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
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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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