Study on the Application of CRISPR/Cas9 Technology in Development of Tomato (Solanum lycopersicum) Male Sterile Line
LIU Yu-Chen1,2, QIU Shi-Jun1,2, JIN Man2, DENG Han-Chao2,3, YIN Mei2, CHEN Zhu-Feng2, ZHOU Xiang-Yang3,*, TANG Xiao-Yan1,2,*
1 College of Life Science, South China Normal University, Guangzhou 510631, China; 2 Shenzhen Institute of Molecular Crop Design, Shenzhen 518110, China; 3 Shenzhen Agricultural Science and Technology Promotion Center, Shenzhen 518055, China
Abstract:Utilization of heterosis is an important breeding method, which can greatly improve the yield, disease resistance and stress resistance of tomato (Solanum lycopersicum). The application of male sterile line can reduce the workload of artificial emasculation and it is increasingly adopted by seed companies. However, breeding of male sterile lines is usually time-consuming, which limits the application of male sterile lines. The application of modern gene editing technique in important genes related to stamen development would help better understand the underlying molecular mechanism and should shorten the breeding period of tomato GMS (genetic male sterility) lines. Tomato SlAP3 (Solanum lycopersicum APETALA3) is a homologous gene of AP3 in Arabidopsis thaliana regulating floral organ development. Mutation of this gene causes abnormal stamen development in tomato, resulting in male sterile phenotype. This study used CRISPR/Cas9 genome editing system to create mutation on SlAP3. Two target sites with PAM (protospacer adjacent motifs) sequence were selected from the exon of SlAP3, and CRISPR/Cas9 expression vectors were constructed. Transgenic tomato plants were generated using Agrobacterium tumefaciens. The DNA sequences around the 2 target sites were amplified and sequenced. The flower organ phenotype in gene edited plants was observed. Twenty and eighteen transgenic tomato plants were obtained by transforming two vectors respectively. The DNA sequence around the target sites was amplified, and sequence alignment showed that different deletions of 1~9 nucleotides occurred upstream of PAM, resulting in amino acid deletion and early termination of the SlAP3 protein. The observation revealed that plants carrying homogeneous mutations have homeotic phenotypes and reduced organ number in petals and stamens. These results indicated that the vectors constructed with CRISPR/Cas9 genome editing system could specifically target the tomato SlAP3 gene, resulting in mutation of the gene and the formation of a stamen homeotic mutant phenotype, which provide theory guide and technical support for using CRISPR/Cas9 system to develop tomato male sterile line.
刘玉琛, 丘式浚, 金曼, 邓汉超, 尹梅, 陈竹锋, 周向阳, 唐晓艳. CRISPR/Cas9技术在创制番茄雄性不育株系中的应用研究[J]. 农业生物技术学报, 2019, 27(6): 951-960.
LIU Yu-Chen, QIU Shi-Jun, JIN Man, DENG Han-Chao, YIN Mei, CHEN Zhu-Feng, ZHOU Xiang-Yang, TANG Xiao-Yan. Study on the Application of CRISPR/Cas9 Technology in Development of Tomato (Solanum lycopersicum) Male Sterile Line. 农业生物技术学报, 2019, 27(6): 951-960.
[1] 陈玉辉, 许向阳, 李桂英, 等. 2004. 番茄雄性不育研究进展[J]. 东北农业大学学报, 35(2): 129-134. (Chen Y H, Xu X Y, Li G Y, et al.2004. Review of advances in research of the male sterility in tomato[J]. Journal of Northeast Agricultural University, 35(2): 129-134.) [2] 范鸿凯. 2008. 番茄杂种优势利用研究进展[J]. 农业科技与装备, 177(3): 80-82. (Fan H K.2008. Research development of tomato heterosis utilization[J].Agricultural Science & Technology and Equipment, 177(3): 80-82.) [3] 黄小贞, 曾晓芳, 李建容, 等. 2017. 基于CRISPR/Cas9技术的水稻转录因子tify1a和tify1b突变体的创建与分析[J]. 农业生物技术学报, 25(6):1004-1012. (Huang X Z, Zeng X F, Li J R, et al.2017. Construction and analysis of tify1a and tify1b mutants in rice (Oryza sativa) based on CRISPR/Cas9 technology[J]. Journal of Agricultural Biotech nology, 25(6): 1004-1012.) [4] 王先裕, 于分弟, 陈伟, 等. 2010. 番茄核雄性不育系大107果实总酸杂种优势及遗传效应[J]. 中国蔬菜, (24): 40-43. (Wang X Y, Yu F D, Chen W, et al.2010. Heterosis and hereditary effects of total acid in genic male-sterile tomato (Lycopersicon esculentum L.)-'Da107'[J]. China Vegetables, (24): 40-43.) [5] 谢荧. 2014. 番茄功能雄性不育系形态与生理特性分析及ps基因的精细定位[D]. 硕士学位论文, 东北农业大学, 导师: 王傲雪, pp. 2-13. (Xie Y.2014. Analysis of morphological and physiological characteristics of functional male sterility and fine mapping of ps gene in tomato[D]. Thesis for M.S., Northeast Agricultural University. Supervisor: Wang A X, pp. 2-13.) [6] 张贺, 李景福, 庄磊, 等. 2012. 番茄新型雄性不育系创造及其制种应用初探[J]. 中国蔬菜, (6): 93-95. (Zhang H, Li J F, Zhuang L, et al. 2012. Initial exploration about creation of new tomato male sterile line and its seed production technology[J]. China Vegetables, (6): 93-95.) [7] 郑红艳, 王磊. 2018. CRISPR/Cas基因编辑技术及其在作物育种中的应用[J]. 生物技术进展, 8(3): 185-190. (Zheng h y, wang l.2018. the crispe/cas gene editing technology and application in crop breeding[J]. Current Biotechnology, 8(3): 185-190.) [8] Braatz J, Harloff H J, Mascher M, et al.2017. CRISPR-Cas9 targeted mutagenesis leads to simultaneous modification of different homoeologous gene copies in polyploid oilseed rape (Brassica napus)[J]. Plant Physiology, 174(2): 935-942. [9] Fu Z, Yu J, Cheng X, et al.2014. The rice basic helix-Loop-helix transcription factor TDR INTERACTING PROTEIN2 is a central switch in early anther development. The Plant Cell, 26(2): 1512-1524. [10] Hazra P, Roy T, Choudhury J, et al.2007. Male sterility in tomato (Lycopersicom esculentum Mill.) and brinjal (Solanum melongena)[J]. International Journal of Plant Breeding, 1(1): 41-50. [11] Ito Y, Nishizawa-Yokoi A, Endo, et al.2015. CRISPR/Cas9-mediated mutagenesis of the RIN locus that regulates tomato fruit ripening[J]. Biochemical and Biophysical Research Communications, 467(1): 76-82. [12] Jack T, Brockman L L, Meyerowitz E M.1992. The homeotic gene APETALA3 of Arabidopsis thaliana encodes a MADS box and is expressed in petals and stamens. Cell, 68(4): 683-697. [13] Krizek B A, Meyerowitz E M.1996. The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function[J]. Development, 122: 11-22. [14] Labun K, Montague T G, Gagnon J A, et al.2016. CHOPCHOP v2: A web tool for the next generation of CRISPR genome engineering[J]. Nucleic Acids Research, 44:W272-W276. [15] Lawreson T, Shorinola O, Stacey N, et al.2015. Induction of targeted, heritable mutations in barley and Brassica oleracea using RNA-guided Cas9 nuclease[J]. Genome Biology, 16(1): 258 . [16] Lei Y, Lu L, Liu H Y, et al.2014. CRISPR-P: A web tool for synthetic single-guide RNA design of CRISPR-system in plants[J]. Molecular Plant, 7(9): 1494-1496. [17] Lu Y M, Zhu J K.2016. Precise editing of a target base in the rice genome using a modified CRISPR/Cas9 system[J]. Molecular Plant, 10(3): 523-525. [18] Jeong H J, Kang J H, Zhao M A, et al.2014. Tomato male sterile 1035 is essential for pollen development and meiosis in anthers[J]. Journal of Experimental Botany, 65(22): 6693-6709. [19] Miao J, Guo D S, Zhang J Z, et al.2013. Targeted mutagenesis in rice using CRISPR-Cas system[J]. Cell Research, 23(10): 1233-1236. [20] Quinet M, Bataille G, Dobrev P I, et al.2014. Transcriptional and hormonal regulation of petal and stamen development by STAMENLESS, the tomato (Solanum lycopersicum) orthologue to the B-class APETALA3 gene[J]. Journal of Experimental Botany, 65(9): 2243-2256. [21] Schwarz-Sommer Z, Hue I, Huijser P, et al.1992. Characterization of the Antirrhinum floral homeotic MADS-box gene deficiens: Evidence for DNA binding and autoregulation of its persistent expression throughout flower development[J]. The EMBO Journal, 11(1): 251-263. [22] Shan Q W, Wang Y P, Li J, et al.2014. Genome editing in rice and wheat using the CRISPR/Cas system[J]. Nature Protocols, 9(10): 2395-2410. [23] Wang S H, Zhang S B, Wang W X, et al.2015. Efficient targeted mutagenesis in potato by the CRISPR/Cas9 system[J]. Plant Cell Reports, 34(9): 1473-1476. [24] Xing H L, Dong L, Wang Z P, et al.2014. A CRISPR/Cas9 toolkit for multiplex genome editing in plants[J]. BMC Plant Biology, 14: 327. [25] Zong Y, Wang Y P, Li C, et al.2017. Precise base editing in rice, wheat and maize with a Cas9-cytidine deaminase fusion[J]. Nature Biotechnology, 35(5): 438-440.