Plant Cas9 variant protein VQR as well as encoding gene and application thereof
A variant, plant technology, applied in the direction of plant gene improvement, application, genetic engineering, etc., can solve the problem of not fully meeting the needs of the scope of plant gene editing, and achieve the effect of expanding the scope of plant gene editing and broad application prospects.
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Embodiment 1
[0036] Embodiment 1, the acquisition of Cas9 variant VQR
[0037] Mutations were introduced at specific sites in the Cas9-encoding gene by overlap-extension PCR. The complete Cas9 needs to be divided into 3 segments with the 1135th amino acid and 1336th amino acid as the boundary, and then its coding sequence is amplified by PCR. The primers used are shown in Table 1; KODFX DNA polymerase (purchased from Toyobo Biotechnology Co., Ltd.) PCR amplified the target band, and the reaction conditions were as follows:
[0038]
[0039]
[0040] Remarks: pC1300-Cas9 can come from application number 201510485573.2.
[0041] Reaction program: 94°C, denaturation for 2 minutes; then denaturation at 98°C for 10 seconds, annealing at 58°C for 30 seconds, extension at 68°C (1000bp / min), 35 cycles of amplification; finally extension at 68°C for 5 minutes.
[0042] Sections 1, 2 and 2, 3 contain a 20bp overlapping region; the first section and the third section contain regions homologou...
Embodiment 2
[0053] Example 2, Design of GL1-1 Gene sgRNA and Construction of Recombinant Plasmid
[0054] Select the GL1-1 gene sequence "5'-ATCGCCCCTCTACCT CTGCAG GA" is the target sequence, "GGA" is the PAM sequence, and the underline is the PstI digestion recognition site. Synthesize positive and negative two oligonucleotide chains, as shown in Table 2. 100 μM g++ and g--primers (ie, Mix 20 μL each of the oligonucleotide primers (5'-3') in Table 2, place at 100°C for 5 minutes, then place at room temperature, gradually cool, denature and anneal to form fragments with sticky ends (i.e., g++ / g --primer annealing product).
[0055] The SK-gRNA was digested with AarI (the enzyme and the reagents used in the system were all purchased from Ferment Company) to form a vector with cohesive ends. The enzyme digestion reaction system was as follows:
[0056]
[0057] Remarks: SK-gRNA can come from application number 201510485573.2.
[0058] Digestion at 37°C for 3 hours, followed by purifi...
Embodiment 3
[0073] Embodiment 3, the acquisition of transgenic plants
[0074] The above-mentioned final knockout rice binary expression vector pC1300-VQR-gRNA GL1-1 The Agrobacterium strain EHA105 was transformed into Agrobacterium strain EHA105 by electric shock, and the binary expression vector was transformed into mature embryo callus of rice Nipponbare by using Agrobacterium-mediated method. The specific method of transformation is to cut out mature embryos of Nipponbare seeds after sterilization, and inoculate them into the culture medium for inducing callus. After culturing for 1 week, vigorously growing, pale yellow, relatively loose embryogenic calli were selected to be used as recipients for transformation. with pC1300-VQR-gRNA containing GL1-1 The EHA105 strain of the plasmid infects the rice callus, and after culturing at 25°C in the dark for 3 days, the resistant callus and transgenic plants are selected on the selection medium containing 50mg / L hygromycin. The transgenic ...
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