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Preparation method of rice Osspear2 mutant plant
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A technology of mutants and plants, applied in the field of genetic engineering, can solve problems such as unclear functions
Active Publication Date: 2021-12-24
SHENZHEN UNIV +1
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[0005] It has been reported that the SPEAR gene plays a key role in the formation of male and female reproductive organs in Arabidopsis, but the function of the OsSPEAR family in maintaining rice fertility in rice is still unclear
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Embodiment 1
[0050]1. Log in to the website http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html, and screen the sgRNA target sequence of the rice gene SPL-like, EAR-containing protein 2 (SPEAR2: LOC_Os01g11430), the sgRNA target The dot sequence is shown in SEQ ID NO.1, specifically: 5'-TGCAGTCGAGGTCCGCCGC-3', and the PAM (protospacer adjacent motif) sequence at the 3' end of the sequence is CGC.
[0051] 2. Design the upstream primer shown in SEQ ID NO.2 and the downstream primer shown in SEQ ID NO.3 according to the sgRNA target sequence, wherein the sequence of the upstream primer shown in SEQ ID NO.2 is specifically: sgRNA-F: 5′-ggcgTGCAGTCGAGGTCCGCCGC-3′; the downstream primer sequence shown in SEQ ID NO.3 is specifically: downstream primer sgRNA-R: 5′-aaacGCGGCGGACCTCGACTGCA-3′.
[0052] 3. Mix 5 μL of upstream and downstream primers sgRNA-F and sgRNA-R (final concentration 10 μM), anneal at 65°C for 5 minutes, and slowly lower the temperature to room temperature to form complemen...
[0065] 1. After the plasmid sequence is identified correctly, transform the plasmid into Agrobacterium. The specific steps are as follows:
[0066] Thaw Agrobacterium competent GV3101 stored at -80°C on ice;
[0067] Add 4 μL of plasmid to 100 μL of competent cells, mix well, and place on ice for 5 minutes, liquid nitrogen for 5 minutes, 37°C water bath for 5 minutes, and ice bath for 5 minutes;
[0068] Add 600 μL of AB liquid medium, and incubate at 28°C, 200 rpm, in the dark for 2 hours;
[0069] Spread the above bacteria solution on AB solid medium (containing kanamycin, rifampicin, hygromycin), and culture upside down at 28°C until a single colony is formed.
[0070] 2. Transformation of callus: Transformation by Agrobacterium transformation method, the specific steps are:
[0071] 1) Seed disinfection
[0072] ①Put the mature rice seeds in 75% ethanol for 1 min after shelling, discard the ethanol,...
Embodiment 3
[0091] Screening and Identification of OsSPEAR2 Gene Mutants
[0092] 1. Use the TPS method to extract the genomic DNA of the transgenic plant, and the preparation method of the TPS extract is as shown in Table 1:
[0093] Table 1 TPS extract
[0094] Reagent 1L system 1M Tris-HCl (pH8.0) 100mL 0.5M EDTA (pH8.0) 20mL 2M KCl 500mL dd water 380mL
[0095] ①Put a small piece of rice leaf into a 2mL centrifuge tube, put in small steel balls, add 500 μL TPS extract to fully crush.
[0096] ② Place the centrifuge tube in a 70°C water bath for 30 minutes, then set it at 12000 rpm for 10 minutes.
[0097] ③ Transfer the extracted supernatant to a new centrifuge tube, add an equal volume of isopropanol, mix thoroughly and let stand for 30 minutes, then set aside at 12000 rpm for 10 minutes.
[0098] ④ Discard the supernatant, add 1mL of 75% ethanol to wash, 12000rpm, 5min.
[0099] ⑤ Discard the supernatant, dry the precipitate and add 50...
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Abstract
The invention discloses a preparation method of a rice Osspear2 mutantplant, which comprises the following steps of: screening an sgRNA target sequence of a rice gene OsSPEAR2; designing an upstream primer and a downstream primer according to the sgRNA target sequence; mixing the upstream primer and the downstream primer, and performing annealing treatment to form double-stranded DNA; performing enzymedigestion on plasmids by using restriction endonuclease to obtain linear plasmids; connecting the linear plasmids and the double-stranded DNA by using T4DNA ligase to obtain a connection product, and converting and screening the connection product to obtain recombinant plasmids; introducing the recombinant plasmids into corresponding dip-dyed bacteria to obtain dip-dyed bacteria containing the recombinant plasmids, and then infecting rice calluses by using the dip-dyed bacteria containing the recombinant plasmids; and inducing the rice calluses to obtain regenerated seedlings, and performing screening to obtain a transgenic positive plant. According to the invention, the rice Osspear2 mutant with important application value is obtained; and the Osspear2 mutant has a pollenabortion phenomenon and has important application value for the production of hybrid rice.
Description
technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a method for preparing a rice Osspear2 mutant plant. Background technique [0002] The CRISPR / CAS9 system is a genomic DNA editing technology developed in recent years. Its principle is that the nuclease Cas9 protein forms a complex with a single guide RNA (small guide RNA, sgRNA), and the sgRNA determines the specificity of the target sequence through complementary base pairing. , Cas9 protein acts as a nuclease to cutgenomic DNA complementary to sgRNA, causing double-stranded DNA damage, and then introduces gene mutations through the NHEJ (nonhomologous end joining) repair mechanism in vivo. CRISPR / Cas9 technology is simple and fast in design and synthesis, and can edit multiple genes at the same time, which doubles the efficiency of gene editing. Therefore, it has become the most important targeted gene editing technology and is widely used in various animals and ...
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