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Cunninghamia virus induced gene silencing system and construction method thereof

A technology of gene silencing and construction methods, applied in the field of genetic engineering of forest trees, can solve problems such as few tests

Active Publication Date: 2019-03-01
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Virus-induced gene silencing (VIGS) has proven to be an effective tool for studying gene function in herbaceous plant species, but has rarely been tested on forest trees

Method used

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  • Cunninghamia virus induced gene silencing system and construction method thereof
  • Cunninghamia virus induced gene silencing system and construction method thereof
  • Cunninghamia virus induced gene silencing system and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] The cultivation of embodiment 1 Chinese fir plant material

[0030] Choose fir seeds with plump particles and uniform texture, soak them in clear water for three days, and remove the shriveled and incomplete seeds floating on the water. Planted in the environment of nutrient soil ratio vermiculite 2:1 under greenhouse conditions, after the seeds are evenly spread on the soil, cover 2cm of soil with the same texture. After culturing for 30 days, the seeds opened and the cotyledons of the pods were fully stretched to 3 cm before carrying out the post-infestation treatment.

Embodiment 2

[0031] Cloning of embodiment 2 target gene fragment

[0032] (1) Extraction of Chinese fir genomic DNA

[0033] Cut the tender leaves at the growth point of the green and strong branches of fir, wrap them in tin foil and store them in liquid nitrogen, and then use a crusher to crush them. The whole process is in a liquid nitrogen environment, and then divide the crushed powder into centrifuge tubes.

[0034] Genomic DNA of Chinese fir plant was extracted according to instructions of TIANGEN Company's Plant Genomic DNA Extraction Kit Spin Column Type Catalog No. DP305. Specific steps are as follows:

[0035] 1) Add 700 μL of drug GP1 preheated in a 65°C water bath to the centrifuge tube (confirm that the volume percentage concentration of mercaptoethanol is 0.1%), then use a vortex shaker to mix well, and then place the centrifuge tube in a 65°C water bath Heat in a water bath for 20 minutes. Mix up and down in the water bath to facilitate full dissolution.

[0036] 2) Add ...

Embodiment 3

[0067] Example 3 Construction of Recombinant Virus Plasmid

[0068] (1) Use the gel-recovered product with the correct band size for BP reaction. Gateway® BP Clonase™ IIEnzyme mix was purchased from Baiquan Bio. The reaction system is shown in Table 2.

[0069] Table 2 Gateway® BP Clonase Reaction System

[0070]

[0071] Add the sample into a 200 μL centrifuge tube, then use a vortex to mix, and store in an electric heater at 25°C for 8 hours to obtain the BP product.

[0072] (2) Transformation of DH5α competent cells by heat shock of the ligated product: Transformation was carried out according to the instructions of Shanghai Weidi Biotechnology Co., Ltd. model DL1001 competent cell DH5α, and the positive clone was verified by amplifying the target fragment by bacterial liquid PCR. The specific steps are:

[0073] 1) After taking out the DH5α competent cells from the -80°C refrigerator, immediately put them in the ice box and let them stand for 5 minutes. Use a pipett...

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Abstract

The invention provides a Cunninghamia virus induced gene silencing system and a construction method thereof and belongs to the field of forest gene engineering. The construction method includes the steps of cultivating a Cunninghamia plant material; cloning a target gene fragment; constructing a recombinant viral plasmid vector; infecting the Cunninghamia plant with an Agrobacterium-mediated silencing vector; verifying silencing effect results. The system and its construction method are suitable for studying the functional mechanism of a target gene according to changes in plant phenotype; theCunninghamia VIGS (virus induced gene silencing) system is of pioneer significance to the research on functional genes of forest gymnosperms.

Description

technical field [0001] The invention belongs to the field of tree genetic engineering, and in particular relates to a Chinese fir virus-induced gene silencing system and a construction method thereof. Background technique [0002] Chinese fir( Cunninghamia lanceolata ) of the Chinese fir family, the genus Chinese fir, is a unique forest species that mainly grows in southern my country. Chinese fir has high economic value, its tree body is straight, its wood structure is uniform, and its material is hard. Chinese fir is valuable in many ways, and it is a good building material. It is also widely used in shipbuilding and furniture manufacturing. However, the stable genetic transformation system of forest trees is difficult, and there is no relevant report so far. Therefore, it is of great significance to develop a fast and effective technical system for the study of gene functions of Chinese fir. [0003] Virus-Induced Gene Silencing (VIGS) is a very useful research tool fo...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12Q1/6851C12Q1/6895A01H5/00A01H6/00
CPCC12N9/001C12N15/8218C12N15/825C12Q1/6851C12Q1/6895C12Y103/05005C12Q2600/158C12Q2545/101C12Q2531/113
Inventor 顾连峰马祥庆高鹏飞席飞虎魏文桃陈凯胡凯强刘博张航晓赵良真
Owner FUJIAN AGRI & FORESTRY UNIV
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