Trichosanthes kirilowii Maxim mosaic virus, and infectious cloning vector, construction method and application thereof

A technology of cloning vectors and construction methods, applied in the field of genetic engineering, can solve problems such as differences in construction strategies and the influence of plant virus characteristics, and achieve high-efficiency infection, easy operation, and good repeatability

Pending Publication Date: 2022-04-12
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

At present, there are a series of methods for the invasive cloning of plant viruses. Due to the influence of the characteristics of plant viruses, the selected construction strategies are also different.

Method used

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  • Trichosanthes kirilowii Maxim mosaic virus, and infectious cloning vector, construction method and application thereof
  • Trichosanthes kirilowii Maxim mosaic virus, and infectious cloning vector, construction method and application thereof
  • Trichosanthes kirilowii Maxim mosaic virus, and infectious cloning vector, construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Cloning and determination of the complete genome sequence of Trichosanthes mottled mosaic virus

[0045] 1. Extraction of total RNA from diseased leaves of Trichosanthes mellifera and sequencing of small RNAs

[0046] Take 1g of the diseased leaf sample of Trichosanthes mellifera, grind it into powder in liquid nitrogen, add 10ml Trizol, vortex and oscillate to mix, let it stand at room temperature for 5min, centrifuge at 12000rpm for 20min at 4°C, take the supernatant, add 0.3 times the volume of chloroform, vortex Vortex to mix, and place at room temperature for 5 min. Centrifuge at 12000rpm for 15min at 4°C. Take the uppermost liquid phase, add an equal volume of isopropanol, invert and mix well, and let stand at room temperature for 20 minutes. Centrifuge at 12000rpm for 20min at 4°C. Remove the supernatant, and wash the precipitate twice with 70% ethanol. After the last centrifugation, centrifuge the alcohol on the tube wall to the bottom and suck it o...

Embodiment 2

[0049] Example 2 Construction of Trichosanthes mottled mosaic virus infective clone pCB301-TrMMV

[0050] According to the full sequence obtained in Example 1, primers TrMMV-insert1F, TrMMV-insert1R, TrMMV-insert2F, TrMMV-insert2R were designed; using the cDNA synthesized in Example 1 as a template, primer pairs TrMMV-insert1F / TrMMV-insert1R and The primers amplified TrMMV-insert2F / TrMMV-insert2R to obtain fragments TrMMV-insert1 and TrMMV-insert2. 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 with StuI and BamHI double enzymes, and reacts in 20 μL system, of which 5×CEⅡBuffer 4 μL , StuI and BamHI double digestion linearized cloning vector pCB301 50ng, TrMMV-insert1 and fragment TrMMV-insert2 each 70ng, 5×CEⅡBuffer 4μL, ExnaseⅡ2μL, ddH 2 O make up the system to 20 μL. Recombination reaction conditions were 37°C for 30 min. The ligation pro...

Embodiment 3

[0051] Example 3 Inoculation of pCB301-TrMMV Infectious Clones

[0052] Pick a single colony of pCB301-TrMMV (EHA105) transformed by electric shock and inoculate it in LB medium containing kanamycin (50 mg / L) and rifampicin (50 mg / L) resistance, and culture it at 28°C with shaking at 240 rpm until the OD600 is 0.5 -0.7; then collect the thalli through centrifugation at 4000rpm for 10min; use infiltration Buffer (final concentration is 10mM MgCl 2 , 10 mM MES (pH 5.6), 200 μM acetosyringone (Ace)) to resuspend, adjust the concentration of the bacterial solution to OD600 of 1.0; place at room temperature in the dark for 2 to 3 hours. Use a 1mL syringe without a needle to infiltrate about 400 μL of the bacterial suspension onto the back of the upper two leaves of Trichosanthes mellifera that have grown for three weeks. With 4-week-old Nicotiana benthamiana as the test material, 3 fully expanded upper leaves were infiltrated. For 5 species of Cucurbitaceae (melon, cucumber, wate...

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Abstract

The invention discloses a trichosanthes kirilowii mottle mosaic virus as well as an infectious cloning vector, a construction method and application thereof, the microbial preservation number of the trichosanthes kirilowii mottle mosaic virus is CGMCC NO: 21930, and the sequence of the trichosanthes kirilowii mottle mosaic virus is shown as SEQ ID NO: 15. The infectious cloning vector of the virus is prepared by fusing the complete sequence of the snake gourd mottle mosaic virus to a vector pCB301 with a 35S promoter and ribozyme Rz. After agrobacterium tumefaciens-mediated transformation, transcription, replication and infection in a host are carried out, so that the method is used for research on functional genomes of viruses. The invention provides a mature system for researching the genome structure and function of the virus and the interaction between the virus and a host.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a new virus of the genus Tobacco mosaic virus, Trichosanthes mottle mosaic virus (TrMMV) and its invasive cloning vector, construction and application. Background technique [0002] Trichosanthes is an important traditional Chinese medicine in my country. Almost all parts of Trichosanthes fruit, peel, seeds and roots have pharmacological activity. In clinical treatment, trichosanthes plays an important role in the treatment of chest cavity obstruction, angina pectoris, heart failure, myocardial infarction, cor pulmonale, cerebral ischemia and other diseases. In addition, modern pharmacological experiments have also confirmed its pharmacological effects in anti-tumor, anti-oxidation, neuroprotection, lowering blood fat, and reducing kidney damage. With the expansion of cultivation scale, Trichosanthes is vulnerable to a variety of diseases and insect pests. Previous s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/00C12N15/84C12N15/66C12N1/21C12Q1/70C12R1/01C12R1/94
CPCY02A50/30
Inventor 杨秀玲周雪平陈诚
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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