[1]唐维,后猛,宋炜涵,等.甘薯U6启动子克隆及其转录活性分析[J].江苏农业学报,2024,(06):969-974.[doi:doi:10.3969/j.issn.1000-4440.2024.06.002]
 TANG Wei,KOU Meng,SONG Weihan,et al.Cloning and transcriptional activity analysis of U6 promoter in sweetpotato[J].,2024,(06):969-974.[doi:doi:10.3969/j.issn.1000-4440.2024.06.002]
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甘薯U6启动子克隆及其转录活性分析()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2024年06期
页码:
969-974
栏目:
遗传育种·生理生化
出版日期:
2024-06-30

文章信息/Info

Title:
Cloning and transcriptional activity analysis of U6 promoter in sweetpotato
作者:
唐维后猛宋炜涵闫会王欣李臣高闰飞张允刚李强
(江苏徐淮地区徐州农业科学研究所/农业农村部甘薯生物学与遗传育种重点实验室,江苏徐州221131)
Author(s):
TANG WeiKOU MengSONG WeihanYAN HuiWANG XinLI ChenGAO RunfeiZHANG YungangLI Qiang
(Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai District/Key Laboratory of Sweet Potato Biology and Genetic Breeding, Ministry of Agriculture and Rural Affairs, Xuzhou 221131, China)
关键词:
甘薯U6启动子克隆基因编辑转录活性
Keywords:
sweetpotatoU6 promotercloninggene editingtranscriptional activity
分类号:
S531
DOI:
doi:10.3969/j.issn.1000-4440.2024.06.002
摘要:
在CRISPR/Cas9基因编辑系统中,U6启动子可以通过驱动sgRNA的表达来编辑目的基因,因而其转录活性会影响基因编辑效率。尽管人们已经在拟南芥、玉米、大豆、棉花等植物中开展了U6启动子的克隆与应用研究,然而目前在甘薯中U6启动子的相关研究还未见报道。本研究利用拟南芥的AtU6 SnRNA的保守序列在三浅裂野牵牛(Ipomoea trifida)基因组数据库中搜索候选U6 RNA,然后在其上游搜索到2个不同的候选U6启动子,长度分别为526 bp(IbU6p-1)和532 bp(IbU6p-2);序列比对分析结果显示,甘薯的U6启动子具有上游序列元件(USE)以及TATA-box,其序列也与拟南芥高度相似。然后,利用获得的U6启动子核酸序列构建了能够驱动萤火虫荧光素酶基因(LUC)表达的重组框U6::LUC。最后,将含有上述重组载体的根癌农杆菌瞬时转化到本氏烟草叶片和甘薯愈伤组织中,并通过荧光成像技术分析荧光素酶活性。结果发现,在烟草及甘薯愈伤组织中2个甘薯U6启动子均能驱动LUC基因表达,具有转录活性。同时,IbU6p-2的转录活性无论是在烟草叶片中还是在甘薯愈伤组织中都显著高于拟南芥U6启动子。本研究结果为进一步发展甘薯基因编辑技术提供了参考。
Abstract:
In the CRISPR/Cas9 gene editing system, the U6 promoter can edit target genes by driving sgRNA expression, so its transcriptional activity can affect the efficiency of gene editing. Although studies on cloning and application of U6 promoter have been carried out in Arabidopsis thaliana, corn, soybean, cotton and other plants, there aren’t any reports on relative research of U6 promoter in sweetpotato. In this study, we searched the Ipomoea trifida genome database for candidate U6 RNA, using the conservative sequence of AtU6 SnRNA in A. thaliana, and then found two different candidate U6 promoters with lengths of 526 bp (IbU6p-1) and 532 bp (IbU6p-2), respectively. Through sequence alignment analysis, we found that the U6 promoter of sweetpotato has upstream sequence element (USE) and TATA box, and its sequence was highly similar to that of A. thaliana. Then, a recombinant box U6 :: LUC was constructed using the obtained U6 promoter nucleic acid sequence to drive the expression of firefly luciferase gene. Finally, Agrobacterium tumefaciens containing the recombinant vector was genetically transformed into Nicotiana benthamiana leaves and sweetpotato callus tissues by transient expression, and luciferase activity was analyzed using fluorescence imaging technology. The results showed that both sweetpotato U6 promoters could drive the expression of LUC gene and had transcriptional activity in tobacco and sweetpotato calli. At the same time, the transcriptional activities of IbU6p-2 in tobacco leaves and sweetpotato calli were significantly higher than that of A. thaliana U6 promoter. The results of this study lay a foundation for further development of sweetpotato gene editing technology.

参考文献/References:

[1]MILLS E M, BARLOW V L, LUK L Y P, et al. Applying switchable Cas9 variants to in vivo gene editing for therapeutic applications[J]. Cell Biology and Toxicology,2020,36(1):17-29.
[2]TANIHARA F, HIRATA M, OTOI T. Current status of the application of gene editing in pigs[J]. Journal of Reproduction and Development,2021,67(3):177-187.
[3]YIN K Q, GAO C X, QIU J L. Progress and prospects in plant genome editing[J]. Nature Plants,2017,3:17107.
[4]GANTZ V M, AKBARI O S. Gene editing technologies and applications for insects[J]. Current Opinion in Insect Science,2018,28:66-72.
[5]ADIEGO-PREZ B, RANDAZZO P, DARAN J M, et al. Multiplex genome editing of microorganisms using CRISPR-Cas[J]. FEMS Microbiology Letters,2019,366(8):86.
[6]CˇERMK T, BALTES N J, CˇEGAN R, et al. High-frequency, precise modification of the tomato genome[J]. Genome Biology,2015,16:232.
[7]BANDYOPADHYAY A, YIN X, BISWAL A, et al. CRISPR-Cas9-mediated genome editing of rice towards better grain quality[J]. Methods in Molecular Biology,2019,1892:311-336.
[8]JIANG W Z, HENRY I M, LYNAGH P G, et al. Significant enhancement of fatty acid composition in seeds of the allohexaploid,Camelina sativa,using CRISPR/Cas9 gene editing[J]. Plant Biotechnology Journal,2017,15(5):648-657.
[9]张威,许文静,许亚男,等. 基于CRISPR/Cas9基因编辑的高油酸大豆品系创制[J]. 江苏农业学报,2023,39(2):321-327.
[10]PICKAR-OLIVER A, GERSBACH C A. The next generation of CRISPR-Cas technologies and applications[J]. Nature Review Molecular Cell Biology,2019,20(8):490-507.
[11]CONG L, RAN F A, COX D, et al. Multiplex genome engineering using CRISPR/Cas systems[J]. Science,2013,339(6121):819-823.
[12]WEI Y D, QIU Y, CHEN Y H, et al. CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter[J]. RNA,2017,23(1):1-5.
[13]GAO J P, WANG G H, MA S Y, et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicotiana tabacum[J]. Plant Molecular Biology,2015,87(1/2):99-110.
[14]卞书迅,韩晓蕾,袁高鹏,等. 苹果U6启动子的克隆及功能分析[J]. 中国农业科学,2019,52(23):4364-4373.
[15]唐志强,李小丽,许小涵,等. 金银花U6启动子的克隆及转录活性分析[J]. 山东科学,2022,35(2):11-17.
[16]李强,揭琴,刘庆昌,等. 甘薯基因组DNA高效快速提取方法[J]. 分子植物育种,2007,5(5):743-746.
[17]SHAN Q W, WANG Y P, LI J, et al. Targeted genome modification of crop plants using a CRISPR-Cas system[J]. Nature Biotechnology,2013,31(8):686-688.
[18]SATHEESH V, ZHANG H, WANG X T, et al. Precise editing of plant genomes-prospects and challenges[J]. Seminars in Cell & Developmental Biology,2019,96:115-123.
[19]JIANG W Z, ZHOU H B, BI H H, et al. Demonstration of CRISPR/Cas9/sgRNA-mediated targeted gene modification in Arabidopsis,tobacco,sorghum and rice[J]. Nucleic Acids Research,2013,41(20):e188.
[20]FAUSER F, SCHIML S, PUCHTA H. Both CRISPR/Cas-based nucleases and nickases can be used efficiently for genome engineering in Arabidopsis thaliana[J]. The Plant Journal,2014,79(2):348-359.
[21]LI J F, NORVILLE J E, AACH J, et al. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9[J]. Nature Biotechnology,2013,31(8):688-691.
[22]ZHOU H B, LIU B, WEEKS D P, et al. Large chromosomal deletions and heritable small genetic changes induced by CRISPR/Cas9 in rice[J]. Nucleic Acids Research,2014,42(17):10903-10914.
[23]BORTESI L, FISCHER R. The CRISPR/Cas9 system for plant genome editing and beyond[J]. Biotechnology Advances,2015,33(1):41-52.
[24]MA X L, ZHANG Q Y, ZHU Q L, et al. A robust CRISPR/Cas9 system for convenient,high-efficiency multiplex genome editing in monocot and dicot plants[J]. Molecular Plant,2015,8(8):1274-1284.
[25]BELHAJ K, CHAPARRO-GARCIA A, KAMOUN S, et al. Plant genome editing made easy:targeted mutagenesis in model and crop plants using the CRISPR/Cas system[J]. Plant Methods,2013,9(1):39.
[26]WANG M B, HELLIWELL C A, WU L M, et al. Hairpin RNAs derived from RNA polymerase Ⅱ and polymerase Ⅲ promoter-directed transgenes are processed differently in plants[J]. RNA,2008,14(5):903-913.
[27]LONG L, GUO D D, GAO W, et al. Optimization of CRISPR/Cas9 genome editing in cotton by improved sgRNA expression[J]. Plant Methods,2018,14:85.
[28]DI Y H, SUN X J, HU Z, et al. Enhancing the CRISPR/Cas9 system based on multiple GmU6 promoters in soybean[J]. Biochemical and Biophysical Research Communications,2019,519(4):819-823.
[29]BERNARD G, GAGNEUL D, ALVES DOS SANTOS H, et al. Efficient genome editing using CRISPR/Cas9 technology in chicory[J]. International Journal of Molecular Sciences,2019,20(5):1155.
[30]LIU H Y, WANG K, JIA Z M, et al. Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system[J]. Journal of Experimental Botany, 2020, 71(4): 1337-1349.
[31]WANG H X, WU Y L, ZHANG Y D, et al. CRISPR/Cas9-Based mutagenesis of starch biosynthetic genes in sweetpotato (Ipomoea Batatas) for the improvement of starch quality[J]. International Journal of Molecular Sciences,2019,20(19):4702.
[32]刘霞宇. 甘薯CRISPR/Cas9基因编辑系统的建立及miR2111调控甘薯块根中花青素积累的功能研究[D]. 太原:山西农业大学,2020.

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备注/Memo

备注/Memo:
收稿日期:2023-07-29基金项目:徐州市现代农业重点专项(KC21116);国家甘薯产业技术体系项目(CARS-10)作者简介:唐维(1982-),男,湖北武汉人,博士,副研究员,主要从事甘薯分子遗传育种研究。(E-mail)tangweilhr@163.com通讯作者:李强,(E-mail)instrong@163.com
更新日期/Last Update: 2024-07-15