乳突果组培苗中脂氧合酶基因的诱导及克隆
其他题名Inducement and Clone of Lipoxygenase Gene from Cultural Seedlings of Adelostemma gracillimum
李靖
学位类型硕士
导师沈月毛
2004
学位授予单位中国科学院昆明植物研究所
学位授予地点中国科学院昆明植物研究所
学位专业植物学
关键词脂氧合酶 乳突果 几丁质 Cdna克隆 云南美登木 水杨酸 多羟基脂肪酸 杜仲 抗真菌蛋白
摘要本论文分三部分,第一部分为综述。通过查阅文献详述了当前植物脂氧合酶的研究进展,包括植物脂氧合酶基因的表达调控、高等植物中脂氧合酶途径的生化研究进展、脂氧合酶初产物的色谱分析,并展望了植物脂氧合酶的研究趋势。第二部分为乳突果组培苗中脂氧合酶基因的诱导及克隆。脂氧合酶是植物十八碳酸途径中一个很重要的酶,该酶作用的产物在植物的生长发育过程中以及在植物对环境胁迫反应中起着重要的作用。先前,我们从乳突果愈伤组织中分离了两个三羟基十八碳烯酸,经分析MS和NMR波谱数据分别鉴定为9,12,13-三羟基-10-十八碳烯酸和9,10,11-三羟基-12-十八碳烯酸,它们具有相同的分子式和分子量(m/z330),而在LC-ESI-MS实验中准分子离子峰m/z 353[M十Na]+的保留时间分别为7和10min,从结构推断它们为脂氧合酶途径的产物。目前尚未见有关乳突果甚至萝摩科植物中脂氧合酶的研究报道。用几丁质对乳突果组培苗进行诱导,检侧氧脂含量,并分离脂氧合酶基因,可以探索几丁质诱导下的十八碳酸代谢途径,并为酶蛋白的分离及研究脂氧合酶的功能奠定基础。本论文用150 mg/L的几丁质对乳突果组培苗进行诱导,通过LC-ESI-MS测定几丁质诱导下悬浮培养系中三羟基十八碳烯酸的含量变化,发现加入几丁质12小时后,悬浮培养物中积累较高水平的9,10,11-三羟基-12-十八碳烯酸,该水平持续到诱导后24小时。粗酶活性鉴定结果显示,该酶可催化亚油酸酷生成9,10,11-三羟基-12-十八碳烯酸。根据诱导数据结合文献资料,对用几丁质诱导6小时的乳突果苗进行总RNA的提取,根据从植物中己克隆到的脂氧合酶基因的保守区合成引物,用RT-PCR技术克隆得到约600 bp脂氧合酶基因片段并测序。从总RNA中纯化出mRNA,通过RACE-PCR的方法得到脂氧合酶全长cDNA序列。该序列是一个新的植物脂氧合酶基因序列,在萝摩科植物中属于首次报道。根据序列分析结果,该酶和烟草的LOX具有最高的氨基酸同源性(72%),其次是土豆(70%),核苷酸序列同源性最高的分别是番茄(79%)和烟草(78%),它们都属于茄科植物。根据氨基酸序列分析结果,该蛋白分子量为98.0kD,pI为5.94,该酶在氨基酸581~582位置含有TV保守区,该区决定了产物的特异性,该酶具有9-LOX活性。第三部分为硕士期间其它两方面的工作:(1)水杨酸诱导美登木悬浮细胞产生脂氧合酶及多羟基脂肪酸的研究。水杨酸(SA)诱导云南美登木。(Maytenushookeri)悬浮培养细胞产生9-LOX,该酶可催化亚油酸生成9,12,13-三羟基-10-十八碳烯酸。通过Lc-ESI-MS测定SA诱导下悬浮培养系中多羟基脂肪酸的含量变化,发现用浓度为800协molfL的sA诱导培养18小时,悬浮细胞产生最大量的三羟基十八碳烯酸。同时,通过对比不同羟基十八碳烯酸含量的变化,发现在SA诱导下,9-Lox介导的十八碳酸途径中有环氧中间体的生成。(2)杜仲抗真菌蛋白(EAFP)时空表达特性的研究。通过分离纯化杜仲抗真菌蛋白来监测该蛋白在杜仲中的部位分布(皮、叶、根)和不同时间的分布。同时利用平板抑菌实验来检测杜仲抗真菌蛋白的抗菌活性。实验结果显示,杜仲抗真菌蛋白主要分布在树皮中,根中分布较少,而在叶中未被检测到。而且该蛋白的分布较为稳定,不随生长周期而改变。
其他摘要Three parts were included in this thesis. The first part was a review about the recent advances in plant lipoxygenases, including gene expression and regulation of plant lipoxygenases, recent developments in biochemistry of higher plant lipoxygenase pathway, chromatographic analysis of lipoxygenase products and the prospect of study trends in plant lipoxygenases. The second part was about the inducement and cloning of 9-LOX gene from the seedling cultures of Adelostemma gracillimum (Asclepiadaceae). Lipoxygenases are important enzymes in plant octadecanoic acid pathway. Products of this enzyme have important functions in plant growth and development, as well as in response to environmental stress. We isolated two trihydroxy octadecenoic acids in the calli of Adelostemma gracillimum before, which were identified to 9,12,13-trihydroxy-10-octadecenoic acid and 9,10,11-trihydroxy-12-octadecenoic acid by MS and NMR analysis. They had the same molecular formula and the same molecular weight (m/z 330), but the retention time were 7min and lOmin respectively according to LC-MS detection. From the structure, they were predicted as products of LOX pathway. No research about lipoxygenase in Asclepiadaceae was reported before. In this thesis, inducement experiment on Adelostemma gracillimum seedlings and isolation of lipoxygenase gene were conducted to explore the octadecanoic acid pathway induced by chitosan. The results also contributed to the isolation of lipoxygenase and the function analysis of lipoxygenase. In the inducement experiment, chitosan of 150 mg /L was used. Through LC-ESI-MS studies on content variation of trihydroxy fatty acid, we found that suspension cultures accumulated maximum 9,10,11-trihydroxy-12-octadecenoic acid after being treated with 150 mg /L chitosan for 12 hours and lasted to the 24th hour. The enzyme can catalyze linoleic acid to 9,10,11-trihydroxy-12-octadecenoic acid. Seedlings of Adelostemma gracillimum induced by chitosan for six hours were used for RNA extraction. A 600bp fragment of LOX gene was amplified by RT-PCR and sequenced. mRNA was isolated from RNA and the full cDNA of LOX was cloned by RACE-PCR. This new LOX sequence was the first time to be reported in Asclepiadaceae. The sequence showed highest identity ,at the amino-acid level, with common tobacco (72%) and next with potato (70%) .At the nucleic acid level, it showed the highest identity with tomato (79%) and common tobacco (78%) .All the plants concerned belonged to Solanaceae. The molecular weight of this protein was 98.0kD and the predicted pi was 5.94.The LOX contained a conserved TV motif in the expected position(581~582) ,which determined the product specificity. The enzyme functioned as 9-LOX. The third part were two other experiments in the course of master degree study. The first experiment concerned about studies on lipoxygenase and polyhydroxy fatty acid in SA-elicited Maytenus hookeri suspension cells. The production of 9-LOX was induced by salicylic acid (SA) in the suspension cell cultures of Maytenus hookeri, which can catalyze linoleic acid to 9,12,13-trihydroxy-10-octadecenoic acid. Through LC-ESI-MS studies on content variation of polyhydroxy fatty acid induced by SA, we found that suspension cells accumulated maximum trihydroxy octadecenoic acid after being treated with 800 μmol/L SA for 18 hours. At the same time, by comparing the content variation of different hydroxy octadecenoic acid, we can conclude that the octadecanoic acid pathway mediated by 9-LOX included the production of epoxy intermediate. The second experiment concerned about research into the accumulation of the antifungal protein in Eucommia ulmoide Oliv (EAFP).The distribution of EAFP in different parts and during different growth phases of Eucommia ulmoide was detected by protein isolation and purification. At the same time, antimicrobial activity was determined by agar diffusion assay. The results indicated that the EAFPs mainly distributed in barks, a little in roots, whereas no EAFPs were detected in leaves. And the trend of the protein distribution did not vary with the plant life cycle, remaining comparatively steady.
页数60
语种中文
文献类型学位论文
条目标识符http://ir.kib.ac.cn/handle/151853/738
专题昆明植物所硕博研究生毕业学位论文
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李靖. 乳突果组培苗中脂氧合酶基因的诱导及克隆[D]. 中国科学院昆明植物研究所. 中国科学院昆明植物研究所,2004.
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