[1]熊伟,等.CRISPR-Cas9介导靶向突变拟南芥ERF1-1基因[J].深圳大学学报理工版,2021,38(5):504-509.[doi:10.3724/SP.J.1249.2021.05504]
 XIONG Wei,,et al.Targeted mutation of ERF1-1 gene in Arabidopsis using CRISPR-Cas9 gene editing system[J].Journal of Shenzhen University Science and Engineering,2021,38(5):504-509.[doi:10.3724/SP.J.1249.2021.05504]
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CRISPR-Cas9介导靶向突变拟南芥ERF1-1基因()
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《深圳大学学报理工版》[ISSN:1000-2618/CN:44-1401/N]

卷:
第38卷
期数:
2021年第5期
页码:
504-509
栏目:
生物工程
出版日期:
2021-09-15

文章信息/Info

Title:
Targeted mutation of ERF1-1 gene in Arabidopsis using CRISPR-Cas9 gene editing system
文章编号:
202105009
作者:
熊伟1 2 3朱成新1 2王玉婷1 2郑媛1 2刘琳1 2莫蓓莘1 2
1) 深圳大学生命与海洋科学学院,广东省植物表观遗传学重点实验室,广东深圳 518071
2)深圳大学龙华生物产业创新研究院,广东深圳 518110
3)深圳大学物理与光电工程学院,光电子器件与系统教育部/广东省重点实验室,广东深圳 518060
Author(s):
XIONG Wei1 2 3 ZHU Chengxin1 2 WANG Yuting1 2 ZHENG Yuan1 2 LIU Lin1 2 and MO Beixin1 2
Targeted mutation of ERF1-1 gene in Arabidopsis using CRISPR-Cas9 gene editing system
XIONG Wei1, 2, 3, ZHU Chengxin1, 2, WANG Yuting1, 2, ZHENG Yuan1, 2, LIU Lin1, 2, and MO Beixin1, 2
1) College of Life Sciences and Oceanography, Guangdong Provincial Key Laboratory for Plant Epigenetics, Shenzhen University, Shenzhen 518071, Guangdong Province, P.R.China
2) Longhua Bioindustry and Innovation Research Institute, Shenzhen University, Shenzhen 518110, Guangdong Province, P.R.China
3) College of Physics and Optoelectronic Engineering, Shenzhen University, Key Laboratory of Optoelectronic Devices and Systems of Ministry of Education and Guangdong Province, Shenzhen 518060, Guangdong Province, P.R.China
关键词:
基因工程 蛋白质翻译 翻译终止 真核生物释放因子 CRISPR-Cas9基因编辑技术 拟南芥
Keywords:
genetic engineering protein translation termination of protein translation eukaryotic release factor 1 CRISPR-Cas9 gene editing technology Arabidopsis
分类号:
Q812
DOI:
10.3724/SP.J.1249.2021.05504
文献标志码:
A
摘要:
利用CRISPR-Cas9基因编辑技术对拟南芥真核生物释放因子1-1(eukaryotic release factor 1-1, ERF1-1)基因进行高效率的靶向突变以获得该基因突变体,观察突变体表型并进一步探索ERF1-1基因在拟南芥中生长发育中的功能.根据原间隔序列邻近模块序列(protospacer adjacent motif, PAM)的特异性,选择1对位于ERF1-1基因内部的靶点序列,并设计1对向导核糖核酸(guide ribonucleic acid, gRNA),将与这对gRNA序列碱基互补配对的脱氧核糖核酸(deoxyribonucleic acid, DNA)序列构建到pHEE401载体中. 将构建好的重组质粒转化到农杆菌GV3101菌株,接着利用含有重组质粒的农杆菌对拟南芥花序进行滴花法侵染. 用含有潮霉素的培养平板筛选转基因T1代阳性苗并传代培养,通过测序检测每一代植株靶点附近是否发生编辑.采用目前最有效的CRISPR/Cas9基因靶向编辑技术突变拟南芥ERF1-1基因,成功获得了一系列不同形式的ERF1-1基因突变植株,在正常生长条件下ERF1-1突变植株与野生型相比具有轻微的生长发育缺陷表型.根据erf1-1突变植株的表型推测ERF1基因家族的成员ERF1-1、 ERF1-2和ERF1-3可能在功能上冗余. 研究成果可为后续拟南芥ERF1基因功能研究提供遗传学方面的数据.
Abstract:
The CRISPR-Cas9 gene editing technology is used to edit eukaryotic release factor 1-1 (ERF1-1) gene in Arabidopsis thaliana. After obtaining erf1-1 mutants, the phenotypes of which are observed and the function of ERF1-1 gene is explored in Arabidopsis thaliana. A pair of target sequences inside the ERF1-1 gene coding region, with the protospacer adjacent motif (PAM) sequence flanking the 3′ end, is chosen on a CRISPR target screening platform website and a pair of guide ribonucleic acids (gRNAs) is designed accordingly. The target segments are amplified by PCR and cloned into pHEE401 vector. The recombinant plasmid is transformed into Agrobacterium tumefaciens strain GV3101 and then the Arabidopsis inflorescence is infected with Agrobacterium tumefaciens using the floral dip method. The putative transgenic plants are screened in hygromycin-containing culture medium. DNAs from each generation of the transgenic plants are extracted and sequenced. Results show that the ERF1-1 gene in Arabidopsis is successfully mutated using the CRISPR-Cas9 gene editing technology and several different alleles of erf1-1 mutants are obtained. Under normal growth conditions, erf1-1 mutant plants show a slight growth and development phenotype compared with the wild type plants. It is speculated that functional redundancies may exist within homologous ERF1 genes. This work will provide genetic basis for the functional studies of ERF1 genes in Arabidopsis.

参考文献/References:

[1] ZHOURAVLEVA G, FROLOVA L, LE GOFF X, et al. Termination of translation in eukaryotes is governed by two interacting polypeptide chain release factors, eRF1 and eRF3[J].The EMBO Journal,1995,14(16):4065-4072.
[2] BROWNING K S, BAILEY-SERRES J. Mechanism of cytoplasmic mRNA translation[J]. Arabidopsis Book, 2015, 13:e0176.
[3] FROLOVA L, LE GOFF X, RASMUSSEN H H, et al. A highly conserved eukaryotic protein family possessing properties of polypeptide chain release factor[J].Nature,1994,372(657):701-703.
[4] FROLOVA L, LE GOFF X, ZHOURAVLEVA G, et al. Eukaryotic polypeptide chain release factor eRF3 is an eRF1- and ribosome-dependent guanosine triphosphatase[J].RNA,1996,2(4):334-341.
[5] FROLOVA L Y, MERKULOVA T I, KISSELEV L L. Translation termination in eukaryotes: polypeptide release factor eRF1 is composed of functionally and structurally distinct domains[J].RNA,2000,6(3):381-390.
[6] 邹友平,陈胜文.核糖体内多肽链翻译终止机制[J].生命的化学,2002, 22(2):151-154.
ZOU Youping, CHEN Shengwen. The mechanism of the termination of the synthesis of polypeptides in ribosome[J]. Chemistry of Life,2002,22(2):151-154.(in Chinese)
[7] SONG Li, WANG Yuyao, CHAI Baofeng, et al. C-terminal 76 amino acids of eRF3 are not required for the binding of release factor eRF1a from Euplotes octocarinatus[J]. Journal of Genetics and Genomics, 2007, 34(6):486-490.
[8] 张素平, 梁爱华. 第一类肽链释放因子研究进展[J]. 生物化学与生物物理进展,2001, 28(5):619-622.
ZHANG Suping, LIANG Aihua. The advances on release factor (calss 1) in translation temination[J]. Progress in Biochemistry and Biophysics, 2001,28(5):619-622.(in Chinese)
[9] SONG H, MUGNIER P, DAS A K,et al. The crystal structure of human eukaryotic release factor eRF1: mechanism of stop codon recognition and peptidyl-tRNA hydrolysis[J].Cell,2000,100(3):311-321.
[10] DEVER TE, GREEN R. The elongation, termination, and recycling phases of translation in eukaryotes[J].Cold Spring Harbor Perspectives in Biology,2012,4(7):a013706-22.
[11] 陈洁, 柴宝峰, 梁爱华. 第一类肽链释放因子结构与功能研究的新进展[J]. 生物化学与生物物理进展 2009, 36(7):817-822.
CHEN Jie, CHAI Baofeng, LIANG Aihua. New progress in function and structure of the class 1 polypeptide release factors[J]. Progress in Biochemistry and Biophysics. 2009, 36(7):817-822.(in Chinese)
[12] BEISSEL C, NEUMANN B, UHSE S,et al. Translation termination depends on the sequential ribosomal entry of eRF1 and eRF3[J]. Nucleic Acids Research 2019, 47(9):4798-4813.
[13] ELAKHDAR A, USHIJIMA T, FUKUDA M,et al. Eukaryotic peptide chain release factor 1 participates in translation termination of specific cysteine-poor prolamines in rice endosperm[J]. Plant Science 2019, 281:223-231.
[14] TASSAN J P, LE GUELLEC K, KRESS M,et al. In xenopus laevis, the product of a developmentally regulated mRNA is structurally and functionally homologous to a saccharomyces cerevisiae protein involved in translation fidelity[J].Molecular and Cellular Biology,1993,13(5):2815-2821.
[15] TASSAN J P, GUELLEC K L, KRESS M,et al. In Xenopus laevis, the product of a developmentally regulated mRNA is structurally and functionally homologous to a Saccharomyces cerevisiae protein involved in translation fidelity[J]. Molecular and Cellular Biology, 1993, 13(5):2815-2821.
[16] KARAMYSHEV A L, KARAMYSHEVA Z N, ITO K,et al. Overexpression and purification of recombinant eRF1 proteins of rabbit and Tetrahymena thermophila[J]. Biochemistry. 1999, 64(12):1391-1400.
[17] KURILLA A, SZOKE A, AUBER A,et al. Expression of the translation termination factor eRF1 is autoregulated by translational readthrough and 3′UTR intron-mediated NMD in Neurospora crassa[J]. FEBS Letters, 2020, 594(21):3504-3517.
[18] CHAPMAN B, BROWN C. Translation termination in Arabidopsis thaliana: characterisation of three versions of release factor 1[J]. Gene, 2004, 341:219-225.
[19] PETSCH K A, MYLNE J, BOTELLA J R.Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division[J].Plant Physiology,2005,139(1):115-126.
[20] ZHOU Xiangjun, COOKE P, LI Li.Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development[J].Journal of Experimental Botany,2010,61(2):357-367.
[21] WANG Zhiping, XING Huili, DONG Li, et al. Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation[J]. Genome Biology, 2015, 16(1):144.
[22] 李琳,罗淋淋,罗光宇,等.一种植物基因组DNA快速提取方法的建立与评估[J].深圳大学学报理工版,2020,37(1):1-8.
LI Lin, LUO Linlin, LUO Guangyu, et al. Establishment and evaluation of a method for rapid extraction of plant genomic DNA[J]. Journal of Shenzhen University Science and Engineering,2020,37(1):1-8.(in Chinese)
[23] 黄瑛,李小连,殷剑波,等.3个基因共调控下莱茵衣藻的转录组分析[J].深圳大学学报理工版,2021,38(2):194-200.
HUANG Ying,LI Xiaolian,YIN Jianbo,et al.Transcriptome analysis of Chlamydomonas reinhardtii under the co-regulation of three genes[J].2021,38(2):194-200.(in Chinese)
[24] AN Yan, LOU Yongfeng, XU Yingwu. Overexpression, crystallization and preliminary X-ray crystallographic analysis of release factor eRF1-1 from Arabidopsis thaliana[J]. Acta Crystallographica Section F, 2013, 69:1295-1298.

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备注/Memo

备注/Memo:
Received:2021-04-19;Accepted:2021-06-07;Online(CNKI):2021-08-24
Foundation:National Natural Science Foundation of China(31870287);Shenzhen Basic Research Foundation (JCYJ20190808115005598)
Corresponding author:Professor MO Beixin.E-mail: bmo@szu.edu.cn
Citation:XIONG Wei, ZHU Chengxin, WANG Yuting,et al.Targeted mutation of ERF1-1 gene in Arabidopsis using CRISPR-Cas9 gene editing system[J]. Journal of Shenzhen University Science and Engineering, 2021, 38(5): 504-509.(in Chinese)
基金项目:国家自然科学基金资助项目(31870287);深圳市基础研究计划资助项目 (JCYJ20190808115005598)
作者简介:熊伟(1987—),深圳大学博士后研究人员.研究方向:植物核糖体与蛋白质翻译.E-mail:hbxfnzhxw@126.com
引文:熊伟,朱成新,王玉婷,等.CRISPR/Cas9介导靶向突变拟南芥ERF1-1基因[J]. 深圳大学学报理工版,2021,38(5):504-509.
更新日期/Last Update: 2021-09-30