Functional Identification of Cotton U3 and U6 Promoters in the CRISPR/Cas9 Genome Editing System |
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| Authors: | Zang Xuyang Dai Peihong Li Jiyang Pu Yan Gu Aixing Liu Xiaodong |
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| Institution: | College of Agronomy, Xinjiang Agricultural University, Laboratory of Agricultural Biotechnology of Xinjiang Agricultural University, Urumqi, Xinjiang 830052, China |
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| Abstract: | Objective] The function of U3 and U6 promoters that were cloned from sea-island cotton were identified in order to provide more available U3 and U6 promoters for the construction of cotton CRISPR/Cas9 multi-sites gene editing system. Method] Two CRISPR/Cas9 gene editing vectors were constructed, in which sgRNA were driven by GbU6-7P and GbU3-2P, respectively, and GGB, a negative regulator in drought tolerance, was used as target gene. The function of the vector were identified in cotton leaf protoplast of Xinhai 16. The core fragment of CRISPR/Cas9 gene editing vector were enriched by Polymerase chain reaction method and were delivered into protoplast through PEG transient transformation. Then genomic DNA were extracted from protoplast. Gene mutations were analyzed using Enzyme digest/Polymerase chain reaction and sequenced. Finaly the mutation efficiencies of the CRISPR/Cas9 system were calculated and the frequency distribution of the mutation in target site were drawn in order to confirm the authenticity of the mutation. Result] All type of mutation in target loci were base substitution. Conclusion]Both CRISPR/Cas9 gene editing systems based on GbU3-2P and GbU6-7P promoters could successfully edit the sequence of cotton GGB gene and cause gene mutation. |
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| Keywords: | cotton CRISPR/Cas9 genome editing U3/U6 promoter protoplast |
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