【作者】 汪阳东；

【导师】 张守攻； 齐力旺；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2007， 博士

【摘要】 木本植物油脂资源丰富，是制造生物柴油的重要原料。提高单不饱和脂肪酸含量，是改善植物油脂品质，改良油脂植物，降低生物柴油成本的关键。同时，脂肪酸也是植物细胞膜组成的重要部分，对于维持植物正常的生长发育和抗逆能力至关重要。利用基因工程手段改变油脂植物的脂肪酸成分，提高木本油料植物单不饱和脂肪酸比例，是创制抗逆性强的优质生物柴油原料植物的重要途径。油酸脱氢酶(油酰磷脂酰胆碱去饱和酶，FAD2)催化油酸脱氢合成亚油酸，调控多不饱和脂肪酸合成，是植物脂肪酸代谢过程中的关键元素。因此研究该酶基因的结构与功能，揭示其调控脂肪酸代谢的机理，探索表达调控方式和时空变化具有重要的理论和实际意义，但是，迄今有关木本油料植物脂肪酸合成分子生物学研究较少。本研究应用分子生物学手段，从木本油料植物—中间锦鸡儿(Caragana intermedia)中克隆获得FAD2基因(CaFAD2)，利用生物信息学分析研究了CaFAD2蛋白质的结构和功能，成功构建了该基因的反义表达和正义表达载体，利用根癌农杆菌介导法成功获得脂肪酸不饱和度明显改变的转基因烟草株系，结合表型、生理生化和分子生物学对转基因烟草株系进行了综合分析，验证分析了CaFAD2基因的功能。1、利用RT-PCR和RACE方法，从中间锦鸡儿茎叶中克隆了CaFAD2基因cDNA的全长序列(Genebank No．AY957393)，并验证分析了其功能，为中间锦鸡儿遗传改良提供了试验和技术基础。2、生物信息学分析结果表明，CaFAD2基因编码区全长为1053bp，编码350个氨基酸。所编码的蛋白质与大豆、芝麻、莲的FAD2同源性均达到80％以上；与拟南芥、烟草、油菜的同源性高于70％，属于油酸脱氢酶类。推测该蛋白质酶具有脂肪酸去饱和酶的6个跨膜区域和3个组氨酸保守区域。3、定量PCR检测结果表明，CaFAD2基因在中间锦鸡儿茎和叶中都有表达，但在不同器官中表达模式不同，叶中表达量高于茎中。4、利用CaFAD2基因保守区域片段构建成功CaFAD2基因反义表达载体，通过根癌农杆菌系统导入烟草，筛选获得CaFAD2基因抑制程度不同的阳性转基因烟草株系RTC0系。与对照烟草相比，转CaFAD2基因烟草叶片和种子总脂中多不饱和脂肪酸含量分别降低约20％和10％，而油酸含量分别升高约16％和7％，说明CaFAD2基因在烟草能够反义表达，抑制烟草内源FAD2基因表达。5、成功构建了CaFAD2基因全长正义表达载体，并利用根癌农杆菌表达系统转化烟草，获得阳性转CaFAD2全长基因的烟草株系TC0系。开展了转基因烟草和对照烟草低温过冬(8-12℃)培养试验，结果发现，对照烟草存活率(20％)明显低于转基因烟草TC0系(90％)。6、RT-PCR检测和脂肪酸分析结果显示，CaFAD2全长基因在TC0系烟草中能够表达，提高烟草中FAD2基因表达量，促进了多不饱和脂肪酸合成，转基因阳性烟草种子和叶片中多不饱和脂肪酸分别增加了7.77％和10.96％。7、通过生物信息学和转基因表达结合方式，探索分析了CaFAD2基因和SoyFAD2基因结构和功能差异。两个基因均来自茎叶，编码的蛋白质氨基酸同源性达90％，具有3个组氨酸保守区和6个跨膜区，但是前者氨基酸比后者少33个，组氨酸和跨膜区位置也有所不同。转基因研究发现，CaFAD2基因和SoyFAD2基因在烟草中均能正义表达，促使烟草种子和叶片中多不饱和脂肪酸含量增加，CaFAD2正义表达促进作用高于SoyFAD2，但是差异不显著。CaFAD2和SoyFAD2基因结构和表达调控机制复杂，可能存在转录和转录后综合调控机制。本研究结果为进一步研究脂肪酸合成酶基因表达规律和开展木本油料植物转基因改良提供了试验技术基础。更多还原

【Abstract】 As an important renewable energy, Biodiesel develops fast in recent years with high price, lacking of plant oil and low quality. Because of low combusting value, high viscosity and instability of plant-derived biodiesel, thus it’s important to cultivate oil plants with suitable components of fatty acids.According the report(Kevin J. Harrington, 1986), the ideal material plant oil contianing high unsaturated fatty acids features a long, unbranched carbon chain with only one double bond at the end of the molecule, on the other side, more polyunsaturated fatty acids can protect the plant from cold.FAD2 is an important enzyme in the first step of polyunsaturated fatty acid synthey, located in endoplasmic reticulum. It catalyzes conversion of oleic acid to linoleic acid. Caragana intermedia is an important bio-energy plant distributing widly in the north China and its seeds can be made biodiesel, and it must be improved to decrease linoleic acid to increase oleic acid for polyunsaturated fatty acid of its oil is as high as 60.1%. And increasing the polyunsaturated fatty acid can protect it from low temperatures. Furthermore, the structure, function and regulation of FAD2 enzyme are far from being elucidated.In this work, According to published sequences, CaFAD2, an FAD2 homolog, was cloned by RT-PCR and RACE from total RNA of Caragana intermedia. The full-length of CaFAD2 is 1411bp (accession No. AY957393), and the longest open reading frame in the CaFAD2 encodes a polypeptide of 350 amino acids. The deduced amino acid sequence of CaFAD2 shows high identity to other plant delta-12 fatty acid desaturases, including the three histidine motifs, and two long stretches of hydrophobic residues which indicates of an integral membrane protein spanning membrane six times. To verify the function of CaFAD2, sense- and antisense of CaFAD2 were transformed into tobacco via Agrobacterium tumefaciens., and some transgenic plants with CaFAD2 were established in the population of the primary transformants. Although transgenic tobacco grew as wildetype tobacco, the analyses of fatty acid profile of the transgenic line RTCO(antisense-CaFAD2) and TC0(sense CaFAD2) showed that CaFAD2 has important effect on the polyunsaturated fatty acid synthesis. Adrastic increase of monounsaturated fatty acid especially oleic acid in total leaf lipid of RTC0 indicated that antisense-CaFAD2 decreased FAD2 expression in tabacco while CaFAD2 over expressing promoted the synthesis of polyunsaturated fatty acid, increase linolicl. It was possible CaFAD2 expressed constituitively in Caragana intermedia for that CaFAD2 expressed in both transgenic tobacco vegetative and seed tissues while the expressing level is higher in leaf than in stem in Caragana intermedia.To compare FAD2 from different plant species, SoyFAD2 was cloned from soybean. CaFAD2 and SoyFAD2 share 90% identity in deduced amino acid sequence. The longest ORF of SoyFAD2 encodes a polypeptide of 380 amino acids, including the three histidine motifs and two long stretches of hydrophobic residues. Sense of SoyFAD2 was transformed into tobacco. And in the transgenic line TS0, the amount of polyunsaturated fatty acid increased, which is the same of that in TC0.Oil biosynthesis is a complex biological process. There must be exist a network of genes playing different roles to control the components of fatty acids. In this study, the function of FAD2 from oil plant was investigated in detail, which may give more understanding in oil synthesis and provides genes to improve fatty acids components suitable for biodiesel.更多还原