Construction and Genetic Transformation of Tobacco (Nicotiana tabacum) NtMYB4a Gene CRISPR/Cas9 Knockout Vector
JIANG Yue1, LUO Qian1, YANG Qin2, LI Hong-Qiang1, XIE Rui-Ying1, NIE Qiong1,2,*
1 College of Tobacco Science/Key Laboratory of Tobacco Quality in Guizhou Province, Guizhou University, Guiyang 550025, China; 2 College of Agriculture, Guizhou University, Guiyang 550025, China
Abstract：MYB transcription factors play an important role in plant growth and development and resistance to stress. In order to carry out the functional study of the tobacco (Nicotiana tabacum) NtMYB4a gene and the creation of a new germplasm, this study took the tobacco NtMYB4a as the target gene, selected two targets Y1 and B1 on exons 1 and 3 based on its CDS sequence, synthesized oligo dimers separately and connected with CRISPR vector, transformed Escherichia coil competent DH5α, extracted the plasmid and sequenced, and the correct Y1-1 and B1-1 were digested with LguⅠ and then recombined with T4 ligase. The CRISPR/Cas9 editing system double target knockout vector was successfully constructed, and through Agrobacterium tumefaciens mediated leaf disc method to transform tobacco. After PCR and sequencing, 29 positive plants containing Cas9 and Hyg resistance genes and target genes were obtained, and the positive rate was 24.37%. The PCR products of 29 positive tobacco strains were sequenced. Among them, 15 strains had different degrees of mutations at the target and non-target sites. There were 4 mutation types, and the mutation rate was 51.72%. The results showed that plants in the edited NtMYB4a T0 generation appeared dwarfing and flower color variation, and some even died. qRT-PCR analysis showed that the expression of NtMYB4a gene was down-regulated in the flowers, stems and leaves of tobacco plants of the T0 generation to varying degrees. This study provides basic information for the further study on the function of NtMYB4a and the molecular regulation mechanism of NtMYB4a.
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