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Codon-optimized cry1ca # Genes and recombinant vectors and methods of altering crop resistance

A technology of recombining vectors and genes, applied in the field of agricultural biology, can solve the problems of low expression level of Bt insecticidal protein and poor insect resistance effect

Active Publication Date: 2017-11-17
INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a defect that overcomes the low expression level and poor insect resistance effect of the original Bt insecticidal protein in plants, and provides a Bt insecticidal protein with high expression level and good insect resistance effect in plants

Method used

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  • Codon-optimized cry1ca <sup>#</sup> Genes and recombinant vectors and methods of altering crop resistance
  • Codon-optimized cry1ca <sup>#</sup> Genes and recombinant vectors and methods of altering crop resistance
  • Codon-optimized cry1ca <sup>#</sup> Genes and recombinant vectors and methods of altering crop resistance

Examples

Experimental program
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Effect test

Embodiment 1

[0043]This embodiment is used to illustrate the Cry1Ca of the present invention # Genetic codon optimization methods.

[0044] Based on the insecticidal protein gene Cry1Ca5 of Bacillus thuringiensis, the original gene sequence of Cry1Ca5 is from NCBI (http: / / www.ncbi.nlm.nih.gov / ), and its query number is X96682. The rice variety Nipponbare Genome Database was selected as the object of analysis, and the URL is http: / / rgp.dna.affrc.go.jp / giot / INE.html. Firstly, the coding sequence of the gene with high protein expression and detailed functional annotation was screened as the analysis object, using CodonW (http: / / mobyle.pasteur.fr / cgi-bin / portal.py?form=codonw) and SeqnConverter (http: / / www.cibj.com / seqnconverter.zip) analysis software, the codon usage frequency analysis was performed on the 101 complete protein coding sequences screened, and the results are shown in Table 1.

[0045] Under the premise of keeping the amino acid sequence of the Cry1Ca5 protein unchanged, the ...

Embodiment 2

[0052] This embodiment is used to illustrate the Cry1Ca of the present invention # genetic optimization

[0053] For the Cry1Ca obtained after codon optimization in Example 1 # The gene is modified as follows: a signal peptide DNA sequence is added at the 5' end, which is beneficial to the transport of the polypeptide, that is, the sequence shown in SEQ ID No. 3 (the signal peptide sequence PR1a of the tobacco virus-related protein gene); The DNA sequence of the signal peptide localized and accumulated in the subcellular organelle, that is, the sequence shown in SEQ ID No.4 (the DNA sequence of the endoplasmic reticulum retention signal peptide KDEL), and the translation stop codon (sequence tgataa) to terminate the translation of the protein. Cry1Ca # The sequence of Cry1Ca is shown in SEQID No.2, which can encode Cry1Ca with a PR1a signal peptide at the N-terminus and a KDEL endoplasmic reticulum retention signal peptide at the C-terminus, which has lethal activity against...

Embodiment 3

[0055] This embodiment is used to illustrate the Cry1Ca of the present invention # genetic optimization

[0056] Cry1Ca optimized in Example 2 # The acc sequence is added to the 5' end of the gene sequence to form the Kozak characteristic sequence accatgg together with the existing sequence; then, the restriction endonuclease SmaI recognition sequence cccggg is added to the 5' end of the above combined sequence, and the SacI recognition sequence is added to the 3' end gagctc finally forms the DNA sequence shown in SEQ ID No.6. This sequence is a new sequence, which was synthesized by Dalian Bao Biological Co., Ltd. and connected to the pMD19-T vector to obtain a Cry1Ca # The pMD19-T vector of the gene is designated as pMD19-T-Cry1Ca.

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Abstract

The invention provides an artificially optimally designed Cry1Ca<#> gene. Specifically, the Cry1Ca<#> gene has a sequence shown as SEQ ID No.1, and can encode polypeptide with lethal activity on Lepidoptera insects. The invention also provides a recombinant vector, wherein the recombinant vector is inserted with the Cry1Ca<#> gene, and the inserted with the Cry1Ca<#> gene can be expressed. Furthermore, the invention also provides a method for improving crop resistance. The method includes transforming the recombinant vector into the crops and expressing the gene inserted into the recombinant vector in the crops. According to the technical scheme, the expression quantity of insecticidal protein is significantly improved, thus being able to providing rice high resistance to rice leaf roller and other rice pests.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a Cry1Ca artificially optimized codon design # Genes, a recombinant vector and a method of altering crop resistance. Background technique [0002] The insecticidal crystal protein encoded by the Bt gene from Bacillus thuringiensis (Bt) has specific insecticidal activity against various insects such as Lepidoptera, Diptera, Coleoptera, and Hymenoptera. Bt gene has become the most widely used and most effective insect resistance gene in plant genetic engineering and transgenic breeding. In 1981, Schnepft and Whiteley successfully cloned the first Bt insecticidal protein gene, Cry1Aa, for the first time. So far, more than 600 insecticidal protein genes have been isolated and cloned. The more in-depth study is the Cry1 gene, which encodes a 130kD insecticidal crystal protein, which is mainly toxic to Lepidoptera insects. Among them, the 134kD crystal protein encoded by the...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/325C12N15/63C12N15/82A01H5/00
CPCC07K14/325C12N15/8286
Inventor 肖国樱邓力华邓晓湘魏岁军
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI