絲瓜簇葉病植物菌質體相關核醣核酸水解酵素之特性分析

Abstract

以絲瓜簇葉病（loofah witches' broom）之病原植物菌質體（phytoplasma原稱為mycoplasma-like organism; MLO）感染於日日春（Catharanthus roseus (L.) Don.），待病癥出現，抽取蛋白質，再以單向與雙向進行核醣核酸水解酵素（ribonuclease; RNase）的活性電泳分析（substrate-base gel assay）。與未感染植物菌質體的日日春核醣核酸水解酵素比較，發現感染者會明顯多出一條核醣核酸水解酵素活性條帶，稱為phytoplasma-related RNase，其分子量約為6.8 kDa，等電點（isoelectric point, pI）為4到5之間，為內切核醣核酸水解酵素（endoribonuclease），特殊作用序列為5'-G↓U-3'。進一步對這些核醣核酸水解酵素的生化活性進行分析，發現作用最適pH範圍相當大（pH 3-8），EDTA及一價陽離子（Na+，K+）對其皆無抑制或促進效果；然Ca2+、Mg2+則可輕微抑制，對Zn2+和β-mercaptoethanol的存在則相當敏感。為確定此核醣核酸水解酵素是否是植物菌質體或在它感染後產生，吾人取不同品系和不同生長時期（開花、老化）的日日春、細胞間流出液（intercellular washing fluids, IWFs）以及各種逆境（缺水，熱休克和乙醇），植物激素（2-chloro-ethyl phosphonic acid；離層酸（abscisic acid））等處理未感染的日日春，結果顯示該核醣核酸水解酵素活性不會因此而表現，故初步確定此核醣核酸水解酵素為植物菌質體感染所造成，唯其是植物菌質體本身所有或因植物菌質體的感染寄主而誘導出來及其功能尚待進一步探討。我們也分析比較其他非植物菌植體感染才產生的核醣核酸水解酵素之生化特性，發現主要是在26.7 kDa與21.5 kDa的位置有活性表現，前者的活性為所有核醣核酸水解酵素同功酵素中最高，稱major group，後者次之，為minor group。兩者特性相近，Ca2+與Mg2+對其活性沒有影響，Zn2+與β-mercaptoethanol則可有效抑制，pI值為4到5之間。惟EDTA可促進major group的活性，而抑制minor group，另外低濃度的一價陽離子（Na+，K+）即可抑制minor group，而major group則有較高的忍受力。

To understand the mechanism of pathogenesis of the phytoplasma, we inoculated periwinkles with phytoplasma of witches' broom associating loofah. When symptoms appeared, we extracted protein from leaves and analyzed their RNase activity by two-dimensional substrate-base electrophoresis. Compared with uninoculated periwinkles, inoculated ones expressed at least one new RNase, called phytoplasma-related RNase with a molecular weight of 6.8 kDa and an isoelectric point between 4-5. In the flowers and yellow wilting leaves (natural senescent state) of healthy periwinkles, the activity was not observed. Because the phytoplasmas are always found in the plant phloem sieve tubes, the RNase activities of intercellular washing fluids (IWFs) from noninoculated- and infected- periwinkles were also tested. However, the RNase activity is present only in the infected IWFs. Further analyses indicated that the phytoplasma-related RNase exhibits a broad pH optimum (pH 3-8), is inhibited by CaCl2, MgCl2 and ZnCl2, or β-mercaptoethanol, but is insensitive to EDTA, KC1 and NaCl. In order to examine if the phytoplasma-related RNase can be induced by other factors, we treated healthy periwinkles with various environmental stresses including heat shock, drought stress and ethanol stress. In addition, phytohormone abscisic acid (ABA) and 2-chloro-ethyl phosphonic acid were tested, neither stress or phytohormone induced the phytoplasma-related RNase. At the limits of detection this new RNase is found only in infected periwinkles cells. We also characterized the non-phytoplasma-related RNases: a major activity group of several bands and a minor activity one with several bands with molecular weights of about 26.7 kDa and 21.5 kDa, respectively. This isoelectric point is about 4-5, and they are both insensitive to Ca2+ and Mg2+, and are inactivated by Zn2+ and β-mercaptoethanol. However, while the major group is enhanced by EDTA, the minor group is inactivated. Furthermore, unlike the minor group, the major group exhibits higher tolerance to Na+ and K+.