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

Abstract

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

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.

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