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Application of hypovirulence CMV vector in expression of pest-resistant gene and enhancement of pest resistance of plant

An insect-resistant gene and virulence technology, which is applied to the application field of cucumber mosaic virus attenuated virulence vector in enhancing plant insect resistance, can solve problems such as no report on enhancing plant insect resistance, insect RNA interference and the like

Inactive Publication Date: 2016-08-31
HUNAN UNIV OF HUMANITIES SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0026] In summary, we found that the application of plant virus vectors in enhancing plant insect resistance is only RNA interference to insects. Cucumber mosaic virus vectors have no reports on enhancing plant insect resistance. Cucumber mosaic virus Attenuated vectors have not been reported to enhance plant insect resistance by expressing insect-resistant genes

Method used

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  • Application of hypovirulence CMV vector in expression of pest-resistant gene and enhancement of pest resistance of plant
  • Application of hypovirulence CMV vector in expression of pest-resistant gene and enhancement of pest resistance of plant
  • Application of hypovirulence CMV vector in expression of pest-resistant gene and enhancement of pest resistance of plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0143] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence; specifically, the steps are as follows:

[0144] (1) Design forward and reverse primers using CMV sequence as a template.

[0145] Primer NcoIF2:CATG with Nco I restriction site Ccatgg ctgagtttgcctg;

[0146] Primer 2aORF1R:ga with Stu I restriction site AGGCCT T CTAs aattctttcgctgtttgttgg.

[0147] Using the plasmid pCB-CMVF209 as a template, the PCR product was amplified and synthesized, and the PCR product was digested with Nco I and Stu I.

[0148] Purify and reserve.

[0149] The PCR amplification system is:

[0150]

[0151] The PCR amplification program is:

[0152]

[0153]

[0154] Note: 1. Plasmid pCB-CMVF209 is an invasive clone of CMVF209 sequence. For the specific construction method, see Chinese Agricultural Sciences 2011, 44(14): 3060-3068).

[0155] (2) Design the forward primer 2bORF333F:GA with enzyme cutting sites Stu I, Mlu I, BamH I AG...

Embodiment 2

[0179] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence.

[0180] (1) Design forward and reverse primers using CMV sequence as a template.

[0181] Forward primer NcoIF2 with enzyme cutting site Nco I: CATGCcatggctgagtttgcctg;

[0182] Reverse primer 2aORF1R:gaAGGCCTT with enzyme cutting site Stu I CTAs aattctttcgctgtttgttg.

[0183] 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.

[0184] (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.

[0185] (3) Plasmid pCB-CMVF209 w...

Embodiment 3

[0201] 1. Transform the virus CMV into a weak virus expression vector that lacks the 2b gene sequence.

[0202] (1) Design forward and reverse primers using CMV sequence as a template.

[0203] Forward primer NcoIF2 with enzyme cutting site Nco I: CATGCcatggctgagtttgcctg;

[0204] 2aORF1R:gaAGGCCTT with restriction site Stu I CTAs aattctttcgctgtttgttg.

[0205] 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.

[0206] (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.

[0207] (3) Plasmid pCB...

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Abstract

The invention discloses an application of a cucumber mosaic virus (CMV) hypovirulence vector in enhancement of pest resistance of a plant. A construction method of the CMV hypovirulence vector comprises the following steps: 1), transforming a CMV into a hypovirulence expression vector having a deleted 2b gene sequence, particularly, transforming one of constructed CMV infectious cloning plasmids, namely pCB-CMVF209, by an enzyme digestion connection method, making the 2b gene deleted, and adding an enzyme digestion site, and constructing to obtain the hypovirulence virus expression vector-hypovirulence vector; and 2), inserting a pest-resistant gene encoding region (ORF) sequence into the hypovirulence vector obtained in the step 1), and constructing a recombinant hypovirulence expression vector; and applying the constructed recombinant hypovirulence expression vector containing the pest-resistant gene encoding region sequence in enhancement of the pest resistance of the plant.

Description

technical field [0001] The invention relates to the application of virus vector, especially the application of cucumber mosaic virus (cucumber mosaic virus, CMV) attenuated vector in enhancing plant insect resistance. 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...

Claims

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Application Information

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IPC IPC(8): C12N15/83C12N15/84A01H5/00
CPCC12N15/8205C12N15/8203C12N15/8286
Inventor 竺锡武吴娟王强刘津李晓超王青陈亚妮
Owner HUNAN UNIV OF HUMANITIES SCI & TECH
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