狂犬病病毒G蛋白在水稻中的表达及遗传稳定性鉴定

doi:10.3969/j.issn.1674-7968.2019.02.002

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Abstract The Rabies virus (RV) G protein is the only structural protein that stimulates the body to produce cellular immunity and induces the production of neutralizing antibodies. In this study, the optimized rabies G gene sequence was inserted into the intermediate vector pMP3, and the intermediate vector pMP3-G was successfully constructed. Then the intermediate vector pMP3-G and the plant vector pCAMBIA1300 were double-digested and ligated, and the plant expression vector pCAMBIA1300-G was successfully constructed. The blank non-transgenic rice (Oryza sativa) seed TP309 was selected and disinfected. The callus was grown on the callus induction medium for 3 d at 31 °C, and the callus was inoculated with Agrobacterium tumefaciens EHA105 which had been transferred to the rabies G gene. The callus was co-cultured for 3 d, and the rabies G gene was introduced into rice callus. The callus was further screened, differentiated, germinated, rooted, and 313 plants were obtained, and the leaves of 313 plants were extracted by hexadecyl trimethyl ammonium bromide (CTAB) method. A total of 79 strains were positively identified by leaf genomic DNA amplification PCR. A total of 50 positively expressed plants were obtained after refining and planting. The seeds of 50 positive plants were randomly selected, cut in half, labeled, and half of the endosperm-free seeds were ground. The extract was extracted from the protein and detected by antigen test strips and western blot. The results showed that G protein was in the transgenic rice seeds. Successfully expressed and has good reactogenicity. Four strains with high expression and best reactivity were selected from 50 strains, and the other half of the seeds containing the endosperm corresponding to the positive test results in the T₁ generation of the 4 strains were planted and planted in the plant. Before and after planting the field, the genomic DNA of T₁ plants was extracted by CTAB method, and the endogenous gene of rice (rice starch branching enzyme, RBE4) was used as an internal reference. The homozygous plants in positive plants were screened by relative fluorescence quantitative PCR. Absolute real-time PCR was used to calculate the copy number of homozygous plants. As a result, 35 out of 132 plants were homozygous plants. The copy number of the homozygous plants were 1, 2, 3, 5, 7, 11, and 16, respectively. This study is dedicated to the expression of rabies G gene in rice and its genetic stability, laying the foundation for the preparation of new rabies subunit vaccines in the future.

Key words： Rabies virus G gene Transgenic rice Fluorescence quantification Homozygote Copy number

Received: 10 October 2018

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Corresponding Authors: *, zhanggaiping20003@163.com

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	SHEN Xue-Jing
	ZHANG Er-Qin
	XU Qian-Ru
	LIU Lin-Ke
	WAN Bo
	LIU Yun-Chao
	SUN Ya-Ning
	ZHAO Dong
	NIU Xiang-Xiang
	DENG Rui-Guang
	ZHANG Gai-Ping

Cite this article:

SHEN Xue-Jing,ZHANG Er-Qin,XU Qian-Ru, et al. Expression of Rabies virus G Protein in Rice (Oryza sativa) and Identification of Its Genetic Stability[J]. 农业生物技术学报, 2019, 27(2): 204-211.

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http://journal05.magtech.org.cn/Jwk_ny/EN/10.3969/j.issn.1674-7968.2019.02.002 OR http://journal05.magtech.org.cn/Jwk_ny/EN/Y2019/V27/I2/204

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