水稻叶鞘原生质体转化体系的构建及Pik-H4和AvrPik-H4蛋白的瞬时表达

【目的】探索水稻叶鞘原生质体最佳游离时间以及转化时间,提高瞬时表达效率,在蛋白水平对目的基因进行检测且大批量表达。探索该系统表达抗稻瘟病蛋白Pik1-H4、Pik2-H4及无毒蛋白Avr Pik-H4的可行性,对目的基因的功能进行分析。【方法】以高抗稻瘟病品种H4及对照品种中二软占作为试验材料,利用1/2 MS培养基种植水稻幼苗25℃恒温培养7—10 d。利用纤维素酶及离析酶对水稻叶鞘进行酶解,血球计数板分别统计游离1、2、3、4、5、6、7、8、9、10、11和12 h的细胞数目获得最佳的酶解时间。将目标基因Pik1-H4、Pik2-H4及Avr Pik-H4分别与GFP融合,构建瞬时表达载体,利用PEG介导转入水稻叶鞘原生质体。设置转化10、12、14、16、18、20、22和24 h,分别提取细胞总RNA,UBQ管家基因为对照,设计GFP特异性扩增引物,利用实时荧光定量PCR(q RT-PCR)检测GFP的相对表达量,探索最佳的转化时间。通过激光共聚焦扫描显微镜对目标基因进行亚细胞定位观察,推测基因功能。提取细胞总蛋白,以Anti-GFP为一抗用Western blot对目标蛋白表达进行验证。【结果】相对室温土壤栽培,营养丰富且均衡的1/2 MS培养基恒温种植的水稻幼苗质量更优质,活力更高。游离时间的长短对游离效率影响较大,游离的最佳时间为4—6 h,在3—4 h细胞游离数目增长速度最快,4—6 h细胞数量趋于平稳,6 h以后细胞总量呈现下降趋势,特别是7 h以后,显微下细胞碎片增多,细胞死亡速度加快。检测GFP的相对表达量,获得最佳转化时间为14—16 h,16 h达到最高值,之后逐渐下降。随着时间的推移,荧光显微镜下观察到GFP蛋白发出的荧光逐渐淬灭。亚细胞定位观察发现Avr Pik-H4蛋白主要被定位于水稻细胞膜上,初步推测是一种膜蛋白,通过某种形式运输到宿主细胞作为激发子触发一系列反应。Pik-H4是高效广谱抗稻瘟病基因,分为Pik1-H4和Pik2-H4两个部分,Pik1-H4主要定位在内质网,Pik2-H4主要定位在质体,从定位结果初步推定Pik1-H4可能主要参与Avr Pik-H4蛋白的识别反应,Pik2-H4主要起到调控下游抗病的作用。Western blot结果显示目标蛋白表达成功,分子大小正确。Pik1-H4和Avr Pik-H4的表达量高于Pik2-H4,说明分子量的大小不是影响转化效率的关键因素。【结论】水稻叶鞘原生质体瞬时表达系统具有高效快速的特点,通过对游离及转化时间的探索为水稻瞬时表达体系的广泛实践应用提供参考。目标基因的成功表达为Pik-H4与无毒蛋白互作机制的研究提供了有价值的理论依据。

Construction of Rice Leaf Sheath Protoplast Transformation System and Transient Expression of Pik-H4 and AvrPik-H4 Proteins

【Objective】 The objective of this study is to obtain the suitable digestion and transformation time of protoplasts of rice sheath, improve the efficiency of transient expression, the target gene can be detected at the protein level and expressed in large quantities. To explore the feasibility of transient expression of rice blast resistance protein Pik₁-H4, Pik₂-H4 and avirulence protein AvrPik-H4 in protoplasts of rice leaf sheath, and to analyze the function of above target genes.【Method】High blast resistance rice variety H4 and control variety Zhonger Ruanzhan were used as experimental materials. Rice seedlings were cultured with 1/2 MS medium at 25℃ for 7-10 d. The protoplasts were isolated by cellulase and macerozyme enzymatic action. The optimal time of digestion was obtained by counting the number of cells in 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12 h using hemocytometer. The target genes Pik₁-H4, Pik₂-H4 and AvrPik-H4 were fused with GFP to construct the transient expression vector. The total RNA was extracted from transformed protoplasts in 10, 12, 14, 16, 18, 20, 22 and 24 h, respectively. Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of GFP to obtain the best transformation time, UBQ housekeeping gene was used as control and GFP specific amplification primers were designed. The method of subcellular localization of the target gene by laser confocal scanning microscopy was used to estimate the gene function. The total protein was extracted and anti-GFP was used as the primary antibody, verified that the target protein successful expression by Western blot. 【Result】 rice seedlings were grown better quality and vitality in constant temperature 1/2 MS medium with rich and balanced nutrients, compared with soil planted at room temperature. Digested time had a greater impact on protoplast isolation efficiency. The results showed that the best time to digest was 4-6 h. The number of cells grew fastest at 3-4 h, tended to be stable at 4-6 h, showed a downward trend after 6 h, cell death rate accelerated and observed debris increased in the microscopic cell after 7 h. By detecting the relative expression of GFP, it was found that the most suitable time for transformation was 14-16 h, reached the highest value at 16 h, and then gradually decreased. Subsequently, fluorescence of the GFP protein was observed to be quenched by fluorescence microscopy. Subcellular localization observation of AvrPik-H4 protein was mainly located in the cell membrane, presumably this is a membrane protein that is transported by some form to the host cell as an exciton to trigger a series of reactions. Pik-H4 is composed of Pik₁-H4 and Pik₂-H4, which is highly efficient broad-spectrum rice blast gene. Pik₁-H4 and Pik₂-H4 were mainly located in the endoplasmic reticulum and plastid, respectively. From the subcellular localization results, it was presumed that Pik₁-H4 might be mainly involved in the recognition of Avr-Pik protein and signal transmission, Pik₂-H4 mainly play a role in changing the energy transmission and regulation of downstream disease caused hypersensitive reaction. Western blot results showed that the target protein was successfully expressed and the molecular size was correct. The expression of Pik₁-H4 and AvrPik-H4 was higher than that of Pik₂-H4, indicating that the size of the molecular weight is not a key factor affecting the transforming efficiency. 【Conclusion】 The protoplast transient expression system of rice leaf sheath has the characteristics of high efficiency and rapidity, the exploration of protoplast isolation and transformation time has laid a foundation for the extensive practice of rice transient expression system. The successful expression of the target gene has provided a valuable theoretical basis for the study of the interaction mechanism between Pik-H4 and Avr protein.