【作者】 王敬文；

【导师】 沈大棱；

【作者基本信息】 复旦大学 ， 遗传学， 2006， 博士

【摘要】 1水稻是我国最主要的粮食作物之一，水稻的播种主要分布于热带和亚热带地区，其地理分布主要受温度的影响。不饱和脂肪酸是植物膜系统的重要组份之一，它的含量的变化是植物缓解外界温度压力的一种重要手段。本论文中用RT-PCR的方法从水稻Oryza sativa L．中克隆到编码水稻叶绿体ω-3脂肪酸脱氢酶的开放读框，命名为Osfad8。Southern杂交显示水稻中的Osfad8基因至少有两个拷贝，或者是由紧密连锁的一个小的基因家族组成。RT-PCR和原位杂交显示，Osfad8主要在叶中转录，而在根中几乎检测不到任何mRNA的积累。较低温度下(15-20℃)Osfad8的转录水平较常温(25℃)明显增高。原位杂交显示当水稻在15℃下低温处理5天和10天后，在叶片栅栏组织和海绵组织中的能检测到较明显的Osfad8的转录信号。为了进一步研究此基因的功能，我们分别正向和反向将Osfad8的ORF转入烟草中，产生8S-52和8S-101两个正向的转化品系，以及8A-35反向转化品系。在正向转化品系中，Osfad8的ORF以正向插入到CaMV 35S启动子的下游，导致16：3和18：3三烯酸的含量显著增高。低温2℃处理7天发现，对照烟草(pBI 121空载转化植株)的叶片已经明显受到伤害，并且已经萎缩，而在8S-52和8S-101中并没有明显的叶片损伤。在8A-35中，三烯脂肪酸的含量较对照相比降低约40.2％，44℃高温处理3天后，对照植株已经枯萎死亡，而8A-35仍能正常存活。以上数据显示Osfad8为编码低温诱导型的叶绿体ω-3脂肪酸脱氢酶。2蝴蝶兰又名蝶兰，原产亚洲热带和澳大利亚，为热带兰中之珍品被益为热带兰皇后。类黄酮-3’5’-羟化酶(F3’5’H)是合成3’，5’-带羟基的花色素(例如翠雀素)的关健酶，此色素是蓝花和紫花色素形成的前体。本研究从蝴蝶兰花瓣中克隆到了类黄酮-3’5’-羟化酶的cDNA，命名为Phf35h，序列号为DQ148458 in GenBank／EMBL／DDBJ。Phf35h的DNA克隆是通过基于PCR的一系列方法获得的。对Phf35h进行序列分析表明，该基因的DNA序列含有一个内含子，两个外显子，含有一个编码507个氨基酸残基的开放读框。Southern杂交表明在蝴蝶兰中只存在一个拷贝的f35h基因。通过RT-PCR的方法发现Phf35h的mRNA是在花朵发育的中期开始转录，和花青素的积累保持一致；在紫色花瓣中有较高的mRNA的积累，在根中和叶中没有检测到转录。同源模拟PhF3’5’H蛋白的三维结构表明此蛋白主要含有两个结构域：α结构域(含有大量的α螺旋和三个小的β折叠)和β结构域(含有很多β折叠和三个α螺旋)。PhF3’5’H蛋白与底物二氢黄酮醇dihydrokaempferol的结合位点通过分子对接的手法进行分析表明，在此蛋白中存在这一个高度保守的HPPTPLSLPH氨基酸序列，可能和二氢黄酮醇的芳香环相互作用。更多还原

【Abstract】 1A cDNA, designated 0sfad8, encoding a chloroplast ω-3 fatty acid desaturase responsible for trienoic fatty acid formation, was isolated from the leaves of Oryza sativa L. by RT-PCR. Southern hybrization analysis indicated that a small gene family composed of two copies or closely linked genes exist. RT-PCR and RNA in situ hybridization showed that the accumulation of 0sfad8 mRNA was abundant in leaves but hardly detectable in roots. The 0sfad8 transcript level in leaves was much higher at 15 ℃ than that at normal temperature (25 ℃). In situ hybridization also showed particularly prominent expression of 0sfad8 in the palisade layer and spongy parenchyma cells of leaves when exposed to 15 C conditions for 5 days and 10 days. Two transgenic lines (8S-52 and 8S-101) harboring the 0sfad8 ORF in sense orientation under the control of the CaMV 35S promoter, contained increased levels of hexadecatrienoic (16:3) and linolenic (18:3) fatty acids. When exposed to 2 ℃ for 7 days, the damage observed to the control plants was significantly alleviated in the 8S-52 and 8S-101 lines. The amounts of trienoic fatty acids in an 0sfad8 antisense line (8A-35) declined 40.2% compared to the control plants. The 8A-35 plants survived after growth at 44 ℃ for 3 days while the control plants died. These data suggest that 0sfad8 encodes a temperature-dependent chloroplast ω-3 fatty acid desaturase.2 A novel cDNA for the flavonoid-3’ , 5’ -hydroxylase (F35H) gene was cloned from petals of Phalaenopsis, and designed to be Phf35h (accession number DQ148458 in GenBank/EMBL/DDBJ). The genomic clone of Phf35h was isolated by a PCR-based strategy. Nucleotide sequence analysis revealedthat its genomic clone contains one intron and an open reading frame encoding a polypeptide of 507 amino acid residues. Southern hybridization analysis indicated the presence of a single gene coding for Phf35h. RT-PCR analysis showed that the Phf35h mRNA is transcribed in late phase of petal development, which is concomitant with the appearance of anthocyanins in petal tissue. The transcript is abundant in the purple petals but not in leaves or roots. The three-dimensional model of PhF3’ 5’ H protein is classified into an a -domain which contains most of the α -helixes with three small β -sheets, a β -domain that contains the larger β -sheets with three small α -helixes by homology modeling. The substrate binding site for dihydrokaempferol on PhF3’ 5’ H protein was determined by molecular docking algorithm. A highly conserved HPPTPLSLPH sequence was predicted to contact the aromatic ring of dihydrokaempferol.更多还原