【作者】 宋晔华；

【导师】 王兴智；

【作者基本信息】 东北师范大学 ， 遗传学， 2008， 硕士

【摘要】 马铃薯薯块受到损伤使自身所含的多酚氧化酶与酚类物质的空间隔离被打破,多酚氧化酶在空气中氧分子的作用下催化酚类物质为苯醌类物质,苯醌类物质稳定性较差,容易在不依赖酶的作用下,自身聚合后与体内的亲和性氨基酸等发生作用生成褐色、黑色物质,严重影响了马铃薯在加工过程中的品质。目前,应用于植物中基因沉默的最新策略是体外将目的基因反向重复连接到内含子两端,转入植物基因组中,转录后产生自我互补的双链RNA,诱导同源基因的转录后沉默。这种RNA干扰技术具有沉默效率高、特异性好的优点。在本实验中,我们克隆了马铃薯的多酚氧化酶基因POT33,并将其高度保守的部分序列反向重复连接到载体pKANNABAL上,获得了中间载体pKANNABAL-ihpPOT33;然后将反向重复的序列连同它们之间的内含子序列一同连入植物双元载体pMBW330和pGB121s上,构建了分别由35S启动子启动的组成型沉默载体pMBW330-ihpPOT33和由GBSS启动子启动的薯块特异性沉默载体pGB121s-ihpPOT33,利用农杆菌LBA4404介导,转化加工型马铃薯大西洋。通过胚性愈伤组织诱导再生途径获得75株再生植株。对转化后的植株进行PCR和Western blot检测。结果显示,两株再生植株的叶片中分子量略小于66 kD的多酚氧化酶的表达被抑制。利用软件Bandscan 5.0分析Western检测结果,数据显示两株低表达多酚氧化酶的再生植株的沉默效率分别为86.02 %和66.10 %。关于在薯块中检测多酚氧化酶基因的表达以及表达降低后对马铃薯薯块褐化的影响还需要进一步的研究。更多还原

【Abstract】 Polyphenol oxidase (PPO) is the major cause of enzymic browning in potato. In vivo, the PPO localized in plastids, while the phenolic substrates of PPO are localized in the vacuole. The browning reaction only occurs as a result of tuber damage leading to a loss of this subcellular compartmentation. PPO catalyzes the conversion of monophenols polyphenols to o-dihydroxyphenols or to quinones. The quinone products can then polymerize and react with amino acid groups of cellular proteins, resulting in brown or black pigment deposits. Such damage causes considerable economic and nutritional losses in the process of potato.Recent work has focused on designing the constructs which require two copies of the target sequence in an inverted-repeat orientation encoding self-complementary `hairpin’ RNA with a intron between the two copies (ihpRNA), in order to produce duplex RNA to efficiently silence the genes in vivo (called RNAi technology). RNAi technology allows silencing all members of a multigene family or homoeologous gene copies in polyploids by targeting sequences that are unique or shared by several genes.In this study, we cloned one member of PPO genes-POT33 and constructed a constitive RNAi vector promoted by 35S promoter carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pMBW330-ihpPOT33), and a tissue-specifical RNAi vector promated by GBSS carrying two copies of part sequences of POT33 in an inverted-repeat orientation (called pGB121s-ihpPOT33). Agrobacterium LBA4404 harboring the binary vector pMBW330-ihpPOT33 or pGB121s-ihpPOT33 was employed to transform Potato ’Atlantic’, and 75 potato plantlets were regenerated after transformation. The transformed potato plants were confirmed by PCR and Western hybridization successfully. The results suggested that one member of the target protein was silenced in two transformed potato plants, and the silencing efficiency of them was 86.02 % and 66.01 % analyzed by software Bandscan, respectively.更多还原