摘要
番茄为呼吸跃变型果实,伴随呼吸跃变产生大量乙烯,即系统II乙烯,易使番茄果实过熟,导致腐烂变质。SlACS2是番茄系统II乙烯合成的限速酶,通过CRISPR-Cas9基因组编辑系统修饰该基因,调控系统Ⅱ乙烯过量表达,将迟滞番茄过熟。本研究基于RNA-seq建立了SlACS2基因的数字表达谱,表明该基因呈果实特异性表达,在植株的根、茎、叶等部位不表达。SlACS2位于番茄1号染色体,含4个外显子和3个内含子。利用在线工具CRISPRdirect和CRISPR-P发现第1、2、3外显子分别具有18、9和11条sgRNA。其中,sgRNA1-14和sgRNA3-8及二者的近PAM的12 nt种子序列在番茄基因组是唯一序列,GC含量高于40%,不存在TTTT终止序列。BLAST结果表明,sgRNA1-14和sgRNA3-8与GenBank公布的8条SlACS2同源序列高度一致,位于该基因的保守区,而与SlACS4和SlACS6的同源序列存在多个SNP,预示这2条sgRNA可用于番茄不同品种SlACS2基因的靶向编辑,并可规避对SlACS家族其他同源基因的脱靶效应。
Tomatoes are typical climacteric fruits and produce large amounts of ethylene, i.e. system II ethylene with the climacteric, which makes the tomato fruits overripe and Perishable. ACC synthase 2 in tomato （SlACS2） is the key enzymy during the process of biosynthesis of the system II ethylene. Suppressing the expression of the system II ethylene will postone the fruits overripen by using CRISPR-Cas9 genome editing system to modify the SlACS2 gene. In this study the digital expression profile of SlACS2 based on RNA-seq shows that SlACS2 gene is expressed specifically in fruit. SlACS2 gene is located in chromosome 1 of tomato, containing four exons and three introns. In the first, second and third exon, 18, 9 and 11 sgRNAs are found by using related online tools CRISPRdirect and CRISPR-P respectively, sgRNAI-14 and sgRNA3-8, and their 12nt seed sequences proximal the protspacer adjacent motif （PAM） all are unique and no completely homologous sequences in other location of tomato genome is found. The GC content is higher than 40% and there is no termination sequence TTTT in sgRNAI-14 and sgRNA3-8. The BLAST results show that sgRNAI-14 or sgRNA3-8 is highly consistent with the sequences of the homologous sites of eight SlACS2 homologous genes, suggesting that the two sgRNAs are located in the conserved regions of SlACS2 gene. Multiple single nucleotide polymorphisms （SNPs） are found between sgRNAI-14 or sgRNA3-8 or their respective seed sequence and the sequences of the homologous sites of SlACS6 or SlACS4, indicating that the two sgRNAs can be used to edit the SlACS2 gene in different varieties with minimal off-target effects for other members in SlACS family.
作者
白云凤
张爱萍
闫建俊
贺飞燕
张维锋
冯瑞云
刘江娜
张西英
BAI Yunfeng ZHANG Aiping YAN Jianjun HE Feiyan ZHANG Weifeng FENG Ruiyun LIU Jiangna ZHANG Xiying(Key Laboratory of Crop Gene Resources and Germplasm Ehhaneement on Loess Plateau, Ministry of Agriculture, Shanxi Province Key Laboratory of Crop Genetics and Molecular Improvement （ Institute of Crop Science, Shanxi Academy of Agricultural Sciences）, Taiyuan 030031, China Institute of Agricultural Science, Sixth Division, Xinjiang Production and Construction Corps, Wujiaqu 831300, China College of Bioengineering, Shanxi University, Taiyuan 030006, China)
出处
《生物信息学》
2017年第1期7-15,共9页
Chinese Journal of Bioinformatics
基金
国家自然科学基金项目(No.30971838)
山西省应用基础研究项目(No.201601D011075)
