药学学报, 2022, 57(4): 1187-1192
引用本文:
许小涵, 李小丽, 唐志强, 刘谦, 李佳, 刘振华, 张永清, 蒲高斌. 忍冬U6启动子的克隆及功能验证[J]. 药学学报, 2022, 57(4): 1187-1192.
XU Xiao-han, LI Xiao-li, TANG Zhi-qiang, LIU Qian, LI Jia, LIU Zhen-hua, ZHANG Yong-qing, PU Gao-bin. Cloning and functional verification of a U6 promoter with high transcriptional activity in Lonicera japonica[J]. Acta Pharmaceutica Sinica, 2022, 57(4): 1187-1192.

忍冬U6启动子的克隆及功能验证
许小涵1, 李小丽1, 唐志强1, 刘谦1,2, 李佳1,2, 刘振华1,2, 张永清1,2, 蒲高斌1,2*
1. 山东中医药大学, 山东 济南 250355;
2. 山东省中药质量控制与全产业链建设协同创新中心, 山东 济南 250355
摘要:
以忍冬基因组DNA为模板,克隆并筛选出具有较高转录活性的忍冬U6启动子。采用PCR方法,从忍冬基因组中克隆到4个LjU6启动子,长度分别为336、708、359、602 bp,PlantCARE分析发现4个启动子中均含有TATA框以及CAAT框等典型的启动子顺式元件,且包含与光响应、胁迫响应等相关的调控元件;克隆产物经测序正确后,将LjU6启动子连接至携带β-葡萄糖苷酸酶(GUS)基因的pBI121载体,成功构建4个LjU6-pBI121融合表达载体,通过农杆菌瞬时转化法转化烟草叶片,并对叶片进行GUS组织化学染色,染色结果显示LjU61-F1转录活性最高,本研究初步筛选出转录活性较高的忍冬U6启动子,为忍冬CRISPR/Cas9基因组编辑技术的建立奠定了基础。
关键词:    忍冬      U6启动子      瞬时表达      β-葡萄糖苷酸酶染色      基因编辑     
Cloning and functional verification of a U6 promoter with high transcriptional activity in Lonicera japonica
XU Xiao-han1, LI Xiao-li1, TANG Zhi-qiang1, LIU Qian1,2, LI Jia1,2, LIU Zhen-hua1,2, ZHANG Yong-qing1,2, PU Gao-bin1,2*
1. Shandong University of Traditional Chinese Medicine, Jinan 250355, China;
2. Shandong Provincial Collaborative Innovation Center for Quality Control and Construction of the Whole Industrial Chain of Traditional Chinese Medicine, Jinan 250355, China
Abstract:
Using Lonicera japonica genomic DNA as a template, we cloned Lonicera japonica U6 promoters. Four LjU6 promoters, 336, 708, 359 and 602 bp in length, were cloned by PCR from Lonicera japonica genomic DNA. PlantCARE analysis found that the four promoters contained typical promoter cis-elements, such as a TATA box and CAAT box, and contained regulatory elements related to light response and stress response. After the cloning products were sequenced, the LjU6 promoter was ligated to the pBI121 vector carrying the β-glucuronidase (GUS) gene to construct four LjU6-pBI121 fusion expression vectors. Nicotiana tabacum leaves were transformed by the Agrobacterium transient transformation method and GUS histochemical staining was performed on the leaves. The staining results showed that LjU61-F1 had the highest transcriptional activity. This study thus identified a U6 promoter with high transcriptional activity, providing a basis for the establishment of CRISPR/Cas9 genome editing technology in Lonicera japonica.
Key words:    Lonicera japonica    U6 promoter    transient expression    β-glucuronidase staining    gene editing   
收稿日期: 2021-10-08
DOI: 10.16438/j.0513-4870.2021-1454
基金项目: 国家自然科学基金资助项目(81872963);中央本级重大增减支项目“名贵中药资源可持续利用能力建设”(2060302);山东省高等学校优秀青年创新团队支持计划(2019KJE004);山东省重大科技创新工程(2019JZZY011020).
通讯作者: 蒲高斌,E-mail:gbpu@163.com
Email: gbpu@163.com
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参考文献:
[1] Cao HX, Wang W, Le HT, et al. The power of CRISPR-Cas9-induced genome editing to speed up plant breeding[J]. Int J Genomics, 2016, 2016:5078796.
[2] Wang Y, Huang G, Yang H, et al. Advances on nonviral vectors of CRISPR/Cas9 system forgenome editing[J]. Acta Pharm Sin (药学学报), 2020, 55:2606-2617.
[3] Nie LZ, Fang YY. Research progress of genome editing technology CRISPR/Cas9 and CRISPR/Cpf1 in crop[J]. J North Agric (北方农业学报), 2019, 47:27-32.
[4] Shan QW, Gao CX. Research progress of genome editing and derivative technologies in plants[J]. Hereditas (遗传), 2015, 37:953-973.
[5] Su YK, Qiu JR, Zhang H, et al. Advances in technology improvement and innovation of CRISPR/Cas9 system in plant genome editing[J]. Chin Bull Bot (植物学报), 2019, 54:385-395.
[6] Ng H, Dean N. Dramatic improvement of CRISPR/Cas9 editing in Candida albicans by increased single guide RNA expression[J]. mSphere, 2017, 2:e00385-16.
[7] Jiang W, Zhou H, Bi H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis, tobacco, sorghum and rice[J]. Nucleic Acids Res, 2013, 41:e188.
[8] Preece R, Georgiadis C, Gkazi SA, et al. 'Mini' U6 Pol III promoter exhibits nucleosome redundancy and supports multiplexed coupling of CRISPR/Cas9 effects[J]. Gene Ther, 2020, 27:451-458.
[9] Miyagishi M, Taira K. U6 promoter-driven siRNAs with four uridine 3' overhangs efficiently suppress targeted gene expression in mammalian cells[J]. Nat Biotechnol, 2002, 20:497-500.
[10] Ren C, Liu Y, Guo Y, et al. Optimizing the CRISPR/Cas9 system for genome editing in grape by using grape promoters[J]. Hort Res, 2021, 8:52.
[11] Wang MB, Helliwell CA, Wu LM, et al. Hairpin RNAs derived from RNA polymerase II and polymerase III promoter-directed transgenes are processed differently in plants[J]. RNA, 2008, 14:903-913.
[12] Bian SX, Han XL, Yuan GP, et al. Cloning and functional analysis of U6 promoter in apple[J]. Sci Agric Sin (中国农业科学), 2019, 52:4364-4373.
[13] Wang C, Rollins JA. Efficient genome editing using endogenous U6 snRNA promoter-driven CRISPR/Cas9 sgRNA in Sclerotinia sclerotiorum[J]. Fungal Genet Biol, 2021, 154:103598.
[14] Ni XY, Lu WJ, Qiao X, et al. Genome editing efficiency of four Drosophila suzukii endogenous U6 promoters[J]. Insect Mol Biol, 2021, 30:420-426.
[15] Sun X, Hu Z, Chen R, et al. Targeted mutagenesis in soybean using the CRISPR-Cas9 system[J]. Sci Rep, 2015, 5:10342.
[16] Long L, Guo DD, Gao W, et al. Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression[J]. Plant Methods, 2018, 14:85.
[17] Wu GF, Cheng HB, Wu YJ, et al. Identification of knockout of BRI1 mutant in Arabidopsis mediated by CRISPR/Cas9[J]. Bull Bot Res (植物研究), 2021, 41:362-371.
[18] Yi Y, Zheng R, Yang B, et al. CRISPR/Cas9 mutants construction of rice zinc finger protein gene and mutation analysis[J]. J South Agric (南方农业学报), 2020, 51:2607-2613.
[19] Sanchez-Leon S, Gil-Humanes J, Ozuna CV, et al. Low-gluten, nontransgenic wheat engineered with CRISPR/Cas9[J]. Plant Biotechnol J, 2018, 16:902-910.
[20] Di YH, Sun XJ, Hu Z, et al. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean[J]. Biochem Biophys Res Commun, 2019, 519:819-823.
[21] Zhang X, Shi YX, Lu BS, et al. Creation of new maize variety with fragrant rice like flavor by editing BADH2-1 and BADH2-2 using CRISPR/Cas9[J]. Sci Agric Sin (中国农业科学), 2021, 54:2064-2075.
[22] Zang XY, Dai PH, Li JY, et al. Functional identification of cotton U6 promoters in the CRISPR/Cas9 genome editing system[J]. J Xinjiang Agric Univ (新疆农业大学学报), 2018, 41:31-35.
[23] Pu Y, Liu C, Li JY, et al. Different SIU6 promoters cloning and establishment of CRISPR/Cas9 mediated gene editing system in tomato[J]. Sci Agric Sin (中国农业科学), 2018, 51:315-326.
[24] Chinese Pharmacopoeia Commission. Pharmacopeia of People's Republic of China (中华人民共和国药典)[S]. Beijing:China Medical Science Press, 2020:52, 230.
[25] Qi LJ, Yuan Y, Wu C, et al. Bioinformatics analysis of DNA demethylase genes in Lonicera japonica Thunb[J]. Acta Pharm Sin (药学学报), 2015, 50:367-371.
[26] Xie DJ, Ye YJ, Yang DM, et al. Cloning and analysis of the DXR gene and its promoter in Morinda officinalis[J]. Acta Pharm Sin (药学学报), 2020, 55:335-344.
[27] Tang F, Tu HZ. Research progress of eukaryotic promoter[J]. China Forest Sci Technol (林业科技开发), 2015, 29:7-12.
[28] Li T, Sun JK, Liu JT. Research advances on plant promote[J]. Biotechnol Bull (生物技术通报), 2015, 31:18-25.
[29] Lei JF, Li Y, Xu XX, et al. Cloning and functional analysis of different truncated GbU6 promoters in cotton[J]. Acta Agron Sin (作物学报), 2016, 42:675-683.
[30] Domitrovich AM, Kunkel GR. Multiple, dispersed human U6 small nuclear RNA genes with varied transcriptional efficiencies[J]. Nucleic Acids Res, 2003, 31:2344-2352.