Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV)

A technology for green mottled flowers and cloning vectors, which is applied in the field of genetic engineering, can solve problems such as economic loss of cucurbit vegetables and melons and fruits, and achieves the effects of simple operation and good repeatability

Inactive Publication Date: 2015-04-08
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the occurrence of CGMMV has been detected in Guangxi, Liaoning, Beijing, Hebei, Gansu, Shandong, Hunan, Shanghai and other regions

Method used

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  • Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV)
  • Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV)
  • Infectious clone vector of cucumber green mottle mosaic virus (CGMMV), agrobacterium strain and preparation method and application of infectious clone vector of cucumber green mottle mosaic virus (CGMMV)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1: Cloning and Determination of the Complete Genome Sequence of Cucumber Green Mottle Mosaic Virus

[0034] 1. Extraction of plant total RNA

[0035]The total RNA of diseased leaves was extracted by the Trizol method, and the specific operation was as follows: about 100㎎ fresh gourd diseased leaf samples were ground into powder in liquid nitrogen, and then 1ml Trizol was quickly added. Transfer to a 2ml sterile centrifuge tube, shake and mix vigorously to fully lyse, and place at room temperature for 5 minutes. Centrifuge at 132000rpm for 5min at 4°C. Take the supernatant, add 1 / 5 volume of chloroform, vibrate vigorously for 15 seconds, and place on ice for 2-3 minutes. Centrifuge at 132000rpm for 30min at 4°C. The bottom layer is a chloroform phase, the middle layer is a protein phase, and the upper layer is a colorless aqueous phase containing RNA. Transfer the supernatant to a new round of centrifuge tube (be careful not to inhale the protein layer), ad...

Embodiment 2

[0043] Example 2: Construction of Cucumber Green Mottle Mosaic Virus Infectious Clone pCB301-CGMMV

[0044] According to the complete sequence obtained in Example 1, primers pCass-CH-CGMMV(+), pCass-CH-CGMMV(-), CGMMV3145(+) and primer CGMMV3145(-) were designed. Using the cDNA synthesized by 3'-RACE in Example 1 as a template, respectively use the primer pair pCass-CH-CGMMV(+) / CGMMV3145(-) and the primer pair CGMMV3145(+) / pCass-CH-CGMMV(-) Two amplified CGMMV complete sequences. Using ClonExpress TM The MultiS Recombination Cloning Kit (Nanjing Nuoweizan Biotechnology Co., Ltd.) recombines the purified amplified target fragment with the pCB301-CH vector digested by Stu I and Sma I, and reacts in 10 μL system, in which 5 × CE MultiS Buffer 2μL, StuI and SmaI double digestion linearized cloning vector pCB301-CH 120ng, fragment p35SCGMMV3164 60ng, fragment pCGMMV3145-RZ 66ng, Exnase TM MultiS 1μl, and finally adjust the system to 10μL with ddH2O. Gently mix the component...

Embodiment 3

[0045] Example 3: Inoculation of pCB301-CGMMV invasive clones

[0046]Electric shock transformation of pCB301-CGMMV was introduced into Agrobacterium strain EHA105. After colony PCR verification, a single spot was picked and inoculated in YEP medium containing Kan (50 mg / L) and Rif (50 mg / L) resistance overnight at 28°C Shaking culture; then transfer to the same resistant LB medium (containing 10 mmol / L MES and 20 μmol / L acetosyringone) at a ratio of 1:100, and grow to the logarithmic growth phase (A600 value is about 0.6-0.8) , collected by centrifugation at 6000 r / min for 5 min; resuspended with sterile water containing a final concentration of 10mM MgCl2, 10mM MES (pH 5.6), and 200uM acetosyringone (Ace), and adjusted the concentration of the bacterial solution to A600 of 1.0 ; at room temperature for more than 3 hours. Use a 2ml syringe (no needle) to draw the Agrobacterium liquid for use. Select Nicotiana benthamiana with 4 to 6 true leaves at 5 to 6 weeks old or gour...

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Abstract

The invention relates to preparation of an infectious clone vector of cucumber green mottle mosaic virus (CGMMV) of RNA plant virus, namely, and tobamovirus and analysis of pathogenicity of agrobacterium strain carrying infectious clone of CGMMV. An infectious clone vector of the CGMMV is prepared by fusing the CGMMV with complete sequence into a vector pCB301-CH carrying 35S promoters and ribozyme Rz; the complete sequence of the CGMMV is shown as Seq ID No: 12. The agrobacterium strain can generate massive infectious clones of the CGMMV; the infectious clones only infect plants, rather than insects or animal and human, so that the safety can be ensured.

Description

technical field [0001] The present invention relates to the field of genetic engineering, specifically, the present invention relates to a kind of RNA plant virus-cucumber green mottle mosaic virus (Tobamovirus) of the genus Tobamovirus ( Cucumber green mottle mosaic virus , CGMMV) infectious cloning vector preparation and pathogenicity analysis of Agrobacterium strains carrying cucumber green mottle mosaic virus infectious clone. Background technique [0002] Cucumber green mottle mosaic virus belongs to the genus Tobacco mosaic virus in the family Tymoviridae Tobamovirus ) member, whose virion shape is rod-shaped, the viral genome is about 6.4kb, including 5'- and 3'-end non-coding regions and 4 open reading frames, encoding 129kDa and 186kDa replication-related proteins, 29kDa Mobile protein and coat protein of 17.4kDa. The virus seriously threatens gourds ( Lagenaria siceraria ),watermelon( Citrullus lanatus ),melon( Cucumis melo ),cucumber( Cucumis sativus Li...

Claims

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Application Information

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IPC IPC(8): C12N15/74C12N1/21C12N15/66C12Q1/70C12Q1/68C12R1/01
Inventor 燕飞郑红英肖彩利韩科雷鲁宇文林林陈剑平
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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