein, wherein, both genes are built into the same plant expression vector; moreover, the plant expression vector of divalent insect-resistant gene is transferred to plant. In addition, the invention provides the method for obtaining transgenic plant of the genes.

Description

field of invention [0001] The present invention relates to a double amaranth agglutinin (ARA) gene derived from Amaranthus retroflexus L. and Bt (Bacillus thuringiesis, Bt for short) gene sequence derived from Bacillus thuringiensis parasporal crystal protein. Construction of plant expression vector for insect-resistant gene and method for obtaining transgenic plants. In the present invention, by cloning the nucleotide sequence of a retroverted amaranthus agglutinin gene (ARA) and the artificially synthesized Bt insecticidal protein gene, the two insecticidal genes are constructed into the same plant expression vector, and the phloem-specific The expression promoter (BSP) drives the specific expression of the ARA gene in the phloem, and the cauliflower mosaic virus (CaMV) 35S promoter drives the constitutive expression of the Bt gene in plants. It has good poisonous effect on Lepidoptera and Lepidoptera insects, and the increase of insect resistance to insecticidal protein ca...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/32C07K14/415C07K14/325C12N15/82
Inventor 刘桂珍周长生陈凌娜陈正华
Owner BEIJING CHANGLE ERSHENG GENE TECH
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