芋頭半胱胺酸蛋白酶抑制劑(Tarocystatin)之基因選殖、表現特性及對微生物抗性的分子機制研究

Abstract

芋頭為一傳統食用作物，主要食用部位為球莖及葉部；一般而言，球莖在發育過程中所含有之貯存性蛋白質除了扮演基因之表現與調控外，亦含有抗病及抗菌功能的蛋白酶抑制劑。本實驗在檢測不同芋頭品種後發現高雄1號品種含有一種硫醇型的蛋白質酶抑制劑， 利用5’-RACE及3’-RACE進行PCR反應，得到全長的tarocystain 基因，命名為CeCPI， CeCPI全長共有1,008 bp，其open reading frame (ORF)為618 bp，可轉譯成205 胺基酸，分子量約29 kDa，其序列特徵為: Gly5 (G)位在N-端序列之第5個胺基酸; 第一個binding loop(L1)區域起始於第49個胺基酸位置,含有一個五胜肽(pentapeptide)序列 Gln49-Val50-Val51-Ser52-Gly53 (Q-V-V-S-G); 第二個binding loop(L2) 則在Trp 80 (W80)之後; 在靠近C-端序列處尚有一個不含cysteine 的contact point -(W114); 此外, 在鄰近N-端第22個胺基酸起含有一個保守性的胺基酸序列ARFAVDEHNKK，因此，認為CeCPI屬於phytocystatin之一種。進行胺基酸演譯序列相似度之比較，就芋頭而言，芋頭為網狀脈葉但屬於單子葉植物，而其CeCPI基因序列之胺基酸演譯及分子量大小，與雙子葉植物之相似度高於單子葉植物。 重組CeCPI 蛋白質當濃度在80 mg/ml時，即可抑制白絹菌(Sclerotium rolfsii)菌核之生長，當重組CeCPI蛋白質濃度達150 ~ 200 mg/ml以上，白絹病菌菌核之菌絲的生長完全的受到抑制。以不同濃度重組CeCPI蛋白質檢測其他真菌性病害，發現重組CeCPI蛋白質對某些真菌性病害，如芸苔鏈格孢菌(Alternaria brassica)，瓜果腐霉(Pythium aphanidermatum)及 立枯絲核菌(Rhizoctonia solani)具有明顯抑制其菌絲生長之毒性。 以水稻OC-I為模組，使用SWISS-Model程式分析CeCPI蛋白質結構模式，由3D構造分析，得到重組CeCPI蛋白質由6個α-helix （α1、α2、α3、α4、α5、α6）及7個antiparallelβ-sheet（β1、β2、β3、β4、β5、β6、β7）組成。檢測定點突變後的數種CeCPI蛋白質(site-directed mutant protein)對木瓜酶的抑制活性是否受影響，結果當保守區(L1) Q49位置一旦突變, 抑制木瓜酶的活性則完全喪失，而且對抑制白絹病菌菌核生長之抗性也消失，但在C-端保守區（W80、W114）的突變體雖然對木瓜酶抑制活性部份減弱，但對白絹病菌菌絲仍顯著抑制其生長; 顯然CeCPI 蛋白質對木瓜酶的抑制活性與抑菌活性是二個不同的分子機制。

Taro (Colocasia esculenta L. Schott) is one of the oldest cultivated crops grown for its edible corms and leaves. Taro corm is a well-differentiated organ and contains approximately 1.4 ~ 3.0% storage proteins on a fresh weight basis. In order to investigate the proteinase inhibitors of storage protein in corm of four local taro cultivars, the affinity analysis of the cystatin/papain was performed by using the mild denaturing gelatin /PAGE. Results from substrate polyacrylamide gel electrophoresis revealed that the cultivar-Kaohsiung No. 1 contains cystatin in corm. Subsequently, a cDNA clone, designated CeCPI, encoding a novel phytocystatin was isolated from corm of taro using both degenerated primers/RT-PCR amplification and 5’- / 3’- RACE extension. The full-length cDNA gene is 1,008 bp in size, encodes 205 amino acid residues, with deduced MW. 29 kDa. It contains a conserved reactive site motif Gln-Val-Val-Ser-Gly of cysteine protease inhibitors, and another consensus ARFAV sequence for phytocystatin. Sequence analysis revealed that CeCPI is phylogenetically closely related to eudicots rather than to monocots, despite taro belongs to monocot. Recombinant GST-CeCPI fusion protein was overexpressed in E.coli and its inhibitory activity against papain was identified on gelatin/ SDS-PAGE. These results confirmed that recombinant CeCPI protein exhibited strong cysteine protease inhibitory activity. The investigation of its antifungal activity clearly revealed the toxic effect on the mycelium growth of phytopathogenic fungi, such as Sclerotium rolfsii Sacc. etc., at the concentration of 80 μg of recombinant CeCPI per ml. Moreover, the mycelium growth was completely inhibited and the sclerotia lysed at the concentration of 150 μg – 200 μg per ml. Further studies have demonstrated that the recombinant CeCPI is capable of acting against the endogenous cysteine proteinase in the fungal mycelium. The predicted tertiary structure of CeCPI was modeled based on the known crystal structure of the rice OC-I, which shares 50.5% (48/95) sequence identity with the taro cystatin. The data indicate that tarocystatin consists of six