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- W2566887637 abstract "The alkaline pH level of plant vacuoles is an important factor for coloration of blue flowers. Tobacco NtPH1 gene is an orthologous gene of PhPH1 that controls the vacuolar pH in petunia petals. To study the function of NtPH1 gene, we constructed a knockout vector with two target sites of NtPH1 gene using plant CRISPR/Cas9 System. The two target sequences in NtPH1 genomic DNA are a 60 bp apart. PCR amplified the small nuclear RNA promoters (AtU6-26 and AtU6-29) and two sgRNAs, respectively. For each sgRNA cassette, the overlapping sequence between promoter and sgRNA was the target sequence of 20 bp. The overlapping PCR was completed to in vitro splice the two sgRNA cassettes. Then the two sgRNA cassettes were cloned into pYLCRISPR/Cas9Pubi-H vector by Golden Gate cloning. After positive colony PCR, restriction enzyme digestion and further sequencing, the knockout vector, pCas9-PH1T1T2, was successfully constructed. This work laid a foundation for further study on the function of NtPH1 gene, and future application of this gene in genetic engineering of blue flowers." @default.
- W2566887637 created "2017-01-06" @default.
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- W2566887637 date "2016-01-01" @default.
- W2566887637 modified "2023-09-24" @default.
- W2566887637 title "Knockout Vector Construction of Tobacco Vacuolar pH-Related NtPH1 Gene by Plant CRISPR/Cas9 System" @default.
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