RNAi plant expression vector for inhibiting cytochrome P450 by utilizing endogenous sequence of rice and application of RNAi plant expression vector
A plant expression vector and cell-inhibiting technology, applied in the direction of cytochrome, introduction of foreign genetic material using vector, application, etc., can solve problems such as very large differences in interference efficiency, and achieve the effect of eliminating risks and worries and preventing transgene escape.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0057] The acquisition of embodiment 1 bacterial strain, plasmid and the synthesis of PCR primer
[0058] (1) Strains and plasmids
[0059] As the backbone vector, the plant binary transformation vector pCAMBIA1300-Ubi-Nos contains hygromycin resistance gene, maize Ubi promoter and NOS terminator. The strain of Escherichia coli is DH5α; the strain of Agrobacterium tumefacieus is EHA105.
[0060] (2) PCR primer sequence
[0061] Design 3 pairs of primers to amplify the DNA fragments shown in SEQ ID No.1-3 respectively. The 5' ends of the upstream and downstream primers of the two adjacent fragments have about 15 nucleotide sequences that overlap with the corresponding connection positions of the fragments or vectors. , in order to reassemble the connection using GibsonAssembly.
[0062] 450i-8-1F tgtttggtgttacttggcgcgccCGACGACATCAGAATGGGC
[0063] P450i-8-1R agcagaagatgaactcacggtaccTCGGCGGAGGACTCGTGC
[0064] P450i-8-2F gcacgagtcctccgccgaggtaccGTGAGTTCATCTTCTGCT
[0065] ...
Embodiment 2
[0068] Example 2 Construction and Transformation of RNAi Plant Expression Vector
[0069] In order to construct the interference vector of the cytochrome P450 gene CYP81A6, the rice cDNA was used as a template, and the P450i-8 gene fragment 1-3 was amplified by PCR using the primers in Example 1. The amplification system and procedures are as follows:
[0070] Program: Pre-denaturation at 94°C for 5-10min, denaturation at 94°C for 30s, annealing at 63°C for 30s, extension at 72°C for 30s, 30-35 cycles, extension at 72°C for 5min; end at 16°C.
[0071]
[0072] Program: Pre-denaturation at 94°C for 5-10min, denaturation at 94°C for 30s, annealing at 63°C for 30s, extension at 72°C for 30s, 30-35 cycles, extension at 72°C for 5min; end at 16°C.
[0073]
[0074]
[0075] The rice DNA was used as a template, and the above primers were used to amplify the P450i-8 gene fragment 2 by PCR. The amplification system and procedures are as follows:
[0076] Program: Pre-denat...
Embodiment 3
[0086] Embodiment 3 Agrobacterium transformed rice and identification of transgenic plants
[0087] The pC1300-UN-P450i-8 recombinant plasmid was introduced into the Agrobacterium EHA105 strain, and transformed into the callus of japonica rice Zhonghua 11, screened for resistance to hygromycin, differentiated, and rooted to obtain 20 regenerated transgenic lines. The transgenic plants were identified by PCR The hygromycin resistance gene transferred in the test showed that 17 PCR-positive plants and 3 weakly positive plants could be considered as negative plants ( image 3 ), the positive plants were transplanted into the soil, and 14 survived.
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More