作者：杨满业
院校：四川大学
关键词：
学位：博士
专业：植物学
摘要：
苦瓜(Momordica charantia L.)隶属于葫芦科(cucurbiteae)苦瓜属，它不仅是我国及东南亚各国人民喜爱的传统蔬菜，也是一种重要的药用植物。苦瓜具有清热、解毒、降血糖、抗生育、抗癌和抗艾滋病等功效。已分离纯化出苦瓜凝集素(Momordica Charantia Lectin，MCL)；α—苦瓜素(α—Momorcharin)；β—苦瓜素(β—Momorcharin)；苦瓜抑制剂(Momordica Charantia Inhibitor，MCI)以及核糖体失活蛋白(Ribosome Inactivating Protein，RIP)，和苦瓜免疫缺陷病素Ⅰ型抑制剂(Inhibitor of VIH—Ⅰ)等多种苦瓜种仁蛋白。近年来，又从新鲜苦瓜原汁中，提纯出一些新的化学成分，主要为苦瓜素甙、胡萝卜甾醇和抗生育活性成分。但有关苦瓜组织培养的研究很少，其离体繁殖技术至今尚未得到解决，严重影响了珍稀濒危品种的保存和繁殖。本试验通过组织培养首次由外植体形成愈伤组织，愈伤组织分化出不定芽，不定芽产生试管苗，初步建立了一个完整的苦瓜离体繁殖体系。为苦瓜的遗传转化奠定了基础，开辟了新的品种改良途径。并分离得到了苦瓜开花相关基因BAG。试验结果如下： 1．苦瓜容易诱导出愈伤组织。外植体在含激素的培养基上产生愈伤组织的过程中对激素种类，用量不敏感，愈伤组织的诱导频率一般为90.0％左右。在愈伤组织起始发生的时间上，幼根、上胚轴、下胚轴、子叶、幼叶等各营养器官的外植体间无明显差异，一般外植体接种四天后开始膨大。愈伤组织的生长速度上，不同组织器官间存在着显著差异。实验表明，幼根、下胚轴、子叶等外植体愈伤组织的生长速度较快；上胚轴、幼叶等外值体的愈伤组织生长速度较慢。 四川大学博士学位论文 2.苦瓜外植体所形成的愈伤组织从形态学上可分为三种不同的类型:绿色 愈伤组织;黄绿色愈伤组织和黄色的愈伤组织。在愈伤组织分化形成不定芽的 过程中，植物激素的种类，用量及所用愈伤组织的类型非常重要。绿色愈伤组 织和黄绿色愈伤组织在培养基(MS十6一BA+KT)上成功地诱导分化出不定芽，最 高分化频率是66.7%;黄色愈伤组织即使在适宜条件下也不能分化出不定芽。 3.筛选出培养基(MS+zT 5.0 mgl一‘+K T o.smgl一今较为适合芽的增殖。增 殖系数为5一6，且幼芽的基部产生较少的愈伤组织。 4.培养基1/2 MS+ZT 0.02 mgl一‘或1/2 MS有助于幼芽离体生根，这两种 培养基既有利于幼芽长出新根，又不至于在幼芽基部产生太多的愈伤组织，影 响试管苗的移栽成活率，是苦瓜幼芽离体生根较为适合的培养基。 5.试管苗经大田移栽试验，成活率约为70.既，其性状与种子苗无明显差异。 6.采用RT一PCR和5’末端快速扩增法成功地克隆了苦瓜开花相关基因BA民 cDNA全长1001个碱基，包含一个完整的，编码228个氨基酸的开放读码框(Open Reading Frame)。5’末端非翻译区由50个碱基组成。3’末端非翻译区含267 个碱基，其中包括由22个碱基组成的ploy(A)‘尾巴及位于833bp处的ploy(A)‘ 加尾信号(从T拟认)。GenBank登录号为:AY 178837。BA‘基因是拟DS一box基因， 含有完整的MADS~box(氨基酸序号1一58)和K一box(氨基酸序号88一193)，与 多种植物的MADS一box蛋白具有很高的同源性。与黄瓜(cucu刃jssatz’憋) 以DS一box Protein‘Z服l口的同源性为95%，与棉花(曲ss动ier hlr万ut姗)MADS box Protein份旅爪7S二2的同源性为84%，与拟南芥栩厂日bz’故沪Sjs动ali日na) 拟DS一box Protein AGLn的同源性为72%;同典型的毗DS一box基因AP3、A夕活0、 夕乙沪讨、J倾二脚、‘5万沪，的毗DS一box区域分别有46、38、44、45、34个氨基酸残基相 同。同源性分别为80%、66%、76%、78%、59%。Southern杂交分析标明， BA口基因在苦瓜基因组中有2或3个拷贝，属于低拷贝数的基因。Northern杂 交和RT一PCR检测证实，BA吞基因在苦瓜花的雄蕊、心皮中大量表达，杂交信号 强烈，在其它的组织、器官中没有明显的杂交信号。其中心皮的表达量最为丰 富。这种表达模式与控制黄瓜花发育的MADS一box基因cA必的表达模式相同。 7.提取苦瓜基因组总DNA，构建DNA步移文库，根据BA口基因的已知序列设 计引物，通过染色体步移技术克隆出别口基因起始密码子上游调控序列BAGP。 四川大学博士学位论文 对BAGP的鉴定和分析表明其具备大多数高等植物启动子的保守元件，预测它对 别‘基因的表达具有一定的作用。为鉴定BA‘基因的基本启动子元件，将基因5’ 侧翼序列做缺失片段分析，利用PCR方法从BAGP中得到三个大小不等两端带有 Hz’ndIII、山斑厅I酶切位点的片段BAGPI，BAGPZ和BAGP3，定向插入载体 pMGFP4(pBI221改建，报告基因为GFP)中，取代原有的C蒯V35S启动子，构建 了由驱动报告基因 GFP的植物表达载体SAGPVI，BAGPVZ和BAGPV3，用于农杆 菌介导的拟南芥遗传转化。为进一步在模式植物中研究其表达功能奠定了基础。 8.将苦瓜开花相关基因(BA必的编码区片段定向克隆到pQE一30质粒后， 获得重组质粒pQE一BAG。用该重组质粒转化大肠杆菌M15菌株，所获得的阳性转 化子经菌落Western检测表达产物。结果表明，在IPTG诱导条
Abstract:
As a member of Citcurbiteae, bitter melon is not only a vegetable crop but also an important medical herb in China and East Asia. These years, phytochemists have isolated a number of potential medical components from this plant, such as the ribosome inactivating protein (RIP) , MAP30 (Momordica anti-Hiv protein), which suppresses HIV (human immunodeficiency virus) activity, momordica charantia lectin (MCL), momordica charantia inhibitor (MCI) and momordicoside A and B, both of which can inhibit tumor growth. However, few materials related to tissue culture and the in vitro regeneration system has not been established yet. These were harmful to the conservation and propagation of rare varieties. Here we reported the establishment of in vitro regeneration system of bitter melon and developed a new way for improving the varieties of bitter melon and laid the base for establishment of genetic transformation system of bitter melon. At the same time, we cloned a flower specific MADS-box gene from bitter melon. The results as following: 1 Explants of bitter melon were easily induced to grow callus. The frequency of callus formation was about 90.0%. The kind and proportion of phytohormones had not noticeably influenced on callus formation of different vegetative organs. The callus of young root, hypocotyls and cotyledon grew faster than those of epicotyl and young leaf. The kind of organs and phytohormones did not make remarkable difference on the time when the callus were produced. In general, explants began to swell in four days after being inoculated on media. 2. Explants of bitter melon could produce three types of callus: green, yellow green and fragile yellow callus. In the process of callus differentiating adventitious bud, the kind, proportion and quantity of phytohormone and the type of callus were very important. The adventitious buds had been induced successfully on medium (MS+6-BA 4.0 mgl"l+KT 2.0 mgl-1) by yellow green callus. The frequency was about 66.7%. Yellow callus had not IX differentiated adventitious buds even under the most suitable conditions. 3. The medium (MS + ZT 5.0 mgl"1 +KT 0.5 mgl'1) was suitable for the proliferation of bud; the coefficient of proliferation was about 5-6. 4. The media (1/2 MS + ZT 0.02 mgl"1 or 1/2 MS) were suitable for rooting in vitro of shoot, the shoot on them could produce new young root 6-7 in three weeks. 5. After plantation test, the survival rate of tube plantlets was about 70%; their characteristics were the same as those from seed by field test. 6. The flower specific gene of bitter melon, named BAG (GeneBank accession number AY 178837), was cloned from flower bud. A full-length cDNA of lOOlbp contained a complete open reading frame of 228 amino acids, 5' and 3' nontranslated regions and a long ploy (A) tail. The predicted amino acids sequence of BAG showed 95% sequence identity to MADS-box protein CUM10 [Cucwnis sativus], 93% to CAG1 [Cucumis sativjts], 84% to MADS box protein GHMADS-2 [Gossipier hirsutum], 83% to MADS-box protein 5 [Vitas vinifera], 76% to MADS-box protein [Mains x domestica], 72% to MADS-box protein AGL11 [Arabidopsis thaliana], respectively. A comparison between the putative translation product of BAG and the other MADS-box related proteins demonstrated that the highest conservation domain is found within the putative MADS-box DNA-binding domain. A second domain, the K-box involved in dimerization, which is conserved among the MADS-box proteins, is also found. Genomic Southern blot analysis suggested the existence of at least two or three copies of the gene. Northern blot and reverse transcriptase polymerase chain reaction indicated that the BAG cDNA is carpels and stamens specific in bitter melon. 7. One pairs of specific nested PCR primers were designed according to the pubilished sequence of MADS-box gene "BAG" in bitter melon. The promoter of Mads-box gene (BAGP) with a length of 664 bp was cloned by DNA walking technology. The sequence of BAGP was analyzed by software DNATools 5.1 and CallMat, which