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  

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

作　　者：刘维[1] 刘浩[1] 董双玉 古丰玮 陈志强[1] 王加峰[1] 王慧[1] 

机构地区：[1]华南农业大学国家植物航天育种工程技术研究中心,广州510642

出　　处：《中国农业科学》2017年第23期4575-4584,共10页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2017YFD0100100);国家"863"计划(2012AA101201);国家自然科学基金(31401722);广东省省级科技计划(2016A020210064)

摘　　要：【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-H【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 Avr Pik-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 Avr Pik-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（q RT-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 downwa

关 键 词：水稻 原生质体 亚细胞定位 Western杂交 

分 类 号：S435.111.41[农业科学—农业昆虫与害虫防治]