百合灰黴病菌之絲胺酸蛋白酶 BeSerp 與植物防禦相關蛋白 LsGRP1 之關係探討

Exploration of the relationship between Botrytis elliptica subtilase BeSerp and plant defense-related protein LsGRP1

林姿均(Tzu-Chun Lin)

指導教授 ： 陳昭瑩

國立臺灣大學/生物資源暨農學院/植物病理與微生物學研究所/碩士(2019年)

https://doi.org/10.6342/NTU201902648

摘要

LsGRP1 為葵百合的植物防禦相關蛋白，經由水楊酸澆灌處理葵百合，可增加LsGRP1的表現量，並能增加植物對百合灰黴病菌(Botrytis elliptica)的抗性。利用免疫共沉澱法釣取與 LsGRP1 交互作用之百合灰黴病菌蛋白時，找到大小接近 35 kDa 的蛋白，經高效液相層析電灑法串聯式質譜分析，初步鑑定可能為似枯草桿菌蛋白酶(subtilisin-like protease, subtilase)，屬於絲胺酸蛋白酶(serine protease)，並且在本實驗室建立之感染百合灰黴病菌之百合轉錄體資訊比對到百合灰黴病菌 subtilase 部分序列(劉，2016)，因此本研究旨在探討百合灰黴病菌之推定subtilase與 LsGRP1 之間的交互作用關係。首先取得 subtilase 的cDNA序列，命名為 BeSerp。RT-qPCR分析指出BeSerp 基因在葵百合感染灰黴病菌初期有高的表現量但接種後 48小時的表現量下降。利用 BeSerp之dsRNA 處理病菌孢子後接種百合葉，會使百合灰黴病菌感染的病徵較為嚴重。BeSerp之寄主誘導基因靜默(host-induced gene silencing, HIGS)測試也觀察到BeSerp 與 LsGRP1基因表現以及病徵的差異。藉由農桿菌浸潤處理三天，使百合增量表現 BeSerp後接種百合灰黴病菌，葉片之病徵較為輕微。於病毒誘導基因靜默(virus-induced gene silencing, VIGS) LsGRP1 的百合上，BeSerp 表現也有改變。進一步利用pET-29a(+) 表現 BeSerp，將LsGRP1與 BeSerp 蛋白進行生體外共培養，西方墨漬法偵測到不同於原始蛋白大小的條帶，相對分子量分別為40~50 kDa 及10 kDa 左右。基於一系列的研究認為 BeSerp與 LsGRP1 之間存在著某種關聯，進而影響百合灰黴病菌的感染及百合的病徵表現。

關鍵字

百合灰黴病菌 ； 絲胺酸蛋白酶 ； 似枯草桿菌蛋白酶 ； LsGRP1 ； 百合 ； 病毒誘導基因靜默 ； 短暫表現 ； 寄主誘導基因靜默

並列摘要

LsGRP1 is a plant defense-related protein from Lilium ‘Stargazer’. This protein was found previously to be increased by treating with salicylic acid and capable of enhancing Lilium resistance to the infection of Botrytis elliptica. In order to predict what proteins could interact with LsGRP1, co-immunoprecipitation (Co-IP) was performed and the result showed one protein about 35 kDa might interact with LsGRP1N. Analysis of LC-ESI-MS/MS predicted this putative interacting protein to be a subtilisin-like protease (subtilase), and partial amino acid sequence was found in the transcriptome database of B. elliptica-infected Lilium ‘Stargazer’. Thus, this study aimed to examine the interaction between the putative subtilase of B. elliptica and LsGRP1. Firstly, full-length serine protease cDNA was cloned from B. elliptica and named BeSerp. The gene expression of BeSerp after inoculation with B. elliptica at different times on lily was analyzed, and the result showed that BeSerp transcript appeared in a high amount at initial stage but decreased at 48 hr post fungal inoculation. Then, dsRNA interference and host-induced gene silencing were implemented to examine the involvement of BeSerp in B. elliptica-lily interaction. Transient expression of BeSerp in lily by agroinfiltration was performed and the result indicated symptom of the control was more severe than BeSerp treatment. Thus, BeSerp might have elicitor function or generate elicitor. The gene expression of BeSerp was altered on VIGS-LsGRP1 lily post fungal inoculation. These results indicated the gene expression of BeSerp and LsGRP1 were altered by each other. Afterwards, protein expression vector pET-29a(+) was used to express BeSerp which then co-incubated with LsGRP1 in vitro, and the protein signals about 40~50 kDa and 10 kDa different from the original ones were shown. In brief, the relationship between BeSerp and LsGRP1 was indicated from a series of experiments, which might affect the infection process and symptom development caused by B. elliptica.

並列關鍵字

Botrytis elliptica ； serine protease ； subtilisin-like protease (subtilase) ； LsGRP1 ； lily ； virus-induced gene silencing ； transient expression ； host-induced gene silencing

參考文獻

1. 潘映潔。2015。LsGRP1C 誘導百合灰黴病菌之程序性細胞死亡現象。台灣大學植物病理與微生物學系碩士論文。

2. 劉芳瑋。2016。抗菌蛋白 LsGRP1 之 N 端區段對百合灰黴病之孢子發芽促進作用。台灣大學植物病理與微生物學系碩士論文。

3. 施侑廷。2018。百合防禦相關蛋白 LsGRP1 誘導植物抗灰黴病菌之應用。台灣大學植物病理與微生物學系碩士論文。

4. Baum, J. A., Bogaert, T., Clinton, W., Heck, G. R., Feldmann, P., Ilagan, O., Johnson, S., Plaetinck, G., Munyikwa, T., and Pleau, M. 2007. Control of coleopteran insect pests through RNA interference. Nature Biotechnology 25:1322.

5. Blow, D. M., Birktoft, J. J., and Hartley, B. S. 1969. Role of a buried acid group in the mechanism of action of chymotrypsin. Nature 221:337.