【作者】 潘秋红；

【导师】 张大鹏；

【作者基本信息】 中国农业大学 ， 果树学， 2003， 博士

【副题名】1.苹果果实酸性转化酶的纯化及抗体制备　2.脱落酸对苹果、葡萄果实酸性转化酶的调节

【摘要】 酸性转化酶（β-fructosidase；EC 3.2.1.26）包括了定位于液泡的可溶性酸性转化酶（SAI）和定位于胞外空间的细胞壁结合酸性转化酶（CWI），它们在经济库细胞中蔗糖的代谢贮藏和作物库强调节中起着十分关键的作用，因此探讨果实发育过程酸性转化酶的调节机制是阐明果实品质形成理论的一个重要方面。本文首先从苹果果实中纯化了SAI和CWI，并制备了抗体，在此基础上，首次探讨了促进果实碳水化合物积累的重要激素—脱落酸（ABA）对苹果和葡萄果实酸性转化酶调节的酶学机制，为进一步弄清果实发育过程中酸性转化酶活性调节的分子机制及其生物学意义奠定了良好的实验和理论基础。 依次通过（NH₄）₂SO₄沉淀、离子交换层析，凝胶过滤和亲和层析等步骤从苹果果实中分别获得了电泳纯的可溶性酸性转化酶和细胞壁酸性转化酶蛋白，其亚基分子量约为50kD，全酶分子量均为220kD；这两种酸性转化酶有着相似的酶学特性，但它们在某些方面与其它植物的酸性转化酶有所不同，如对棉子糖的Km极高，对水苏糖没有水解活性，果糖对酶活性的抑制方式为混合型抑制；在苹果果实中未发现有酸性转化酶的蛋白抑制因子。分别将这两种酸性转化酶的抗血清通过（NH₄）₂SO₄分级沉淀、DEAE-Sepharose层析和POD-Sepharose层析纯化，根据Western blotting、免疫抑制分析，酶联免疫分析和Protein dot blot，表明所制得的这两种抗体对苹果果实酸性转化酶有良好的特异性和较高的检测灵敏度；利用这些抗体在苹果的叶、花和果实的粗提液中均检测到三条多肽，分子量分别为50kD，68kD和30kD，后两条可能在纯化过程中丢失了，苹果中的这两种酸性转化酶可能各存在三种亚型。 通过应用ABA注射连体的苹果（Malus Domestica Borkh cv．Starkrimson）果实和温育果实圆片，研究证明：ABA可明显地激活果实可溶性酸性转化酶和细胞壁酸性转化酶，且这种激活作用具有温育时间、ABA剂量、介质pH、活体组织以及发育阶段的依赖性，ABA最大的激活效应在pH5.5介质中和盛花后100d的果实；20μmol／LABA温育40min即可观察到果实圆片酸性转化酶活性的增加。酶联免疫法定量测定和Western blotting分析均表明：ABA对酸性转化酶的这种激活作用不是通过促进酶蛋白的合成来实现的，而可能是一种翻译后的调节机制；根据这些结果，我们认为以往的研究中所观测到的ABA对经济库器官库强的增强效应可能与ABA诱导酸性转化酶的激活有关。 应用外源ABA预温育葡萄（V．vinifera．×V．lubrusca L．cv．Kyoho）果实圆片和渗透完整果实的实验体系，证实了ABA在果实发育的各个时期都促进了可溶性酸性转化酶和细胞壁酸性转化酶活性；并应用Western blotting和酶联免疫分析，证明了ABA对酸性转化酶活性的促进作用是通过增加其基因表达量来实现的；ABA的这种激活效应具有介质pH、温育时间、ABA剂量和活体组织的依赖性；蛋白质合成抑制剂环已亚胺和mRNA转录抑制剂放线菌素D均可抵消ABA的效应，这些结果表明ABA可能通过激活酸性转化酶，参与果实发育的调控。更多还原

【Abstract】 Acid invertase ( 3 -fructosidase; EC 3.2.1.26) can be categorized into two kinds, one being located in the vacuole, called soluble acid invertase and the other in the apoplasmic space, called cell wall-bound acid invertase. The enzymes play key roles in sucrose metabolism and storage in economic sink cells and the sink strength modulation in crops. So, the study on regulating mechanism of acid invertases in developing fruit should constitute an important step toward elucidating the theory of fruit quality formation. In this paper, soluble and cell wall acid invertases, respectively, were purified from developing apple fruits and the corresponding polyclonal antisera were prepared. Enzymological mechanism of acid invertase regulation by abscisic acid (ABA), a vital hormone that enhances the accumulation of carbohydrate in fruit, was then studied in developing apple fruit and grape berry, respectively. These results provide a substantial base for the further studies on the molecular mechanism and biological significance of acid invertase modulation in developing fruit.Soluble and cell wall- bound acid invertases were respectively purified to apparent electrophoretic homogeneity from apple fruit by the successive procedures including ammonium sulphate precipitation, ion-exchange chromatography, gel filtration and affinity chromatography. The fractions were both shown to have the same apparent molecular mass with a holoenzyme of molecular mass of 220 kD composed of the identical subunits of 50 kD. The two kinds of isoenzymes had similar enzymological characterization and were distinct from the other plant acid invertases in some properties such as the extremely high Km value for raffinose, no hydrolytic activity for stachyose and a mixed form of inhibition by fructose to their activities. The proteinous inhibitor to acid invertase was not found in apple fruit. The antibodies directed against the soluble and cell wall-bound acid invertases, respectively, were purified by sequential ammonium sulphate precipitation, DEAE-Sepharose FF and POD-Sepharose chromatography. The purified antibodies were shown highly specific for apple acid invertases by both Western blotting and immunoinhibition assays and highly sensitive to the enzymes by both enzyme linked immunosorbent assay and protein dot blot. The antibodies immuno-recognized from the crude extract of apple tissues three classes of apple acid invertases with molecular mass of, respectively, 50 kD, 68 kD and 30 kD. The last two invertases may be lost during the purification. Acid invertase in apple fruit may have three isoforms.The present experiment, using the techniques of the injection of ABA into the intact apple (Mains Domestica Borkh cv. Starkrimson) fruit attached to growing apple tree and the incubation of the excised fruit discs in the ABA-contained medium, showed that ABA strongly activated the fruit soluble acid invertase and cell wall-bound acid invertase. The ABA-induced acid invertase activation was time course, ABA dose-, medium pH-, in vivo tissue- and fruit developmental stage dependent with the maximum induced effects being attained, respectively, in the medium of pH 5.5 and at 100 lhday afterfull bloom. The ABA-induced acid invertase activation appeared after 40 min of the in vivo incubation in contained-20 umol/LABA medium. The results of enzyme linked immunosorbent assay and Western blotting study indicated that the acid invertase activation by ABA could involve a post-translational regulation mechanism but no increase in the synthesis of enzyme proteins. These results suggest that the improvement of sink strength by ABA in the economic sink organs may be attributed, at least partly, to the ABA- induced cell wall-bound acid invertase and soluble acid invertase activation.The present experiment, performed with the in vivo incubation of the grape (Vitis vinifera x K labrnsca L.) berry discs in the ABA-contained medium and the in vivo infiltration of ABA into the entire berries, demonstrated that ABA activated both the solu更多还原