Abstract: 【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.

Key words: rice, protoplast, subcellular localization, Western blot