Scientia Agricultura Sinica ›› 2017, Vol. 50 ›› Issue (19): 3631-3639.doi: 10.3864/j.issn.0578-1752.2017.19.001

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS •     Next Articles

Western Blot Detection of CAS9 Protein in Transgenic Rice

GUO YaLu 1, MA XiaoFei 2, SHI JiaNan 1, ZHANG Liu 1, ZHANG JianShuo 1, HUANG Teng 1, WU PengCheng 2, KANG HaoXiang 2, GENG GuangHui 2, CHEN Hao 2, WEI Jian 1, DOU ShiJuan 1, LI LiYun1, YIN ChangCheng 2, LIU GuoZhen1   

  1. 1College of Life Sciences, Hebei Agricultural University, Baoding 071001, Hebei; 2Beijing Protein Innovation Co., Ltd., Beijing 101318
  • Received:2017-03-20 Online:2017-10-01 Published:2017-10-01

RichHTML

35

PDF

1154

Abstract: 【Objective】The objective of this study is to generate monoclonal antibodies against CAS9 protein and establish immunological method for the detection of CAS9 protein in transgenic plants, and to understand the characters of the expression patterns of CAS9 protein in transgenic rice. 【Method】The 5′ fragment (810 bp) of Cas9 was amplified using plasmid DNA confers Cas9 as template. The amplican was cloned into expression vector pET30a. Restriction enzyme digestion identified recombinant plasmid were sequencing verified. The recombinant plasmid was transformed into E. coli Condon Plus strain. The induced CAS9 protein was purified and used as immunogene to generate monoclonal antibodies. Positive hybridoma cell lines were identified by western blot analysis. PCR amplifications were carried out to identify positive transgenic rice using specific primers of Cas9. Western blot was carried out to detect CAS9 protein in rice. The recombinant CAS9 protein and protein samples extracted from rice seedling were analyzed in parallel by western blot, and standard curves were drawn based on Image J software extracted signals. CAS9 protein in rice tissues were analyzed quantitatively based on the standard curve. Total protein of single rice grain was extracted and the sensitivity of CAS9 protein detectable by western blot was analyzed using diluted samples. The abundance of CAS9 protein in different tissues, including shoot and root at seedling stage, stem, node, sheath and leaf at tillering stage, were compared by western blot. 【Result】The Cas9 was cloned and the plasmid was transformed into E. coli. Recombinant N-terminal portion of CAS9 protein was obtained and used as immunogen to inject mice. Forty-two positive hybridoma cell lines were obtained after immunization. Among them, cell line #12D2 showed higher specificity and sensitivity for the detection of CAS9 protein in rice tissues. Western blot analysis was carried out for the detection of transgenic rice via the antibody of #12D2-derived hybridoma cell lines. The lowest amount of recombinant CAS9 protein detectable by the established western blot protocol was about 0.25 ng. It was also revealed that the CAS9 protein accounted for about 0.00005% of fresh weight in rice seedling, and CAS9 protein in 8% of single grain rice (about 2 mg) was detectable. It was also found that the abundance of CAS9 protein in rice shoot tissues at seedling stage was higher than that in root tissues, the abundance in stem and leaves at tillering stage was higher than that in root and sheath tissues. 【Conclusion】In this study, anti-CAS9 monoclonal antibodies with satisfied specificity and sensitivity were obtained and western blot protocol for the detection of CAS9 protein in transgenic plants was established. The expression patterns of CAS9 protein in different rice tissues were revealed. Moreover, the data also demonstrated the potential of application in other plants.

Key words: rice, transgenic plants, CAS9 protein, monoclonal antibody, western blot

[1]    Garneau J E, Dupuis M E, Villion M, Romero D A, Barrangou R, Boyaval P, Fremaux C, Horvath P, Magadan A H, Moineau S. The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature, 2010, 468(7320): 67-71.
[2]    Ran F A, Hsu P D, Lin C Y, Gootenberg J S, Konermann S, Trevino A E, Scott D A, Inoue A, Matoba S, Zhang Y, Zhang F. Double nicking by RNA-guided CRISPR Cas9 for enhanced genome editing specificity. Cell,2013, 154(6): 1380-1389.
[3]    Lin Y, Yang Z, Xu A, Dong P, Huang Y, Liu H, Li F, Wang H, Xu Q, Wang Y. PIK3R1 negatively regulates the epithelial- mesenchymal transition and stem-like phenotype of renal cancer cells through the AKT/GSK3β/CTNNB1 signaling pathway. Scientific Reports, 2015, 5: 8977.
[4]    Xie F, Ye L, Chang J C, Beyer A I, Wang J, Muench M O, Kan Y W. Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac. Genome Research,2014, 24(9): 1526-1533.
[5]    Feng Y, Sassi S, Shen J K, Yang X, Gao Y, Osaka E, Zhang J, Yang S, Yang C, Mankin H J. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system. Journal of Orthopaedic Research, 2015, 33(2): 199-207.
[6]    Ikeda T, Tanaka W, Mikami M, Endo M, Hirano H Y. Generation of artificial drooping leaf mutants by CRISPR-Cas9 technology in rice. Genes & Genetic Systems,2016, 90(4): 231-235.
[7]    Zheng X, Yang S, Zhang D, Zhong Z, Tang X, Deng K, Zhou J, Qi Y, Zhang Y. Effective screen of CRISPR/Cas9-induced mutants in rice by single-strand conformation polymorphism. Plant Cell Reports,2016, 35(7): 1545-1554.
[8]    Feng Z, Mao Y, Xu N, Zhang B, Wei P, Yang D L, Wang Z, Zhang Z, Zheng R, Yang L. Multigeneration analysis reveals the inheritance, specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis. Proceedings of the National Academy of Sciences of the United States of America,2014, 111(12): 4632-4637.
[9]    Xu R, Li H, Qin R, Wang L, Li L, Wei P, Yang J. Gene targeting using the Agrobacterium tumefaciens-mediated CRISPR-Cas system in rice. Rice,2014, 7(1): 1-4.
[10]   Qi W, Zhu T, Tian Z, Li C, Zhang W, Song R. High-efficiency CRISPR/Cas9 multiplex gene editing using the glycine tRNA- processing system-based strategy in maize. BMC Biotechnology,2016, 16(1): 58.
[11]   张峰华, 王厚鹏, 黄思雨, 熊凤, 朱作言, 孙永华. 两种密码子优化的Cas9编码基因在斑马鱼胚胎中基因敲除效率的比较. 遗传, 2016, 38(2): 144-154.
ZHANG F H, WANG H P, HUANG S Y, XIONG F, ZHU Z Y, SUN Y H. Two kinds of codon optimization Cas9 encoding genes in the zebrafish embryo knockout efficiency comparison. Hereditas, 2016, 38(2): 144-154. (in Chinese)
[12]   Xing H L, Dong L, Wang Z P, Zhang H Y, Han C Y, Liu B, Wang X C, Chen Q J. A CRISPR/Cas9 toolkit for multiplex genome editing in plants. BMC Plant Biology, 2014, 14(1): 1-12.
[13]   Howden S E, McColl B, Glaser A, Vadolas J, Petrou S, Little M H, Elefanty A G, Stanley E G. A Cas9 variant for efficient generation of indel-free knockin or gene-corrected human pluripotent stem cells. Stem Cell Reports,2016, 7(3): 508-517.
[14]   Lin S, Ewen-Campen B, Ni X, Housden B E, Perrimon N. In vivo transcriptional activation using CRISPR/Cas9 in Drosophila. Genetics, 2015, 201(2): 433-442.
[15]   Zhu J, Song N, Sun S, Yang W, Zhao H, Song W, Lai J. Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9. Journal of Genetics and Genomics, 2016, 43(1): 25-36.
[16]   Li X, Bai H, Wang X, Li L, Cao Y, Wei J, Liu Y, Liu L, Gong X, Wu L, Liu S, Liu G. Identification and validation of rice reference proteins for western blotting. Journal of Experimental Botany, 2011, 62(14): 4763-4772.
[17]   牛东东, 郝育杰, 荣瑞娟, 韦汉福, 兰金苹, 史佳楠, 魏健, 李雪姣, 杨烁, 奚文辉, 武鹏程, 刘丽娟, 吴琳, 刘斯奇, 尹长城, 刘国振. 转基因水稻中GUS蛋白质的检测及其表达特征. 中国农业科学, 2014, 47(14): 2715-2722.
NIU D D, Hao Y J, RONG R J, WEI H F, LAN J P, SHI J N, WEI J, LI X J, YANG S, XI W H, WU P C, LIU L J, WU L, LIU S Q, YIN C C, LIU G Z. Detection of GUS protein and its expression pattern in transgenic rice plants. Scientia Agricultura Sinica, 2014, 47(14): 2715-2722. (in Chinese)
[18]   兰金苹, 武鹏程, 郭美岑, 史佳楠, 韦汉福, 荣瑞娟, 郝育杰, 杨烁, 白影影, 李莉云, 吴琳, 刘斯奇, 尹长城, 刘国振. NPTⅡ蛋白质在转基因水稻中的表达特征研究. 生物化学与生物物理进展, 2015, 42(3): 268-276.
LAN J P, WU P C, GUO M C, SHI J N, WEI H F, RONG R J, HAO Y J, YANG S, BAI Y Y, LI L Y, WU L, LIU S Q, YIN C C, LIU G Z.The expression properties of NPTⅡ protein in transgenic rice plant. Progress in Biochemistry and Biophysics, 2015, 42(3): 268-276. (in Chinese)
[19]   Rong R, Pengcheng W U, Lan J, Wei H, Wei J, Chen H, Shi J, Hao Y, Liu L, Dou S. Western blot detection of PMI protein in transgenic rice. Journal of Integrative Agriculture, 2015, 15(4): 726-734.
[20]   杨烁, 郝育杰, 兰金苹, 韦汉福, 魏健, 荣瑞娟, 武鹏程, 刘国振, 尹长城, 李莉云. 转基因水稻中HPT蛋白质的检测及表达特征研究. 中国生物工程杂志, 2015, 35(6): 61-67.
YANG S, HAO Y J, LAN J P, WEI H F, WEI J, RONG R J, WU P C, LIU G Z, YIN C C, LI L Y. HPT protein detection and expression pattern in transgenic rice. China Biotechnology, 2015, 35(6): 61-67. (in Chinese)
[21]   柴帅杰, 武鹏程, 荣瑞娟, 郝育杰, 史佳楠, 郭亚璐, 丁国涛, 韩宇宁, 赵志静, 魏健, 尹长城, 刘国振. 转基因水稻中CP4-EPSPS蛋白质的检测及其表达特征研究. 核农学报,2017, 31(1): 44-50.
CHAI S J, WU P C, RONG R J, HAO Y J, SHI J N, GUO Y L, DING G T, HAN Y N, ZHAO Z J, WEI J, YIN C C, LIU G Z. Detection and expression characteristics of CP4-EPSPS protein in transgenic rice. Journal of Nuclear Agricultural Sciences, 2017, 31(1): 44-50. (in Chinese)
[22]   Cao Y, Sun J, Zhu J, Li L, Liu G. PrimerCE: designing primers for cloning and gene expression. Molecular Biotechnology, 2010, 46(2): 113-117.
[23]   Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry,1976, 72: 248-254.
[24]   楼巧君, 陈亮, 罗利军. 三种水稻基因组DNA快速提取方法的比较. 分子植物育种,2005, 3(5): 749-752.
LOU Q J, CHEN L, LUO L J. Three kinds of rice genome DNA rapid extraction method of comparison. Molecular Plant Breeding, 2005, 3(5): 749-752. (in Chinese)
[25]   Xu W, Wang Y, Liu G, Chen X, Porntip T, Pi L, Song W. The autophosphorylated Ser686, Thr688, and Ser689 residues in the intracellular juxtamembrane domain of XA21 are implicated in stability control of rice receptor-like kinase. The Plant Journal,2006, 45(5): 740-751.
[26]   兰金苹, 李莉云, 贾霖, 曹英豪, 白辉, 陈浩, 刘胜南, 吴琳, 刘国振. 叶绿体基因编码蛋白质在水稻叶片生长过程中的表达研究. 生物化学与生物物理进展,2011, 38(7): 652-660.
LAN J P, LI L Y, JIA L, CAO Y H, BAI H, CHEN H, LIU S N, WU L, LIU G Z. Expression profiling of chloroplast-encoded proteins in rice leaves at different growth stages. Progress in Biochemistry and Biophysics, 2011, 38(7): 652-660. (in Chinese)
[27]   Yao L, Cengic I, Anfelt J, Hudson E P. Multiple gene repression in cyanobacteria using CRISPRi. Acs Synthetic Biology,2015, 5(3): 270.
[28]   单奇伟, 高彩霞. 植物基因组编辑及衍生技术最新研究进展. 遗传,2015, 37(10): 953-973.
SHAN Q W, GAO C X. Research progress of genome editing and derivative technologies in plants. Hereditas, 2015, 37(10): 953-973. (in Chinese)
[29]   Chuai G H, Wang Q L, Liu Q. In silico meets in vivo: Towards computational CRISPR-based sgRNA design. Trends in Biotechnology,2017, 35(1): 12-21.
[30]   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.
ZHU J J. Targeted genome editing in maize using CRISPR-Cas9[D]. Beijing: China Agricultural University, 2015. (in Chinese)
[1] PENG TingShen, LU JiuYan, WU MeiLin, YAN YuXin, LIU HongZhou, NAN WenBin, QIN XiaoJian, LI Ming, GONG JunYi, LIANG YongShu. QTL Analysis of Yield-Related Traits in Both Huangnuo2# and Changbai7# of Perennial Chinese Rice [J]. Scientia Agricultura Sinica, 2026, 59(7): 1361-1379.
[2] CUI JieHao, ZHANG Meng, WANG Qin, YU JiaYan, LIN Kun, LI ShangZe, LAN Heng, GENG YanQiu, ZHANG Qiang, GUO LiYing, SHAO XiWen. Evaluation of Lodging Resistance and Its Physiological Mechanisms in Japonica Rice Resources [J]. Scientia Agricultura Sinica, 2026, 59(7): 1420-1438.
[3] YUAN HaoLiang, NIE Jun, LI Peng, LU YanHong, LIAO YuLin, XU ChangXu, LI ZhongYi, CAO WeiDong, ZHANG JiangLin. Effects of Co-Utilization of Chinese Milk Vetch and Rice Straw on Soil Phosphorus Composition and Phosphorus Activation of Paddy Field in Southern China [J]. Scientia Agricultura Sinica, 2026, 59(7): 1480-1491.
[4] XU YangHaoJun, CHEN LiMing, YANG ShiQi, TANG YiFan, TAN XueMing, ZENG YongJun, PAN XiaoHua, ZENG YanHua. Effects of Long-Term Different Straw Returning Methods on Soil Organic Carbon, Nutrients and Aggregate Formation in Different Soil Layers of Double Cropping Rice Field [J]. Scientia Agricultura Sinica, 2026, 59(7): 1492-1506.
[5] LI XingYu, HUANG Rong, XIAN YiMing, TIAN JiaoJiao, MA XiaoJin, YANG QiaoXi, LI Bing, WANG ChangQuan. Characteristics of Organic Carbon Fractions and Carbon Dioxide Emissions of Different Size Aggregates in Rice Field Soils in Response to Long-Term Fertilization [J]. Scientia Agricultura Sinica, 2026, 59(6): 1255-1271.
[6] MA ZhaoHui, QUAN ChengZhe, CHENG HaiTao, YANG KanJie, LI XinRui, LÜ WenYan. The Breeding Goals and Strategies of Northeast Japonica Rice Under the Background of Zhongke Fa No.5 [J]. Scientia Agricultura Sinica, 2026, 59(5): 927-936.
[7] ZHANG WeiJian, YAN ShengJi, SHANG ZiYin, TANG ZhiWei, WU LiuGe, LI JiaRui, CHEN HaoTian, DENG AiXing, ZHANG Jun, ZHANG Xin, ZHENG ChengYan, SONG ZhenWei. Methane Emissions from Paddy Fields: Not Entirely Attributable to Rice Cultivation [J]. Scientia Agricultura Sinica, 2026, 59(4): 824-833.
[8] CHEN Min, JIAO ZiLan, QIAO ChengBin, XU Hao, ZHANG Bi, MA DongHua, KONG WeiRu, WANG JingWen, SONG JiaWei, LUO ChengKe, LI PeiFu, TIAN Lei. Morpho-Physiological Responses and Adaptive Strategies of Rice Germplasm Accessions from Different Subspecies Under Salt Stress [J]. Scientia Agricultura Sinica, 2026, 59(4): 705-722.
[9] GUO FuCheng, TANG HaiJiang, HAO XinYi, MA GuoLin, YANG JiuJu, HUANG LinFeng, TIAN Lei, WANG Bin, LUO ChengKe. Effects of Different Irrigation Methods on Water-Salt Transport, Rice Yield, and Water Use Efficiency in Saline Soil in Ningxia [J]. Scientia Agricultura Sinica, 2026, 59(4): 750-764.
[10] LUO Wei, YU Hong, YUAN LiXin, WANG LingLing, ZHAO JinPeng, YIN Wei, WANG MingTian, WANG RuLin. Change of Geographic Distributions of Ratoon Rice in Sichuan- Chongqing Under Global Climate Change [J]. Scientia Agricultura Sinica, 2026, 59(4): 765-780.
[11] ZHU Shu, GUO ZhiPeng, SUN Ying. Functional Analysis of Rice Target of Rapamycin OsTOR in Regulating Root Elongation [J]. Scientia Agricultura Sinica, 2026, 59(3): 475-485.
[12] LÜ WenYan, CHENG HaiTao, MA ZhaoHui, TIAN ShuHua. Discussion on Hybridization Breeding Technology and Strategy of Rice in the New Era of Breeding [J]. Scientia Agricultura Sinica, 2026, 59(2): 233-238.
[13] LIAO TingLu, SHI YaFei, XIAO DongHao, SHE YangMengFei, GUO FuCheng, YANG JiuJu, TANG HaiJiang, LUO ChengKe. The Effect of Exogenous Nitroprusside on Sugar Metabolism in Rice Seedlings Under Alkaline Stress [J]. Scientia Agricultura Sinica, 2026, 59(2): 265-277.
[14] LIU TianSheng, LIU GengYuan, ZHAO AnQi, YANG Xu, CAI MingXue, YANG AiWen, LOU MingXuan, LI MuKai, WANG Han, ZHANG YaLing. Pathogenic Population of Rice Bakanae Disease in Heilongjiang Province [J]. Scientia Agricultura Sinica, 2026, 59(2): 305-321.
[15] WANG ZhongNi, LEI Yue, LI JiaLi, GONG YanLong, ZHU SuSong. Functions of ABC Transporter OsARG1 in Rice Heading Date Regulation [J]. Scientia Agricultura Sinica, 2026, 59(1): 1-16.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
No Suggested Reading articles found!
京ICP备09089781号-30
Copyright 2018 © Editorial office of Scientia Agricultura Sinica
Tel: 86-010-82106279    E-mail: zgnykx@mail.caas.net.cn
Supported by Beijing Magtech Co.Ltd