CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline
2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the <i>betaine aldehyde dehydrogenase 2</i> (<i>BADH2</i>) gene results in the accumulation of 2AP. However, the function of <i>BAD...
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MDPI AG
2024-02-01
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| author | Mingli Chen Siyu Shen Zhiyuan Li Huashun Wang Jin Wang Guangyu Yang Wenwu Yang Lele Deng Daping Gong Jianduo Zhang |
| author_facet | Mingli Chen Siyu Shen Zhiyuan Li Huashun Wang Jin Wang Guangyu Yang Wenwu Yang Lele Deng Daping Gong Jianduo Zhang |
| author_sort | Mingli Chen |
| collection | DOAJ |
| description | 2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the <i>betaine aldehyde dehydrogenase 2</i> (<i>BADH2</i>) gene results in the accumulation of 2AP. However, the function of <i>BADH</i> genes in tobacco (<i>Nicotiana tabacum</i> L.) remains poorly understood. In this study, we successfully obtained four <i>betaine aldehyde dehydrogenase</i> (<i>BADH</i>) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (<i>Lycium barbarum</i>) <i>BADH</i> gene (<i>LbBADH1</i>), so we named them <i>NtBADH1a</i> and <i>NtBADH1b</i>, respectively. The other two <i>BADH</i> genes were orthologues of the tomato (<i>Solanum lycopersicum</i>) <i>aminoaldehyde dehydrogenase 2</i> (<i>SlAMADH2</i>) gene, and were named <i>NtBADH2a</i> and <i>NtBADH2b</i>, respectively. Expression analysis revealed that the biological functions of <i>NtBADH1a</i> and <i>NtBADH1b</i> were different from those of genes <i>NtBADH2a</i> and <i>NtBADH2b</i>. We introduced mutations into <i>NtBADH1a</i>, <i>NtBADH1b</i>, <i>NtBADH2a</i> and <i>NtBADH2b</i> in tobacco using the CRISPR/Cas9 system and identified transgenic <i>Ntbadh</i> mutant tobacco lines. Single mutants (<i>Ntbadh1a</i>, <i>Ntbadh1b</i>, <i>Ntbadh2a</i> and <i>Ntbadh2b</i>) and double mutants (<i>Ntbadh1a-Ntbadh1b</i> and <i>Ntbadh2a-Ntbadh2b</i>) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the <i>Ntbadh2a-Ntbadh2b</i> double mutant, but not from any single mutant or the <i>Ntbadh1a-Ntbadh1b</i> double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the <i>Ntbadh2a-Ntbadh2b</i> double mutant. These findings indicate that only the combined inactivation of <i>NtBADH2a</i> and <i>NtBADH2b</i> results in 2AP accumulation in tobacco, which was not related to <i>NtBADH1</i>. |
| first_indexed | 2024-03-07T22:45:27Z |
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| series | Agronomy |
| spelling | doaj.art-562f738864504276a3a299cf00437d4e2024-02-23T15:04:10ZengMDPI AGAgronomy2073-43952024-02-0114232110.3390/agronomy14020321CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrrolineMingli Chen0Siyu Shen1Zhiyuan Li2Huashun Wang3Jin Wang4Guangyu Yang5Wenwu Yang6Lele Deng7Daping Gong8Jianduo Zhang9Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, ChinaKey Laboratory of Natural Products Synthetic Biology of Ethnic Medicinal Endophytes, State Ethnic Affairs Commission, Yunnan Minzu University, Kunming 650031, ChinaTobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, ChinaKey Laboratory of Natural Products Synthetic Biology of Ethnic Medicinal Endophytes, State Ethnic Affairs Commission, Yunnan Minzu University, Kunming 650031, ChinaYunnan Academy of Tobacco Science, Kunming 650231, ChinaYunnan Academy of Tobacco Science, Kunming 650231, ChinaYunnan Academy of Tobacco Science, Kunming 650231, ChinaYunnan Academy of Tobacco Science, Kunming 650231, ChinaTobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, ChinaYunnan Academy of Tobacco Science, Kunming 650231, China2-acetyl-1-pyrroline (2AP) is a highly effective volatile compound that gives fragrance to numerous plant species and food. Mutation(s) in the <i>betaine aldehyde dehydrogenase 2</i> (<i>BADH2</i>) gene results in the accumulation of 2AP. However, the function of <i>BADH</i> genes in tobacco (<i>Nicotiana tabacum</i> L.) remains poorly understood. In this study, we successfully obtained four <i>betaine aldehyde dehydrogenase</i> (<i>BADH</i>) genes from tobacco. Phylogenetic analysis of the protein sequences showed that two of the four BADH genes were closely related to the wolfberry (<i>Lycium barbarum</i>) <i>BADH</i> gene (<i>LbBADH1</i>), so we named them <i>NtBADH1a</i> and <i>NtBADH1b</i>, respectively. The other two <i>BADH</i> genes were orthologues of the tomato (<i>Solanum lycopersicum</i>) <i>aminoaldehyde dehydrogenase 2</i> (<i>SlAMADH2</i>) gene, and were named <i>NtBADH2a</i> and <i>NtBADH2b</i>, respectively. Expression analysis revealed that the biological functions of <i>NtBADH1a</i> and <i>NtBADH1b</i> were different from those of genes <i>NtBADH2a</i> and <i>NtBADH2b</i>. We introduced mutations into <i>NtBADH1a</i>, <i>NtBADH1b</i>, <i>NtBADH2a</i> and <i>NtBADH2b</i> in tobacco using the CRISPR/Cas9 system and identified transgenic <i>Ntbadh</i> mutant tobacco lines. Single mutants (<i>Ntbadh1a</i>, <i>Ntbadh1b</i>, <i>Ntbadh2a</i> and <i>Ntbadh2b</i>) and double mutants (<i>Ntbadh1a-Ntbadh1b</i> and <i>Ntbadh2a-Ntbadh2b</i>) harbored deletion or insertion of nucleotides, both of which led to the production of a frameshift, preventing protein accumulation. A popcorn-like scent was noticeable in tobacco leaves from the <i>Ntbadh2a-Ntbadh2b</i> double mutant, but not from any single mutant or the <i>Ntbadh1a-Ntbadh1b</i> double mutant or the wild type. Consistent with this observation, we only detected 2AP in fresh leaves from the <i>Ntbadh2a-Ntbadh2b</i> double mutant. These findings indicate that only the combined inactivation of <i>NtBADH2a</i> and <i>NtBADH2b</i> results in 2AP accumulation in tobacco, which was not related to <i>NtBADH1</i>.https://www.mdpi.com/2073-4395/14/2/321<i>betaine aldehyde dehydrogenase</i> (<i>BADH</i>)<i>Nicotiana tabacum</i>2-acetyl-1-pyrrolinetargeted mutagenesis |
| spellingShingle | Mingli Chen Siyu Shen Zhiyuan Li Huashun Wang Jin Wang Guangyu Yang Wenwu Yang Lele Deng Daping Gong Jianduo Zhang CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline Agronomy <i>betaine aldehyde dehydrogenase</i> (<i>BADH</i>) <i>Nicotiana tabacum</i> 2-acetyl-1-pyrroline targeted mutagenesis |
| title | CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline |
| title_full | CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline |
| title_fullStr | CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline |
| title_full_unstemmed | CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline |
| title_short | CRISPR/Cas9-Mediated Targeted Mutagenesis of Betaine Aldehyde Dehydrogenase 2 (BADH2) in Tobacco Affects 2-Acetyl-1-pyrroline |
| title_sort | crispr cas9 mediated targeted mutagenesis of betaine aldehyde dehydrogenase 2 badh2 in tobacco affects 2 acetyl 1 pyrroline |
| topic | <i>betaine aldehyde dehydrogenase</i> (<i>BADH</i>) <i>Nicotiana tabacum</i> 2-acetyl-1-pyrroline targeted mutagenesis |
| url | https://www.mdpi.com/2073-4395/14/2/321 |
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