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.
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