【作者】 李萌；

【导师】 张元湖； 孟庆伟；

【作者基本信息】 山东农业大学 ， 植物学， 2005， 硕士

【摘要】 α-法尼烯是存在于植物体内的次生代谢物。α-法尼烯与昆虫的诱导性有关,是苹果蠹蛾幼虫与成虫的引诱剂;α-法尼烯含量与植物的冷害程度呈正相关;α-法尼烯是诱导虎皮病发生的重要因素。虎皮病是苹果、梨在低温贮藏中后期发生的最严重的生理病害之一,果皮中α-法尼烯的生物合成是通过类异戊二烯途径中的甲羟戊酸途径,调控此途径的酶包括3-羟基-3-甲基戊二酸单酰辅酶A还原酶(HMGR),法尼基焦磷酸合成酶(FPPS),α-法尼烯合成酶(AFS)。其中AFS是调控α-法尼烯合成的最直接的限速酶,它催化法尼基焦磷酸(FPP)转变为α-法尼烯。近年来,通过基因敲除或反义抑制的方法调控AFS基因的表达,从而调控α-法尼烯合成的研究及解释虎皮病发生的分子机理成为国内外研究的热点问题。本研究根据Genbank 注册的α-法尼烯合成酶基因序列,设计特异引物,从青香蕉苹果果皮中克隆得到AFS 基因的cDNA 片断,研究了其在苹果低温冷藏(0℃)期间及出库后一段时期内AFS 基因的表达与α-法尼烯释放的关系;构建了原核表达载体,在大肠杆菌中表达了该基因;利用农杆菌介导的叶盘法成功转化烟草,并初步对转基因烟草部分光合指标进行了测定。主要结果如下: 1 采用RT-PCR 方法,从苹果果皮中克隆得到了苹果α-法尼烯合成酶基因(AFS)cDNA,该基因编码区全长1728 个碱基,编码576 个氨基酸。在GenBank 注册该基因(登记号为AY563622)。2 序列比较发现该序列与其他品种苹果和梨的α-法尼烯合成酶基因同源性较高,与其它作物如黄瓜、松、杉植物等同源性较低。Southern 杂交结果显示,AFS 基因在苹果基因组中以单拷贝形式存在。3 对苹果冷藏期间AFS 的表达进行的Northern 杂交结果显示,在对照果实中,AFS 基因在冷藏初期表达量较低,到冷藏的第12 周表达量达到最高,随时间延长表达量降低;用新的乙烯抑制剂1-MCP 处理可以抑更多还原

【Abstract】 α-Farnesene is a secondary metabolite in plant. This compound, among many other components in apple volatiles, has been proven to play major roles in host plant searching and reproduction of codling moth, to be correlated to the degree of plant sufferred from cold, and to the development of superficial scald which is one kind of serious storage disorder of several apple cultivars. The accumulation of α-farnesene in the skin of apple fruit during storage appears to be predominantly through classical mevalonate (MVA) pathway. 3-Hydroxy-3-methylglutaryl CoA reductase (HMGR), farnesyl diphosphate synthase (FPPS) and α-farnesene synthase (AFS) are the rate-limiting enzymes in the mevalonate pathway. AFS is the final, rate-limiting enzyme that converts farnesyl diphosphate (FPP) to α-farnesene in the mevalonic acid (MVA) pathway of terpene biosynthesis. Studies have been focused on the control of α-farnesene synthesis and molecular bases of scald development by gene knockout or antisense RNA of AFS. To elucidate the relationship between AFS expression and α-farnesene synthesis during low temperature storage and after apples warming to room temperature, the cDNA encoding α-farnesene synthase gene (AFS) was cloned from ‘White Pearmain’apple. Specific primers for PCR designed from AFS-1 had been registered in GenBank (AY182241). The results were as followed. 1 According to the specific primer a sequence about 1800bp concluding the ORF of AFS gene was cloned by RT-PCR, the clone contains an open reading frame (ORF) of 1728 bp comprising 576 amino acid residues and was registered in GenBank (Accession No.AY563622). 2 The deduced amino acid sequence shows high identities with other α-farnesene synthase gene from other apple and pear varieties and low identities with crops such as cucumber, pinus and picea. Southern blot analysis indicated that AFS gene is only one copy in genome of apple. 3 Northern blot analysis for elucidating the expression characteristic of AFS gene in cold storage indicated that the expression of AFS gene in control was low at beginning of cold storage, and reached a peak at 12th weeks, and decreased in followed cold storage. The expression of AFS gene in apple peel was inhibited by 1-MCP, and there is no detection from 8th to 20th weeks. The expression of AFS in apple peel treated with DPA was postponed and reached maximal in 16th weeks, and dropped during continued storage. 4 Northern blot analysis showed that AFS expression level of non-treated apple kept increasing gradually when cold storage apple was shifted to room temperature for 10 days. AFS expression in apple peel treated with DPA reached a higher level at first day and dropped gradually then after; there was no AFS expression in fruit treated with 1-MCP at the first day, less expression was detected at the fifth day, and more at the tenth day. 5 Quantification of α-farnesene was performed with HPLC system. The content of α-farnesene in apple peel tissue treated with DPA decreased at 10 days after warming up to room temperature, and the content in apples treated with 1-MCP and in control increased. But the content of α-farnesene in apple peel treated with DPA was higher than that of non-treated apple at the first day.更多还原