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Application of attenuated CMV (cucumber mosaic virus) vector expression resistance gene in enhancing plant herbicide resistance

A herbicide-resistant, herbicide-resistant gene technology, used in applications, plant products, genetic engineering, etc.

Inactive Publication Date: 2016-09-07
HUNAN UNIV OF HUMANITIES SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is no report on the application of cucumber mosaic virus vector in enhancing the herbicide resistance of plants, and there is no report on the attenuated vector of cucumber mosaic virus in enhancing the herbicide resistance of plants by expressing herbicide resistance genes

Method used

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  • Application of attenuated CMV (cucumber mosaic virus) vector expression resistance gene in enhancing plant herbicide resistance
  • Application of attenuated CMV (cucumber mosaic virus) vector expression resistance gene in enhancing plant herbicide resistance
  • Application of attenuated CMV (cucumber mosaic virus) vector expression resistance gene in enhancing plant herbicide resistance

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0083] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence; specifically, the steps are as follows:

[0084] (1) Design forward and reverse primers using CMV sequence as a template.

[0085] NcoIF2 with enzyme cutting site Nco I: CATGCcatggctgagtttgcctg;

[0086] 2aORF1R:gaAGGCCTT with restriction site Stu I CTAs aattctttcgctgtttgttgg.

[0087] The plasmid pCB-CMVF209 was used as a template to amplify the PCR product to synthesize the PCR product. The PCR product was digested with Nco I and Stu I, purified and set aside.

[0088] The PCR amplification system is:

[0089]

[0090] The PCR amplification program is:

[0091]

[0092] Note: Plasmid pCB-CMVF209 is an invasive clone of CMVF209 sequence. For specific construction methods, see Chinese Agricultural Sciences 2011, 44(14): 3060-3068.

[0093] (2) Design the forward primer 2bORF333F:GA with enzyme cutting sites Stu I, Mlu I, BamH I AGGCCT G ACGCGT GACTAGTGGATCCAACc...

Embodiment 2

[0113] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence.

[0114] (1), using the CMV sequence as a template to design forward and reverse primers. NcoIF2 with restriction site Nco I: CATGCcatggctgagtttgcctg; 2aORF1R with restriction site Stu I: gaAGGCCTT CTAs aattctttcgctgtttgttg. The plasmid pCB-CMVF209 was used as a template to amplify the PCR product to synthesize the PCR product. The PCR product was digested with Nco I and Stu I, purified and set aside.

[0115] (2), design the forward primer 2bORF333F:GA with enzyme cutting sites Stu I, Spe I, Apa I, BamH I AGGCCT GGACTAGT gggcccG GGATCCAACctccccttccgcatct; reverse primer 2bAvrIIR: Cttccgaagaaacctagag. The plasmid pCB-CMVF209 was used as a template to amplify the PCR product to synthesize the PCR product, and the PCR product was digested with Stu I and AvrII, purified and set aside.

[0116] (3) Plasmid pCB-CMVF209 was digested with Nco I and Avr II for later use.

[011...

Embodiment 3

[0130] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence.

[0131] (1) Design forward and reverse primers using CMV sequence as a template. NcoIF2 with restriction site Nco I: CATGCcatggctgagtttgcctg; 2aORF1R with restriction site Stu I: gaAGGCCTT CTAs aattctttcgctgtttgttg. Using the plasmid pCB-CMVF209 as a template, PCR amplifies and synthesizes the target gene sequence, purifies the PCR product, digests the PCR product with Nco I and StuI, purifies it, and sets aside.

[0132] (2) Design the forward primer 2bORF333F:GA with enzyme cutting sites Stu I, Mlu I, Spe I, Apa I, BamH I, Sac II AGGCCT G ACGCGT GACTAGT gggcccG GGATCCccgcggAACctccccttccgcatct; reverse primer 2bAvrIIR: Cttccgaagaaacctagag. The plasmid pCB-CMVF209 was used as a template to amplify the PCR product to synthesize the PCR product. The PCR product was digested with Stu I and Avr II, purified and set aside.

[0133] (3) Plasmid pCB-CMVF209 was digested wi...

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Abstract

The invention discloses an application of a cucumber mosaic virus (CMV) attenuated vector expression herbicide-resistant gene in an aspect of enhancing plant herbicide resistance. A construction method of a CMV attenuated vector comprises the following steps: 1) transforming the virus CMV into an attenuated expression vector with a 2b gene sequence deleted, specifically transforming one infectious cloning plasmid, namely pCB-CMVF209, of the constructed virus CMV by virtue of a method of enzyme digestion connection, so that a 2b gene becomes deleted, and adding an enzyme digestion site, so that an attenuated virus expression vector, namely the attenuated virus vector, is constructed; and 2) inserting the sequence of a herbicide-resistant gene coding region (ORF) into the attenuated virus vector obtained in the step 1), so that a recombinant virus expression vector is constructed, wherein the constructed recombinant attenuated expression vector containing the sequence of the herbicide-resistant gene coding region is used for enhancing the resistance of plants to a herbicide.

Description

technical field [0001] The invention relates to the application of virus vector, in particular the application of cucumber mosaic virus attenuated vector in enhancing plant resistance to herbicides. Background technique [0002] The existing research and development of plant virus vectors can be divided into three purposes: one is mainly used to express medicinal proteins, antibodies, etc.; the other is used to analyze or utilize the function of foreign genes; the third is used for virus-induced gene silencing analysis Function of the silenced gene. In terms of the use of viral vectors for the expression of foreign proteins, the purpose of early research and development of viral vectors was mainly for the expression of foreign proteins. There are two main methods for expressing foreign genes using plant virus vectors: one is to transcribe the invasive cDNA clone carrying the target gene in vitro, and to carry out gene substitution, insertion and fusion of the viral genome c...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/84A01H5/00
CPCC12N15/8205C12N15/8203C12N15/8274
Inventor 竺锡武彭日民王强张媛媛吴娟
Owner HUNAN UNIV OF HUMANITIES SCI & TECH
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