利用体外靶点效率检测指导丹参SmPAL1基因的CRISPR/Cas9载体构建
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摘要
为定向编辑丹参苯丙烷代谢途径中关键酶基因SmPAL1构建CRISPR/Cas9载体,通过在线软件设计CIRSPR/Cas9靶位点,采用体外酶切法进行体外靶点效率检测,筛选活性高的序列,构建到CRISPR/Cas9载体中。设计了3个可能的CRISPR/Cas9靶位点(SmPAL1-g1,SmPAL1-g2,SmPAL1-g3),通过体外靶点效率检测,其活性分别为53.3%,76.6%,10.0%,将筛选出活性较高的2个靶位点SmPAL1-g1和SmPAL1-g2构建到载体VK005-03中并命名为VK005-03-g1和VK005-03-g2。测序结果显示,设计的2个CRISPR/Cas靶位点全部插入到VK005-03中,载体构建成功。
To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences(SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.
To construct CRISPR/Cas9 vectors for the editing of SmPAL1 in the phenylpropane metabolic pathway of Salvia miltiorrhiza, CIRSPR/Cas9 target sites of SmPAL1 were designed by online software. Its target efficiencies were detected in vitro by enzyme digestion and sequences with highly efficiency were constructed into CRISPR/Cas9 vectors. Three possible CRISPR target sequences(SmPAL1-g1, SmPAL1-g2, SmPAL1-g3) were designed and the enzyme digestion efficiencies were 53.3%, 76.6% and 10.0%. SmPAL1-g1 and SmPAL1-g2 were constructed into vector VK005-03 named as VK005-03-g1 and VK005-03-g2. The results of sequencing showed that the two CRISPR/Cas target sequences were all constructed into VK005-03. Here we first laid the foundation for the study of SmPAL1 and provided an effective strategy for the screening of sgRNA.
引文
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[2] 曾明,邸晓辉,王金萍.注射用丹参多酚酸盐治疗缺血性脑卒中疗效与安全性的系统评价[J].中国实验方剂学杂志, 2015,21(5):225.
[3] 朱艺平. 静脉滴注注射用丹参多酚酸盐致过敏性休克1例[J]. 中国现代应用药学, 2016, 33(9): 1213.
[4] 宋经元,罗红梅,李春芳,等.丹参药用模式植物研究探讨[J].药学学报, 2013, 48 (7): 1099.
[5] 曾慧婷,宿树兰,朱悦,等.丹参酚酸类成分生物合成途径及调控机制研究进展[J].中草药, 2016, 47(18): 3324.
[6] 侯学敏.丹参迷迭香酸合成途径关键酶基因的鉴定与分析[D].临汾:山西师范大学,2013.
[7] 谢胜松,张懿,张利生,等.CRISPR/Cas9系统中sgRNA设计与脱靶效应评估[J].遗传, 2015, 37(11): 1125.
[8] 宗成堃,宋振巧,陈海梅,等.利用SSR、SRAP和ISSR分子标记构建首张丹参遗传连锁图谱[J].药学学报, 2015, 50(3): 360.
[9] 陶洋,李金城,廖奇.CRISPR相关的生物信息学基础[J].中国生物化学与分子生物学报, 2016, 10(32):1067.
[10] Xu H B, Song J Y, Luo H M, et al. Analysis of the genome sequence of the medicinal plant Salvia miltiorrhiza[J]. Mol Plant, 2016, 9(6): 949.
[11] 左其生,王颖洁,赵瑞丰,等.CRISPR/Cas技术可有效介导家鸡基因敲除[J].畜牧兽医学报, 2016, 47(6): 1266.
[12] Liu X, Wu S R, Xu J, et al. Application of CRISPR/Cas9 in plant biology[J]. Acta Pharm Sinica B, 2017; 7(3): 292.
[13] 朱金洁.CRISPR-Cas9介导的玉米基因组定点编辑研究[D].北京:中国农业大学, 2015.
[14] Liu T, Guo L L, Pan Y L, et al. Construction of the first highdensity genetic linkage map of Salvia miltiorrhiza using specific length amplified fragment (SLAF) sequencing[J]. Sci Rep, 2016, 6: 24070.
[15] 叶兴国,杜丽璞.从转基因技术角度谈转基因植物的安全性[J].中国种业, 2016(9): 12.
[16] Zhang G H, Tian Y, Zhang J,et al.Hybrid denovo genome assembly of the Chinese herbal plant danshen (Salvia miltiorrhiza Bunge)[J]. Giga Sci, 2015, 4: 62.