转基因水稻中CAS9蛋白质的免疫印迹检测

doi:10.3864/j.issn.0578-1752.2017.19.001

摘要/Abstract

摘要： 【目的】制备抗CAS9蛋白质的单克隆抗体，建立转基因水稻中CAS9蛋白质的免疫印迹检测方法，了解CAS9蛋白质在转基因水稻中的表达特征。【方法】以带Cas9的质粒DNA为模板，PCR扩增Cas9 5′端810 bp片段后克隆到pET30a载体中，将酶切验证且序列正确的重组质粒转入表达菌Condon Plus中，经IPTG诱导表达CAS9蛋白质（N端270氨基酸），用纯化的重组蛋白质作为免疫原免疫小鼠，制备单克隆抗体，用免疫印迹分析筛选特异性和灵敏度高的抗体细胞株。用特异性引物PCR扩增转基因水稻的Cas9，用免疫印迹检测转基因水稻中的CAS9蛋白质。将重组CAS9蛋白质和带有CAS9的水稻苗期叶片蛋白质进行平行免疫印迹分析，用Image J软件采集信号绘制CAS9蛋白质的标准曲线，进而对水稻叶片中的CAS9蛋白质进行定量分析。提取单粒稻米的总蛋白质，稀释后用免疫印迹分析CAS9蛋白质的检测下限，提取多个时期、部位的水稻材料的总蛋白质，包括苗期的地上部和地下部，分蘖期的茎、茎节、叶鞘、叶片等，用SDS-PAGE分离后免疫印迹检测比较CAS9蛋白质的表达特征。【结果】通过体外克隆、诱导表达获得了纯化的重组CAS9蛋白质N端片段，免疫小鼠后得到42株阳性杂交瘤细胞株，经筛选鉴定到#12D2细胞株对水稻样品的检测具有较好的特异性和灵敏度。用该抗体通过免疫印迹检测了转基因水稻，所建立的免疫印迹方法对重组CAS9蛋白质的检测下限约为0.25 ng。在水稻苗期叶片中，CAS9蛋白质约占鲜重的0.00005%，可检出单粒稻米8%样品（约2 mg）中的CAS9蛋白质。在苗期地上部CAS9蛋白质的表达丰度高于地下部，分蘖期茎和叶片中表达量较高，根和叶鞘表达量较低。【结论】获得特异性强、灵敏度高的抗CAS9单克隆抗体，建立了检测转基因水稻中CAS9蛋白质的免疫印记方法，揭示了CAS9蛋白质在水稻不同部位的表达特征，并展示了在其他植物中的应用潜力。

关键词: 水稻, 转基因植物, CAS9蛋白质, 单克隆抗体, 免疫印迹

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

郭亚璐，马晓飞，史佳楠，张柳，张剑硕，黄腾，武鹏程，康昊翔，耿广荟，陈浩，魏健，窦世娟，李莉云，尹长城，刘国振. 转基因水稻中CAS9蛋白质的免疫印迹检测[J]. 中国农业科学, 2017, 50(19): 3631-3639.

GUO YaLu, MA XiaoFei, SHI JiaNan, ZHANG Liu, ZHANG JianShuo, HUANG Teng, WU PengCheng, KANG HaoXiang, GENG GuangHui, CHEN Hao, WEI Jian, DOU ShiJuan, LI LiYun, YIN ChangCheng, LIU GuoZhen . Western Blot Detection of CAS9 Protein in Transgenic Rice[J]. Scientia Agricultura Sinica, 2017, 50(19): 3631-3639.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2017.19.001

https://www.chinaagrisci.com/CN/Y2017/V50/I19/3631

参考文献

[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)