| 引用本文: | 白云凤,张爱萍,闫建俊,贺飞燕,张维锋1,冯瑞云,刘江娜,张西英.靶向番茄SlACS2基因CRISPR-Cas9 sgRNA的设计和分析[J].生物信息学,2017,15(1):7-15. |
| BAI Yunfeng,ZHANG Aiping,YAN Jianjun,HE Feiyan,ZHANG Weifeng,FENG Ruiyun,LIU Jiangna,ZHANG Xiying.Design and evaluation for sgRNAs targeting SlACS2gene in CRISPR/Cas9 system[J].Chinese Journal of Bioinformatics,2017,15(1):7-15. |
|
| |
|
|
| 本文已被：浏览 3214次 下载 2360次 |
码上扫一扫！ |
|
|
| 靶向番茄SlACS2基因CRISPR-Cas9 sgRNA的设计和分析 |
|
白云凤1,张爱萍2,闫建俊1,贺飞燕3,张维锋1,冯瑞云1,刘江娜2,张西英2
|
|
(1.农业部黄土高原作物基因资源与种质创制重点实验室,作物遗传与分子改良山西省重点实验室(山西省农业科学院作物科学研究所),太原 030031; 2. 新疆生产建设兵团第6师农业科学研究所,新疆 五家渠831300; 3.山西大学生物工程学院,太原030006)
|
|
| 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。 |
| 关键词: 番茄 ACC合成酶2 sgRNA CRISPR-Cas9 基因组编辑 |
| DOI：10.3969/j.issn.1672-5565.2017.01.201609002 |
| 分类号:Q986;S641.2 |
| 文献标识码:A |
| 基金项目: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)。 |
|
| Design and evaluation for sgRNAs targeting SlACS2gene in CRISPR/Cas9 system |
|
BAI Yunfeng1, ZHANG Aiping2, YAN Jianjun1, HE Feiyan3, ZHANG Weifeng1, FENG Ruiyun1, LIU Jiangna2, ZHANG Xiying2
|
|
(1.Key Laboratory of Crop Gene Resources and Germplasm Ehhancement on Loess Plateau, Ministry of Agriculture, Shanxi Province KeyLaboratory of Crop Genetics and Molecular Improvement (Institute of Crop Science, Shanxi Academy of Agricultural Sciences), Taiyuan 030031, China; 2.Institute of Agricultural Science, Sixth Division, Xinjiang Production and Construction Corps, Wujiaqu 831300, China; 3.College of Bioengineering, Shanxi University, Taiyuan 030006, China)
|
| Abstract: |
| 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 SlACS2gene. In this study the digital expression profile of SlACS2based on RNA-seq shows that SlACS2gene is expressed specifically in fruit. SlACS2gene is located in chromosome 1 of tomato, containing four exons and three introns. In the first, second and third exon, 8,9 and 11 sgRNAs are found by using related online tools CRISPRdirect and CRISPR-P respectively. sgRNA1-14 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 sgRNA1-14 and sgRNA3-8. The BLAST results show that sgRNA1-14 or sgRNA3-8 is highly consistent with the sequences of the homologous sites of eight SlACS2homologous genes, suggesting that the two sgRNAs are located in the conserved regions of SlACS2gene. Multiple single nucleotide polymorphisms (SNPs) are found between sgRNA1-14 or sgRNA3-8 or their respective seed sequence and the sequences of the homologous sites of SlACS6or SlACS4, indicating that the two sgRNAs can be used to edit the SlACS2gene in different varieties with minimal off-target effects for other members in SlACSfamily. |
| Key words: Tomato ACC synthase 2 sgRNA CRISPR-Cas9 Genomic editing |
|
|
|
|
