Codon optimized Cry2Aa gene and recombinant vector as well as crop resistance changing method

A technology of codon optimization and recombinant vector, applied in microorganism-based methods, plant genetic improvement, chemical instruments and methods, etc., can solve the problems of weak insect resistance, low expression of insecticidal proteins, and difficulty in detecting mRNA.

Active Publication Date: 2014-02-12
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

However, the insect resistance of the above-mentioned transgenic plants is very weak, it is difficult to detect the transcription of mRNA, and the expression level of the insecticidal protein is very low, accounting for only 0.001% of the soluble protein by weight.

Method used

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  • Codon optimized Cry2Aa gene and recombinant vector as well as crop resistance changing method
  • Codon optimized Cry2Aa gene and recombinant vector as well as crop resistance changing method
  • Codon optimized Cry2Aa gene and recombinant vector as well as crop resistance changing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] This embodiment is used to illustrate the Cry2Aa of the present invention # Gene codon optimization method

[0041] The original Cry2Aa gene sequence is from NCBI (http: / / www.ncbi.nlm.nih.gov / ), and its accession number is AY496458. The Nipponbare genome database of rice varieties was selected as the analysis object. The website is http: / / rgp.dna.affrc.go.jp / giot / INE.html. First, screen the coding sequences of genes with high protein expression and detailed functional annotations as the analysis objects, using CodonW (http: / / mobyle.pasteur.fr / cgi-bin / portal.py?form=codonw) and SeqnConverter (http: / / www.cibj.com / seqnconverter.zip) analysis software to perform codon usage frequency analysis on the 101 complete protein coding sequences obtained through screening. The results are shown in Table 1.

[0042] Under the premise of keeping the amino acid sequence of Cry2Aa protein unchanged, the RSCU value (that is, the relative usage of synonymous codons, which refers to the ratio...

Embodiment 2

[0051] This embodiment is used to illustrate the Cry2Aa of the present invention # Gene construction method

[0052] Codon optimized Cry2Aa in Example 1 # The 5'end of the gene sequence is added with a signal peptide DNA sequence that facilitates polypeptide transport, that is, the sequence shown in SEQ ID No: 3 (the signal peptide sequence of the PR1a gene of the tobacco disease-related protein). Cry2Aa optimized above # The 3'end of the gene sequence is added with a signal peptide DNA sequence that is conducive to the localization and accumulation of polypeptides in subcellular organelles, that is, Cry2Aa optimized above # The DNA sequence of KDEL endoplasmic reticulum retention signal peptide KDEL is added to the 3'end of the gene sequence (as shown in SEQ ID No: 4). Combine into a new fusion Cry2Aa # The fusion protein Cry2Aa encoded by the gene (SEQ ID No: 2) # (SEQ ID No: 5) It can quickly transport and locate and stay in the endoplasmic reticulum and other organelles to inc...

Embodiment 3

[0054] This embodiment is used to illustrate the Cry2Aa of the present invention # Gene construction method

[0055] Cry2Aa optimized in Example 2 # The acc sequence is added to the 5'end of the gene sequence to form the Kozak characteristic sequence acccatg. In addition, the restriction enzyme SmaI recognition site sequence (cccggg) was added to the 5'end of the above-mentioned combined sequence, and the SacI recognition site sequence (gagctc) was added to the 3'end, and finally the DNA as shown in SEQ ID No: 6 was determined sequence. This sequence is a brand-new sequence, which was entrusted to Dalian Bao Biological Company to synthesize and ligate to pMD19T vector to obtain Cry2Aa # Gene pMD19T vector.

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Abstract

The invention provides a Cry2Aa# gene which is a codon optimized Cry2Aa gene. The Cry2Aa# gene has a sequence as shown in an SEQ ID No:1 and can be used for coding polypeptides having the lethal activity for insects such as Lepidoptera. In addition, the invention also provides a recombinant vector and a crop resistance changing method. The recombinant vector is characterized in that the Cry2Aa# gene is inserted in the recombinant vector which can enable the inserted Cry2Aa# gene to be expressed. The crop resistance changing method is characterized in that the recombinant vector is transformed so as to enter into crops, so that the gene inserted in the recombinant vector is expressed in the crops. Through the technical scheme, the expression quantity of insecticidal proteins is obviously increased, and the high resistance on rice pests such as cnaphalocrocis medinalis can be provided for rice.

Description

Technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a codon-optimized Cry2Aa gene, a recombinant vector and a method for changing crop resistance. Background technique [0002] Bacillus thuringiensis (Bt) is a type of bacteria that is ubiquitous in nature. Bt bacteria and Bt toxin are used as biological pesticides and have a history of more than 70 years of safe use in agriculture. In 1981, Schnepft and Whiteley successfully cloned the first gene encoding Bt insecticidal protein for the first time. So far, more than 600 insecticidal protein genes have been isolated and cloned, such as: CrylA, Cry2A, Cry1F, etc. (Chickmore, 2011 ). Since 1987, Bt genes or modified Bt genes have been successfully introduced into tobacco, corn, cotton, rice and other plants, and a large number of genetically modified plant varieties and germplasm resources with good insect resistance have been obtained. Significant social and economic benefits....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/32C07K14/325C12N15/63C12N15/82A01H5/00C12R1/07
Inventor 肖国樱翁绿水
Owner INST OF SUBTROPICAL AGRI CHINESE ACAD OF SCI
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