Constitutive expression promoter of strawberry vein banding virus

A strawberry inlaid vein virus and constitutive expression technology, applied in the field of plant genetic engineering, can solve the problems of difficult to achieve ideal effects, low target gene expression, low transformation efficiency, etc. The effects of gene silencing

Inactive Publication Date: 2015-05-20
ANHUI AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although strawberry transgenic engineering has developed rapidly, there are still many problems: such as low transformation efficiency, weak expression of target genes, etc.
At present, the cauliflower mosaic virus (CaMV) 35S promoter is mainly used

Method used

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  • Constitutive expression promoter of strawberry vein banding virus
  • Constitutive expression promoter of strawberry vein banding virus
  • Constitutive expression promoter of strawberry vein banding virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] Example 1 Construction of full-length promoter and deletion promoter

[0058] Molecular techniques were performed essentially as described by Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY, 1989).

[0059] Cloning and sequence determination of the promoter fragment of SVBV Chinese isolate:

[0060] The fresh leaves of strawberry infected with SVBV were used as materials, and the total plant DNA was extracted by CTAB method [Murray MG et al., 1987, EMBO J, 6:3901-3907]. See the pre-amplified promoter fragments figure 1 . Using the total plant DNA as a template, the promoter fragments of SVBV Chinese isolates were amplified by specific primers.

[0061] Primers:

[0062] Full-length 984F: 5′-GGATCCGTCATCGCATATGTTCGAGACC-3′,

[0063] Full-length 984R: 5'-TCTAGAATGTAAGCAGTTAGGCCCTGTG-3';

[0064] Deletion 984△660F: 5′-GGATCCCATGGACTCCTTGACTATGTACA-3′,

[0065] Deletion 984△660R:5′-TCTAGA...

Embodiment 2

[0081] The construction of embodiment 2GUS fusion expression vector

[0082] The binary expression vector pINT121 was digested with restriction enzymes Xba I and BamH I, and the digested full-length promoter and various promoter fragments were inserted into this site to construct a promoter expression vector. The schematic diagram of the construction process is shown in figure 2 : The full-length promoter construct is pINT-984, the construct with deletion of promoter 984Δ620 is pINT-Δ660, the construct with deletion of promoter 984Δ165 is pINT-Δ165, and the construct with deletion of promoter 984Δ30 is pINT-Δ30. The vector pINT121 was used as a positive control.

Embodiment 3

[0083] Construction of embodiment 3GFP fusion expression vector

[0084] Digest the binary expression vector pCHF3 with restriction enzymes EcoR I and BamH I, insert the digested full-length promoter and various missing promoter fragments into this site, and construct a promoter expression vector. The schematic diagram of the construction process is shown in figure 2 : The full-length promoter construct is pCHF3-984, the construct with deletion of promoter 984Δ660 is pCHF3-Δ660, the construct with deletion of promoter 984Δ165 is pCHF3-Δ165, the construct with deletion of promoter 984Δ30 is pCHF3-Δ30. The vector pCHF3 was used as a positive control.

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Abstract

The invention provides a constitutive expression promoter of strawberry vein banding virus (SVBV). The promoter is a total-length promoter sequence or deficiency promoter sequence of the strawberry vein banding virus. In the constitutive expression promoter disclosed by the invention, the separated SVBV promoter and the deficiency construction product thereof can stably drive the expression of a target gene with high intensity on a rosaceous plant, a better tool is provided for the genetic engineering improvement of the rosaceous plant, and a new improvement means is provided for the whole plant genetic engineering. Moreover, a huge technical support is provided for the strawberry transgenic engineering, and a beneficial technical guarantee is provided for enriching the virus basic theory and intensifying practical application.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a constitutive expression promoter and DNA construct technology for producing transgenic plants. Background technique [0002] The purpose of plant genetic engineering is to introduce foreign genes into recipient plants and make them express correctly and effectively. However, this process is affected and restricted by many factors, and a suitable promoter is the key factor for the effective expression of foreign genes. [0003] Transgenic technology has not only become an important means to improve strawberry quality, but also opened up a new way for strawberry disease resistance breeding. Although strawberry transgenic engineering has developed rapidly, there are still many problems: such as low transformation efficiency and weak expression of target genes. The expression intensity of the target gene is the bottleneck restricting the efficiency of gene trans...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/82C12N15/84C12N5/10
Inventor 江彤李瑞杨友志李爽陈静
Owner ANHUI AGRICULTURAL UNIVERSITY
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