禽呼肠孤病毒σ3基因在烟草中的表达与鉴定

doi:10.3864/j.issn.0578-1752.2015.09.17

摘要/Abstract

摘要： 【目的】研究禽呼肠孤病毒S1基因编码的σ3基因在烟草中的表达及其表达产物的反应原性。【方法】根据GenBank中公布的禽呼肠孤毒株S1133的基因序列，设计特异引物扩增σ3基因，构建重组植物表达载体pBI121-σ3，热激法转化农杆菌EHA105感受态细胞，筛选含有重组质粒的pBI121-σ3的阳性工程菌株EHA105。采用农杆菌介导的叶盘法转化野生型烟草，卡那霉素筛选获得抗性植株。随后通过σ3基因的特异引物，PCR方法初步筛选获得转σ3基因的烟草植株。进一步对阳性植株通过实时荧光定量PCR方法检测σ3基因在转基因烟草中的拷贝数，以烟草自身单拷贝的内源基因核糖核酸还原酶-RNR2作为内参基因，通过梯度稀释分别构建RNR2及σ3基因的起始模板量和对应C_T值的标准曲线，通过检测样品中σ3基因和RNR2基因模板量对数值的比值估算出σ3基因在转基因烟草中的拷贝数。并通过Western-blotting方法对转基因烟草中表达的σ3融合蛋白的反应原性进行分析。【结果】①通过双酶切和测序验证表明σ3基因插入位置、大小和读码框均正确，表明σ3基因已经成功的构建于植物表达载体pBI121的花椰菜花叶病毒（CaMV）35S启动子的下游，以NOS为终止子，含有卡那霉素抗性标记（NPT II）。其表达的蛋白与下游的绿色荧光蛋白形成融合蛋白，其大小约为61.6 ku；②在转化筛选抗性植株的过程中采用350 mg·L^-1的头孢霉素可以很好地抑制农杆菌的污染，100 mg·L^-1的硫酸卡那霉素筛选压力能够很好地抑制非转化细胞的生长；③随机选取10株转化烟草进行PCR检测发现8株转化烟草可以扩增到清晰的目的条带约994 bp；④内参基因RNR2的标准曲线为y=-3.5352x+15.143，σ3基因的标准曲线为y=-3.5366x+1.8265，两个标准曲线的相关系数均接近于1。在检测的5株转化烟草中σ3基因的拷贝数均为4，阴性对照没有扩增。⑤Western-blotting显示σ3融合蛋白在转化烟草中可以正确表达，目的条带大小约为61.6 kD，与预计大小一致；σ3融合蛋白能够与禽呼肠孤病毒的阳性血清发生特异反应，表明该融合蛋白具有良好的反应原性。【结论】成功地构建了植物表达载体pBI121-σ3，筛选获得低拷贝的转σ3基因的烟草，σ3融合蛋白能够在烟草中表达且具有相应的反应原性，为进一步研究σ3基因的功能和利用植物作为生物反应器生产σ3蛋白口服疫苗的研发奠定了基础。

关键词: 禽呼肠孤, &sigma, 3基因, 植物表达载体, 烟草

Abstract: 【Objective】 An experiment was carried out in laboratory to study on expression of Avian reovirus σ3 gene in tobacco and its genicity reactions of the expression products. 【Method】 A pair of primers was designed according to the major antigen region of the σ3 gene derived from GenBank. A plant vector of pBI121-σ3 constitutively expressed σ3 gene was constructed. The pBI121-σ3 vector was transferred into the Agrobacterium strain EHA105 by thermal activation method and a recombinant Agrobacterium strain EHA105 containing pBI121-σ3 was obtained. After transformation of tobacco plants via Agrobacterium tumefaciens, the resistant plants were selected with kanamycin. The resistant plants were firstly analyzed by PCR, using σ3 gene specific primers, and then real-time fluorescence quantitative PCR was used to further estimate the copy number of the σ3 gene in positive plants, and endogenous RNR2 gene in tobacco was used as a reference gene. With a serial of dilutions, the standard curves of the cycle threshold (C_T) relative to the log of each initial template copy of σ3 and RNR2 genes were obtained. The transgenic copy number was obtained by comparing the initial template copy of σ3 gene with that of RNR2. Western blot analysis was used to examine the σ3 protein expression in transgenic tobacco. 【Result】 The recombinant plasmid was identified by restriction endonuclease analysis and by gene sequencing. It was confirmed by DNA sequencing that the recombinant plasmid pBI121-σ3 contains a complete σ3 gene, a correct insertion site and expected open reading frame (ORF). The plant nuclear vectors expressed the σ3 gene by using CaMV 35S promoter and NOS terminator, and a kanamycin resistance marker (NPT II). The σ3 gene may be expressed as a 61.6 ku green fluorescent fusion protein(GFP) in tobacco. MS medium containing 350 mg cefotaxime·L^-1 inhibited the growth of Agrobacteria and 100 mg kanamycin·L^-1 can provide enough selective pressures. Eight resistant plants containing σ3 gene were obtained by PCR screening. The standard curve of RNR2 gene was expressed as y=-3.5352x+15.143, the standard curve of σ3 gene was expressed as y=-3.5366x+1.8265. Among the five putative transgenic lines, five had four copy numbers, whereas the negative control had none. Western blotting results showed that the 61.6 ku recombinant σ3 protein was successfully expressed, and the protein was specifically recognized by anti ARV positive serum. 【Conclusion】 A plant expression vector of pBI121-σ3 was successfully constructed and the regenerated transgenic σ3 gene tobacco plants were obtained. Recombinant σ3 protein has a good reactivity with anti ARV positive serum. The research findings provide a basis for further analysis on plants as bioreactors development and the production of σ3 oral vaccines.

Key words: avian reovirus, σ3 gene, plant expression vector, tobacco

王盛，谢芝勋，黄莉，谢丽基，邓显文，谢志勤，刘加波，罗思思，曾婷婷. 禽呼肠孤病毒σ3基因在烟草中的表达与鉴定[J]. 中国农业科学, 2015, 48(9): 1836-1844.

WANG Sheng, XIE Zhi-xun, HUANG Li, XIE Li-ji, DENG Xian-wen, XIE Zhi-qin, LIU Jia-bo, LUO Si-si, ZENG Ting-ting. Expression and Identification of the σ3 Gene of Avian Reovirus in Transgenic Tobacco[J]. Scientia Agricultura Sinica, 2015, 48(9): 1836-1844.

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