Infectious cloning vector of tomato brown wrinkled fruit virus and its construction method and application

A cloning carrier, brown technology, applied in the field of genetic engineering, can solve the problems of strong infectivity, long survival period, affecting yield and quality, etc., and achieve the effect of good repeatability and simple operation

Active Publication Date: 2022-04-19
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The virus seriously threatens the production of tomato, pepper and other solanaceous crops. The virus can cause mosaic and narrow tomato leaves and yellow spots on the fruit surface, seriously affecting yield and quality
Because ToBRFV can overcome the Tm-22 resistance gene in tomato varieties, there are currently no tomato and pepper varieties resistant to this virus, and ToBRFV has a long survival period in vitro, strong infectivity, and is easy to spread through seeds and contact

Method used

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  • Infectious cloning vector of tomato brown wrinkled fruit virus and its construction method and application
  • Infectious cloning vector of tomato brown wrinkled fruit virus and its construction method and application
  • Infectious cloning vector of tomato brown wrinkled fruit virus and its construction method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Cloning and determination of the full genome sequence of tomato brown wrinkled fruit virus

[0038] 1. Extraction of plant total RNA

[0039] The total RNA of diseased leaves was extracted by the Trizol method, and the specific operations were as follows:

[0040] Grind about 0.05-0.1g of fresh tomato diseased leaf samples into powder in liquid nitrogen, then quickly add 1ml Trizol, shake vigorously and mix well to fully lyse, and place at room temperature for 5min. Take the supernatant, add 0.3mL chloroform, shake the grinder vigorously for 300s, and place it at room temperature for 20min. Centrifuge at 12000rpm for 15min 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 1.5mL centrifuge tube (be careful not to inhale the protein layer), add an equal volume of isopropanol, invert and mix, and place for 5min. Centrifuge at 1...

Embodiment 2

[0043] Example 2 Construction of tomato brown wrinkled fruit virus infective clone pCB-ToBRFV

[0044] According to the complete sequence obtained in Example 1, primers pCB-ToBRFV-1F, pCB-ToBRFV-1R, pCB-ToBRFV-2F and primer pCB-ToBRFV-2R were designed. Using the cDNA synthesized in Example 1 as a template, the entire ToBRFV sequence was amplified in two segments with the primer pair pCB-ToBRFV-1F / pCB-ToBRFV-1R and the primer pair pCB-ToBRFV-2F / pCB-ToBRFV-2R respectively. use II One Step Cloning Kit Recombination Cloning Kit (Nanjing Nuoweizan Biotechnology Co., Ltd.) recombines the purified amplified target fragment with the pCB301 vector digested by Stu I and SmaI, and reacts in 20 μL system, in which 5×CEⅡBuffer 4 μL, StuI and SmaI double digestion linearization cloning vector pCB301150ng about, fragment ToBRFV-1 and fragment ToBRFV-2 about 80ng each, ExnaseⅡ2μL, ddH 2 O adjust the system to 20 μL. Gently mix the components by pipetting up and down several times. Place ...

Embodiment 3

[0045] Embodiment 3 pCB-ToBRFV invasive clone inoculation

[0046] Electric shock transformation of pCB-ToBRFV was introduced into Agrobacterium strain EHA105, and a single spot was picked and inoculated in LB medium containing kanamycin (50 mg / L) and rifampicin (50 mg / L) resistance and cultured overnight at 28°C with shaking; Then centrifuge at 4000rpm for 10min to discard the supernatant and collect the bacteria; 2 , 10mM MES (pH 5.6), 200μM acetosyringone (Ace) in infiltration buffer to resuspend, adjust the bacterial solution concentration to OD600 of 1.0; place at room temperature in the dark for 2-3 hours. Use a 2ml syringe (without a needle) to draw the Agrobacterium liquid for use. Select 4-6 true leaves of Nicotiana benthamiana, lightly poke a few small holes on the back of the leaves with a needle, and then slowly infiltrate and inject the bacterial solution into the interstitial space. Use a 2ml syringe (with a needle) to draw the Agrobacterium liquid, and inject ...

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Abstract

The invention discloses an infectious cloning vector of tomato brown wrinkled fruit virus and its construction method and application. On the basis of obtaining the complete genome sequence of tomato brown wrinkled fruit virus, an infectious clone of the virus is constructed, which will contain The cDNA sequence of tomato brown wrinkled fruit virus from the 35S promoter was constructed into the binary vector pCB301, and transcribed, replicated, and infected in the host after transformation mediated by Agrobacterium, so as to be used for functional genome research of the virus. The method of the invention provides a mature system for studying the structure and function of the virus genome and the interaction between the virus and the host.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to construction of an invasive clone of a positive-sense single-stranded RNA virus, Tobacco mosaic virus, and Tomato brown rugrose fruit virus (ToBRFV). Background technique [0002] Tomato brown wrinkled fruit virus is a member of the genus Tobamovirus, and its virion is rod-shaped. The ToBRFV genome is a single-stranded positive-sense RNA containing 6390 nucleotides, including 5' and 3' non-coding regions and Four open reading frames encode replication-associated proteins of 126kDa and 183kDa, mobile proteins of 29.8kDa and coat proteins of 17.5kDa, respectively. The virus seriously threatens the production of tomato, pepper and other Solanaceae crops. The virus can cause mosaic and narrowing of tomato leaves and yellow spots on the fruit surface, seriously affecting yield and quality. Because ToBRFV can overcome the Tm-22 resistance gene in tomato varieties, there ar...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12N15/40C12N1/21C12N15/66C12R1/01
CPCC12N15/82C12N15/66C07K14/005C12N2770/14022
Inventor 杨秀玲周雪平马子玥
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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