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.
[1] 曹雨薇, 徐雷锋, 杨盼盼, 等. 2019. 百合花青素苷呈色类型中3种R2R3-MYBs基因的差异表达[J]. 园艺学报, 46(5): 955-963. (Cao Y W, Xu L F, Yang P P, et al.2019. Differential expression of three R2R3-MYBs genes regulating anthocyanin pigmentation patterns in Lilium spp[J]. Acta Horticulturae Sinica, 46(5): 955-963.) [2] 常振仪, 严维, 刘东风, 等. 2015. CRISPR/Cas技术研究进展[J]. 农业生物技术学报, 23(09): 1196-1206. (Chang Z Y, Yan W, Liu D F, et al.2015. Research progress on CRISPR/Cas[J]. Journal of Agricultural Biotechnology, 23(09): 1196-1206.) [3] 程丽. 2020. 苹果属垂丝海棠转录因子MhERF017和MhMYB114-like的克隆及响应缺铁的功能研究[D]. 硕士学位论文, 甘肃农业大学, 导师: 王延秀, pp. 61-77. (Cheng L.2020. Cloning of the transcription factors MhERF017 and MhMYB114-like from Malus halliana and its function in response to iron deficiency[D]. Thesis for M.S., Gansu Agricultural University, Supervisor: Wang Y X, PP. 61-77.) [4] 陈帅. 2017. 烟草类黄酮代谢途径中关键酶CHS基因与R2R3 MYB类转录抑制因子功能研究[D]. 博士学位论文, 四川农业大学, 导师: 潘光堂, 曹墨菊, 杨爱国. pp. 69-71. (Chen S.2017. Function analysis of CHS gene and R2R3 MYB repressors related to flavonoids biosynthesis pathway in Nicotiana tobacum[D]. Thesis for Ph.D., Sichuan Agricultural University, Supervisor: Pan G T, Cao M J, Yang A G. pp. 69-71.) [5] 申玉晓. 2019. 玫瑰MYB转录因子调控类黄酮介导的逆境响应机制研究[D]. 博士学位论文, 华中农业大学, 导师: 王彩云, 宁国贵, pp. 59-65. (Sheng Y X.2019. MYB transcription factors regulate flavonoid-mediated stress response in Rosa rugosa[D]. Thesis for Ph.D., HuaZhong Agricultural University, Supervisor: Wang C Y, Ning G G. pp. 59-65.) [6] 胡若琳, 袁超, 牛义, 等. 2020. 植物MYB转录因子在花药发育中的调控作用[J]. 生物工程学报, 36(11): 2277-2286. (Hu R L, Yuan C, Niu Y, et al.2020. Regulation of plant MYB transcription factors in anther development[J]. Chinese Journal of Biotechnology, 36(11): 2277-2286.) [7] 姜奇彦, 胡正, 孙现军, 等. 2020. CRISPR-Cas9基因编辑的历史[J]. 科技传播, 12(20): 1-5. (Jiang Q Y, Hu Z, Sun X J, et al.2020. The history of CRISPR-Cas9 gene editing[J]. Public Communication of Science, 12(20): 1-5.) [8] 孔晓聪, 邳瑞雪, 石雪鹭, 等. 2019. 基于CRISPR/Cas9技术的水稻OsDUF1475突变体的创建与分析[J]. 农业生物技术学报, 27(3): 393-401. (Kong X C, Qi R X, Shi X L, et al.2019. Obtainment and characterization of rice (Oryza sativa) OsDUF1475 mutants based on CRISPR/Cas9 technique[J]. Journal of Agricultural Biotechnology, 27(3): 393-401.) [9] 李星坤, 潘慧, 李攀, 等. 2020. 基于CRISPR/Cas9系统的拟南芥ugt84a1/ugt84a2双突变体制作及突变位点分析[J]. 江苏农业科学, 48(20): 49-55. (Li X K, Pan H, Li P, et al. 2020. Production and mutation site analysis of arabidopsis ugt84a1/ugt84a2 double mutant based on CRISPR/Cas9 system[J]. Jiangsu Agricultural Sciences48(20): 49-55.) [10] 李君霞, 代书桃, 陈宇翔, 等. 2020a. MYB转录因子在植物抗旱基因工程中的应用进展[J]. 河南农业科学, 49(11): 1-9. (Li J X, Dai S T, Chen Y X, et al.2020a. Progress on application of MYB transcription factor in plant drought tolerance genetic engineering[J].Journal of Henan Agricultural Sciences, 49(11): 1-9.) [11] 李君霞, 王春义, 丁宇涛, 等. 2020b. MYB转录因子在植物耐盐基因工程中的应用进展[J]. 浙江农业学报, 32(10):1910-1920. (Li J X, Wang C Y, Ding Y T, et al.2020b. Progress on application of MYB transcription factor in plant salt tolerance genetic engineering[J]. Acta Agriculturae Zhejiangensis, 32(10): 1910-1920.) [12] 李艳婷. 2020. 转录因子CsMYB77遗传转化功能验证及其互作基因挖掘[D]. 硕士学位论文, 华中农业大学, 导师: 伊华林. pp. 38-44. (Li Y T.2020. Verification of genetic transformation function of transcription factor CsMYB77 and their interaction gene excavation[D]. Thesis for M.S., Huazhong Agricultural University, Supervisor: Yi H L, PP. 38-44.) [13] 刘壮斌. 2019. 低温胁迫下番茄MYB113基因的功能分析[D]. 硕士学位论文, 山东农业大学, 导师: 孟庆伟, 马娜娜. pp. 58-67. (Liu Z B.2019. Functional analysis of MYB113 gene in tomato under chilling stress[D]. Thesis for M.S., Shandong Agricultural University, Supervisor: Meng Q W, Ma N N. PP. 58-67.) [14] 卿冬进, 邓国富, 戴高兴, 等. 2020. 利用CRISPR/Cas9技术获得水稻恢复系Bsr-d1基因突变体[J]. 分子植物育种, 18(16): 5343-5350. (Qing D J, Deng G F, Dai G X, et al.2020. Construction of the Bsr-d1 gene mutants in rice restorer line by CRISPR/Cas9 technology[J]. Molecular Plant Breeding, 18(16): 5343-5350.) [15] 田茂竹. 2019. 应用CRISPR/Cas9技术定向突变烟草CYP71D16基因的研究[D]. 硕士学位论文, 贵州大学, 导师: 刘仁祥. pp. 45-57. (Tian M Z.2019. Study on the gene of CYP71D16 directed mutation by CRISPR/Cas9 technology in tobacco[D]. Thesis for M.S., Guizhou University, Supervisor: Liu R X. PP. 45-57.) [16] 卫秋慧. 2017. 小麦Myb转录因子基因TaODORANT1和TaMyb1D的克隆及功能分析[D]. 博士学位论文, 华中科技大学, 导师: 杨广帅, pp. 100-108. (Wei Q H.2017. Identification and functional characterization of Myb transcription factor genes TaODORANT1 and TaMyb1D in wheat[D]. Thesis for Ph.D., Huazhong University of Science and Technology, Supervisor: Yang G S, pp. 100-108.) [17] 王中, 赵利杰, 刘萍萍, 等. 2021. 烟草NtMYB59基因克隆及过表达对绿原酸含量的影响[J/OL]. 烟草科技: 1-13. (Wang Z, Zhao L J, Liu P P, et al.2021. Cloning and over-expressing tobacco NtMYB59 gene and the effects on chlorogenic acid content[J/OL]. Tobacco Science & Technology: 1-13.) [18] 颜复林. 2020. 转录因子GhMYB201的功能分析[D]. 硕士学位论文, 西南大学, 导师: 肖月华. pp. 45-69. (Yan F L.2020. Function analysis of transcription factor GhMYB201[D]. Thesis for M.S., Southwest University, Supervisor: Xiao Y H. pp. 45-59.) [19] 杨柳, 李晓峰, 祝万万, 等. 2020. 利用CRISPR-Cas9基因编辑技术获得水稻OsMADS56基因突变体[J]. 分子植物育种, 18(11): 3571-3578. (Yang L, Li X F, Zhu W W, et al.2020. Generation of OsMADS56 mutants in rice using CRISPR/Cas9 editing approach[J]. Molecular Plant Breeding, 18(11): 3571-3578.) [20] 杨传凤. 2015. 基于慈竹转录组MYB转录因子克隆及其遗传转化研究[D]. 硕士学位论文, 西南科技大学, 导师: 胡尚连. pp. 103-104. (Yang C F.2015. Cloning and transformation of MYB transcription factors based on Bambusa emeiensis transcriptome[D]. Thesis for M.S., Southwest University of Science and Technology, Supervisor: Hu S L. pp.103-104.) [21] 赵雪. 2020. 转录因子MYC2和MYB305对烟碱合成基因的表达调控研究[D]. 硕士学位论文, 中国农业科学院, 导师: 张洪博, pp. 35-47. (Zhao X.2020. Roles of MYC2 and MYB305 in regulating nicotine synthesis genes[D]. Thesis for M.S., Chinese Academy of Agricultural Sciences, Supervisor: Zhang H B, pp. 35-47.) [22] 赵重燕, 暴会会, 仵亚汝, 等. 2020. 烟草atp2基因CRISPR/Cas9载体构建与遗传转化[J/OL]. 分子植物育种, 1-11. (Zhao C Y, Bao H H, Wu Y R, et al.2020. RNAi vector construction of atp2 gene and genetic transformation in tobacco[J/OL]. Molecular Plant Breeding, 1-11.) [23] 周瑜宇. 2020. 烟草转录因子NtBRC1b的功能研究[D]. 硕士学位论文, 西南大学, 导师: 程道军, pp. 50-65. (Zhou Y Y.2020. Study on the function of transcription factor NtBRC1b in Nicotiana tabacum[D]. Thesis for M.S., Southwest University, Supervisor: Cheng D J, PP. 50-65.) [24] Ambawat S, Sharma P, Yadav R N, et al.2013. MYB transcrip-tion factor genes as regulators for plant responses: An overview[J]. Physiology and Molecular Biology of Plants, 19(3): 307-321. [25] Chen X, Li M H, Ni J, et al.2021. The R2R3-MYB transcription factor SsMYB1 positively regulates anthocyanin biosynthesis and determines leaf color in Chinese tallow (Sapium sebiferum Roxb)[J]. Industrial Crops and Products, 164. DOI: 10.1016/J.INDCROP.2021.113335 [26] Ito M.2005. Conservation and diversification on there-repeat Myb transcription factors in plants[J]. Jouranl of Plant Research, 118(1): 61-69. [27] Jinek M, Chylinski K, Fonfara I, et al.2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity[J]. Science, 337(6096): 816-821. [28] Kirik V, Kolle K, Misera S, et al.1998. Two novel MYB homologues with changed expression in late embryog enesis-defective a rabidopsis mutants[J]. Plant, 37(5): 819-827. [29] Ma X L, Zhang Q Y, Zhu Q L, et al.2015. A robust CRISPR/Cas9 system for convenient, high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant, 8(8): 1274-84. [30] Yao P F, Huang Y J, Dong Q X, et al.2020. FtMYB6, a light-induced SG7 R2R3-MYB transcription factor, promotes flavonol biosynthesis in tartary buckwheat (Fagopyrum Tataricum)[J]. Journal of Agricultural and Food Chemistry, 68(47): 13685-13696. [31] Prashant M, Luhan Y, Kevin M E, et al.2013. RNA-guided human genome engineering via Cas9[J]. Science, 339(6121): 823-826. [32] Pattanaik S, Que K, David Zaitlin, et al.2010. Isolation and functional characterization of a floral tissue-specific R2R3 MYB regulator from tobacco[J]. Planta, 231(5): 1061-1076.
