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Method for constructing cotton leaf curl Multan virus (CLCuMV) infectious vectors

A technology of cotton leaf curl virus and Multan cotton, which is applied in the field of genetic engineering, can solve the problems of expensive consumables, inconvenient promotion, and infecting plants, and achieve the effect of increasing applicability, small replication amount, and enhanced replication amount

Inactive Publication Date: 2012-12-12
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The first is the method of mechanical friction, which is only suitable for plant viruses that can be transmitted mechanically, such as tobacco mosaic virus and cucumber mosaic virus, and most geminiviruses cannot be infected by this method because they cannot be transmitted mechanically. dyed plants
The second is the method of gene gun bombardment, which is easy to operate, but the efficiency is low, and the whole set of equipment and consumables such as gold powder are expensive, so it is not easy to promote

Method used

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  • Method for constructing cotton leaf curl Multan virus (CLCuMV) infectious vectors
  • Method for constructing cotton leaf curl Multan virus (CLCuMV) infectious vectors
  • Method for constructing cotton leaf curl Multan virus (CLCuMV) infectious vectors

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning and Determination of the Full Genome Sequence of Multan Cotton Leaf Curl Virus DNA A and DNAβ

[0041] 1. Extraction of total DNA of susceptible cotton

[0042] Weigh 0.1 g of cotton diseased leaves, add liquid nitrogen and grind to powder, add 1 ml of cell lysate (100 mM pH8.0 Tris-Hcl, 25 mM EDTANa 2 , 2M NaCl, 2% CTAB, 2%), add β-mercaptoethanol 2.3 μl / ml) before use, continue to grind, transfer to a 2 ml centrifuge tube, incubate at 65°C for 30 minutes, add 0.4 ml chloroform, and invert gently Mix well, centrifuge at 12000 rpm at 4°C for 5 minutes, absorb the supernatant, repeat 3 times, transfer the supernatant to a 1.5 ml centrifuge tube, add an equal volume of isopropanol, and let stand at -20°C for 1 hour. Then centrifuge at 12000 rpm for 5 minutes, discard the supernatant, wash the precipitate once with 75% ethanol, discard the ethanol, volatilize the residual ethanol in a vacuum dryer, and dissolve the precipitate in 40 ul ultrapure water. ...

Embodiment 2

[0046] Example 2 Construction of Multan Cotton Leaf Curl Virus DNA A and DNAβ Infectious Vectors

[0047] 1. Construction of Invasive Clones of DNA A Component

[0048] Agrobacterium-mediated invasive cloning of geminiviruses requires a full-length genome with more than 1.1-2.0 repeats and must include the 2 common regions of geminiviruses. Using the full-length DNA A clone as a template, GXA Xba1 F (5'-CCTGAAAGATCCTGTCTAGATTTGCAT-3', SEQ ID NO:9) and GXA sal1 R as primers, the reaction parameters are: 94°C for 3 min; 94°C for 45 sec, Anneal at 55°C for 30 sec, extend at 72°C for 3 min, 35 cycles, and finally extend at 72°C for 10 min, and use the PCR product Sal I and Xba After I double digestion, use the AxgenPCR cleaning kit to clean, and the same process Sal I and Xba The digested pCambia2300 was mixed at a molar ratio of 7:1, then T4 ligated and reacted overnight at 16°C. The next day, heat-shock the ligation reaction solution to transform Escherichia coli DH5α ...

Embodiment 3

[0054] Embodiment 3. Transformation of Agrobacterium by electroporation with infectious vector

[0055] pCambia-1.9A (DH5α), pCambia-1.8β (DH5α) and pCambia-1.9A-1.8β (DH5α) were inoculated with 5 ml of LB liquid medium containing Km (50 mg / L), and cultured overnight at 200 rpm at 37°C Afterwards, the cells were collected by centrifugation, and the plasmids were extracted using the Axgen plasmid extraction kit. Agrobacterium strain EHA105 was inoculated with 5 ml of YEP liquid medium containing Rif (50 mg / L), cultured at 200 rpm at 28°C for 48 hours, discarded the supernatant culture solution after centrifugation, resuspended the bacteria with sterile water, centrifuged again, discarded The supernatant was repeated 3-4 times to obtain EHA105 competent cells.

[0056] Take 2 μL of pCambia-1.9A, pCambia-1.8β, and pCambia-1.9A-1.8β plasmids, mix them with 200 μL of Agrobacterium EHA105 competent cells, and use a Bio-rad gene pluser xcell electric shocker with a voltage of 2400V...

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Abstract

The invention relates to a method for constructing CLCuMV infectious vectors. The method includes performing infectious clone construction on a genome DNAA and satellite DNA belta with the CLCuMV serving as a raw material, 1.9 direct repeat CLCuMV full-length genome DNA A and 1.8 direct repeat satellite DNA beta are respectively or simultaneously constructed in an efficient plant expression vector pCambia2300 which has a high replication capacity through two construction strategies, and introducing recombinant vectors into an agrobacterium strain through an electric shock method to obtain the infectious vectors which are introduced by the agrobacterium and have efficient infection capabilities. According to the method, a mature method and a system are provided for studying structures and functions of virus genomes, the interaction between hosts and virus and plant functional genomes through gene silencing induced by virus.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a method for constructing an infectious vector of a cotton DNA virus. Background technique [0002] Cotton leaf curl disease is a disease that seriously endangers cotton production in the Indian peninsula. Cotton leaf curl disease is mainly caused by Gemini virus, which is transmitted by Bemisia tabaci. The typical symptoms are that the leaves of the plant curl upward or downward, the color of the veins deepens, the veins on the back of the leaves expand, and the auricles proliferate, forming cup-shaped side leaves and dwarfing the plants. , Low yield of cotton fiber, few or no knots in cotton bolls, can cause serious production reduction, or even no harvest. Cotton leaf curl Multan virus (CLCuMV) is the main pathogen that causes cotton leaf curl disease. Cotton leaf curl virus in Multan belongs to the genus Begomovirus in the family Geminiviridae. It has a typical doublet pa...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/66C12Q1/70C12Q1/68C12R1/94
Inventor 周雪平顾周杭谢艳胡高洁
Owner ZHEJIANG UNIV
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