【作者】 官玲亮；

【导师】 吴卫；

【作者基本信息】 四川农业大学 ， 药用植物学， 2011， 博士

【摘要】 亚油酸(Linoleic acid,LA,C18:2Δ9,12)和α-亚麻酸(α-linolenic acid,ALA, C18:2Δ9,12,15为人体必需脂肪酸,具有降低血液粘稠度、降低血液中甘油三酯和胆固醇含量、有效预防心脑血管病的作用。同时18：2和18：3也是植物细胞膜的重要组成部分,在植物抵御外界生物和非生物胁迫过程中起着不可替代的作用,并且也是多种信号分子的前体物质。18：2和18：3的生物合成是由一系列ω-6和ω-3脂肪酸脱氢酶通过原核和真核途径催化完成。其中ω-6脂肪酸脱氢酶催化油酸(Oleic acid,OA,C18:1△9)在碳链的Δ-12位脱氢生产双键生成18：2,而ω-3脂肪酸脱氢酶进一步在18：2的Δ-15位催化脱氢生成18：3。红花素有“亚油酸之王”的美誉,其普通型红花材料籽油中18：2的含量达70%以上。但迄今为止,仍未见任何有关红花亚油酸形成机制的分子生物学研究。本文对油用型红花材料ω-6和ω-3脂肪酸脱氢酶进行了基因克隆、组织表达以及系统进化分析,主要研究结果如下：1.采用RT-PCR和RACE(rapid amplification of cDNA ends)技术,从红花未成熟的种子和叶片中分离到8个FAD2基因和1个FAD6基因,并全部提交至GenBank上。分析其推导的氨基酸序列发现,所有基因均包含有3个组氨酸保守区。其中红花微体ω-6脂肪酸脱氢酶氨基酸序列C-端含有内质网滞留信号,而质体ω-6脂肪酸脱氢酶氨基酸序列N-端有质体信号肽序列。在NCBI中Blast结果显示,CtFAD2-1和CtFAD2-8与向日葵、大豆、棉花等种子特异表达的FAD2-1基因的同源性较高。而CtFAD2-2与其他作物中组成型表达的FAD2-2/ CtFAD2-3有较高的相似性。将ω-6脂肪酸脱氢酶氨基酸序列进行同源性比对发现,CtFAD2-1与CtFAD2-8的同源性最高,为79.9%；其次是CtFAD2-2与CtFAD2-1和CtFAD2-8,分别为70.8%和70.2；而这3个FAD2基因与其余5个拷贝间的序列相似性均较低,在52.0-61.0%之间；CtFAD6与FAD2之间的相似性极低,在18.0-21.9%之间。疏水性和跨膜分析结果显示,除CtFAD2-6以外,其余FAD2均含有6个疏水区,分别跨膜4-6次。蛋白质二级结构预测分析表明,所有红花ω-6脂肪酸脱氢酶基因二级结构均主要包含α螺旋和β折叠。这些基因的成功克隆,为进一步研究不同拷贝FAD2之间的分工、表达模式、调控及对环境的响应规律打下了坚实的基础,为红花亚油酸形成机制及脂肪酸组分的调控提供了一定的理论依据。2.采用RT-PCR和RACE(rapid amplification of cDNA ends)技术,从红花叶片中分离到2个质体类ω-3脂肪酸脱氢酶基因(CtFAD7和CtFAD8)的全长cDNA和1个微体类ω-3脂肪酸脱氢酶基因(CtFAD3)的部分序列,均已提交至GenBank中。CtFAD7和CtFAD8与其他植物的质体类ω-3脂肪酸脱氢酶的相似性分别为61-79%,63-78%。而CtFAD3与其他植物微体类ω-3脂肪酸脱氢酶的同源性较高,为60-93%。氨基酸序列分析表明红花CtFAD3,CtFAD7和CtFAD8均含有3个富含组氨酸的保守结构域,分别为HDCGH,HXXXXXHRTHH和HVIHH,其中CtFAD7和CtFAD8的N-端分别含有56和27aa的质体信号肽序列。疏水性及跨膜分析表明,红花ω-3脂肪酸脱氢酶氨基酸序列均包含4个疏水区域,分别跨膜1-3次。蛋白质二级结构预测结果表明,3个ω-3脂肪酸脱氢酶蛋白主要由α螺旋和p折叠组成。通过对CtFAD7和CtFAD8的cDNA和DNA序列比较发现,2个基因的DNA序列中均包含有7个内含子,8个外显子。各内含子在物种间则表现出丰富的多态性,序列和长度大小均各不相同；而从第2“到7“外显子的长度和序列相似性在物种间非常保守。将各内含子的位置表现在氨基酸序列上发现,在内含子出现的位置,均为该酶的保守区。因此,推测ωo-3脂肪酸脱氢酶基因的内含子对于保证基因在物种进化过程中功能的保守性起着关键作用。3.对红花各组织在不同温度下的脂肪酸组成及各ωo-6和ωo-3脂肪酸脱氢酶基因在对应组织中的mRNA表达量进行分析。结果表明,红花除种子以外的各组织均含有4种脂肪酸,棕榈酸(16：0)、硬脂酸(18：0)、亚油酸(18：2)和亚麻酸(18：3),因不含有棕榈亚麻酸(16：3),因此红花属于“18：3植物”。与其他所有植物不同,红花营养组织中不含有油酸(18：1),而在红花种子中,含有大量18：1但不含有18：3。另外本研究在红花根中检测到大量18：3Δ9,12,15脂肪醇,与该组织中其它脂肪酸共同比较,其组分含量为22.41%。ω-6脂肪酸脱氢酶家族基因的组织表达分析结果显示,所有该家族的基因均组成型表达,并且在不同的组织中表达量不同。红花co-3脂肪酸脱氢酶基因(CtFAD3, CtFAD7, CtFAD8)在不同组织中的表达研究结果表明,CtFAD3在除种子以外的所有组织中表达,在花中的表达量最高,其次是叶片。而CtFAD7和CtFAD8主要在叶片中高表达量。在种子发育的不同时期,16：0和18：0随着种子的发育含量逐渐降低,18：1在早期逐渐增加,而在开花15天后迅速降低,18：2在种子发育早期含量略有降低,但在15天后含量迅速升高。在种子表达量较高的ω-6脂肪酸脱氢酶基因主要有CtFAD2-1、CtFAD2-3和CtFAD2-8,并且3个基因均在开花后第10天的表达量显著高于其他各时期。CtFAD3在种子发育的各时期均不表达。在低温处理下,红花18：2和18：3的含量在茎和叶柄中均有所提高；而在叶片中,18：3的含量有所增加,18：2的含量则相应的减少。表达分析结果表明ω-6和ω-3脂肪酸脱氢酶基因在转录水平和转录后水平上共同调控着18：2和18：3的合成。在根中,ω-6和ω-3脂肪酸脱氢酶基因在低温下的表达量均有显著提高,但18：2和18：3含量却有所减少。而C18：3Δ9,12,15醇在低温的含量极显著增加。推测该脂肪醇由亚麻酸转化而来,对红花的低温抗性有非常重要的作用。4.对来自不同国家的高/低亚油酸红花材料的CtFAD2-1基因序列进行比对分析发现,从低亚油酸红花材料中分离到的CtFAD2-1’基因在起始密码子后+603 bp处存在1个碱基(胞嘧啶)的缺失,从而造成移码突变,使翻译提前终止。为验证CtFAD2-1’基因所编码的蛋白质大小以及活性,本研究将CtFAD2-1’基因与从高亚油酸材料中分离到的CtFAD2-1基因ORF序列分别插入到原核和真核表达载体pET30a和pYES2中,并分别转入大肠杆菌BL21(DE3)pLysS和营养缺陷型酵母INVScl表达系统中。在1mmol/L IPTG的诱导下,含有pETCtFAD2-1质粒的BL21菌体沉淀经SDS-PAGE电泳后,分离到1条约43kDa大小的特异条带,而在含有PETCtFAD2-1’质粒的BL21菌体总蛋白中却没有该特异条带。对含有pYES2FAD2, pYES2FAD2质粒以及空载pYES2.0的酵母细胞抽提脂肪酸,并进行GC/MS分析。结果表明,含有pYES2CtFAD2质粒的菌株中能诱导产生具有活性的油酸脱氢酶,将部分油酸(18：1)转化为亚油酸(18：2)。而在含有pYES2CtFAD2质粒和空载pYES2.0的工程菌中则没有能检测到18：2的生成。因此,从低亚油酸红花材料中分离到的CtFAD2-1’基因不能编码具有活性的油酸脱氢酶,该基因序列中+603 bp处胞嘧啶的缺失突变是低亚油酸红花材料形成的一个重要因素。比较CtFAD2-1基因在高/低亚油酸红花材料种子不同时期的表达量结果显示,在种子发育的各个时期,CtFAD2-1在高亚油酸材料中的表达量均高于在低亚油酸材料中对应时期的表达量。5.利用生物信息学方法对ω-6和ω-3脂肪酸脱氢酶基因家族的氨基酸序列特征、系统进化及功能分化进行分析。结果表明,ω-6和ω-3脂肪酸脱氢酶氨基酸序列均含有3个保守的组氨酸基序(Hisbox),不同生物来源的脂肪酸脱氢酶之间其Hisbox存在一定的差异,物种的进化程度越高,Hisbox的组氨酸(His)残基相对更保守。质体类ω-6和ω-3脂肪酸脱氢酶氨基酸N-端序列均有数目不等的信号肽区域,并且在信号肽区中部发现1个由10个疏水性或中性氨基酸残基组成的相对保守的疏水区,推测为该类酶信号肽的功能区域。而多数植物微体ω-6和ω-3脂肪酸脱氢酶氨基酸C-端均有KKXX-like motif内质网滞留信号,而红花CtFAD2-3、CtFAD2-4、CtFAD2-5、CtFAD2-6和CtFAD2-7中没有检测到该滞留信号,但C-端序列富含芳香族氨基酸,同样具有内质网滞留信号的作用。系统进化分析表明,所有序列分主要分为4大类,类Ⅰ为植物Stearic-ACP脂肪酸脱氢酶；类Ⅱ包括植物质体类ω-6脂肪酸脱氢酶和原核生物ω-6脂肪酸脱氢酶；类Ⅲ为真菌和植物微体ω-6脂肪酸脱氢酶；类Ⅳ由所有ω-3脂肪酸脱氢酶组成,证明ω-3脂肪酸脱氢酶在原核生物中由ω-6脂肪酸脱氢酶基因进化而来。并且植物质体和微体类ω-3脂肪酸脱氢酶亚类间包含单子叶和双子叶2个小类,表明植物质体和微体类ω-3脂肪酸脱氢酶功能的分化早在单子叶和双子叶植物分化之前就已经形成。植物微体类ω-6脂肪酸脱氢酶可细分为种子特异表达型和组成性表达型两类,并且是在双子叶形成之后才开始分化的。对各亚群间的功能分化类型分析结果表明,ω-6/ω-3脂肪酸脱氢酶以及植物质体/微体ω-6脂肪酸脱氢酶间经历过Ⅰ型和Ⅱ型功能分化；而质体/微体ω-3脂肪酸脱氢酶以及种子特异表达FAD/组成性表达FAD2亚群间只存在Ⅰ型功能分化。各亚群间的功能分化位点分析表明,除plant FAD3/plant FAD2外,在所有存在功能分化的亚群间,均存在后验概率值超过0.80的氨基酸位点,而这些位点主要分布在HisboxⅠ的前后两端以及HisboxⅡ的前端。以上结果均证明,ω-6和ω-3脂肪酸脱氢酶基因家族内部在长期进化过程中形成了亚群间的功能分化。更多还原

【Abstract】 The Linoleic acid (LA, C18:2Δ9,12) andα-linolenic acid(ALA, C18:2Δ9,12,15) are essential fatty acids that cannot be synthesized by mammals and therefore must be obtained from dietary sources and have the role of lower the plasma cholesterol levels, low-density lipoproteins and reducing blood cholesterol levels in human body. 18:2 and 18:3 were the main structural components of membrane lipids and storage lipids in plant. They contribute to inducible stress resistance through the remodeling of membrane fluidity when plants encounter the biotic and abiotic stress.18:2 and 18:3 are synthesized through both prokaryotic (chloroplast) and eukaryotic (ER) pathways by a group ofω-6 andω-3 fatty acid desaturases. The membrane-boundω-6 desaturase (codified by the microsomal FAD2 and the plastidial FAD6 genes) inserts a double bond between carbons 12 and 13 of 18:1 to generate di-unsaturated linoleic acid (18:2).ω-3 desaturase (codified by one microsomal FAD3 and two plastidial FAD7 and FAD8 genes) further catalyzes the introduction of a third bond between carbons 15 and 16 to form tri-unsaturated a-linolenic acid (18:3). Safflower oil has been traditionally characterized by a high polyunsaturation level with linoleic acid (18:2) representing more than 70% of total fatty acid. Although the lipid contents of safflower seed oil and its commercial values have been well documented, the molecular regulation of lipid biosynthesis in safflower seeds has not been explored. The process of linoleic accumulation in safflower is still much of a mystery. Our main results about safflowerω-6 andω-3 fatty acid desaturases genes and regulatory mechanisims were described as follows: 1. Eight different microsomalω-6 fatty acid desaturases and one plastidialω-6 fatty acid desaturase cDNA sequences, designated CtFAD2-1, CtFAD2-2, CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6, CtFAD2-7, CtFAD2-8 and CtFAD6 have been isolated from safflower(Carthamus tinctorius L.) using a PCR approach. All of safflower deduced amino acid sequences showed the three histidine boxes characteristic of all membrane-bound desaturases, and CtFAD2 contain a C-terminal ER retrieval motif, where as CtFAD6 possess a putative N-termianl signal peptide. Our Blast searches of the deduced aa sequences revealed that the deduced amino acid sequences of CtFAD2-1 and CtFAD2-8 showed higher similarities to other plant seed type microsomalω-6 fatty acid desaturases, where as OFAD2-2 showed higher similarities to constitutively-expressed type. The other microsomalω-6 fatty acid desaturase genes showed much lower identities to other plant FAD2. The CtFAD6 showed higher similarity with other plant plastidialω-6 fatty acid desaturases. In hydropathy analysis showed that the encoded polypeptide contains six putative membrane- spaning domains. Transmembrane analysis indicated that all the safflower fatty acid desaturases contained four to six putative membrane-spanning domains except CtFAD6, Protein second-structure indicated that safflowerω-6 fatty acid desaturase is composed by a-helix andβ-sheet2. One microsomalω-3 fatty acid desaturase gene fragment and two plastidialω-3 fatty acid desaturases cDNA and genomic sequences, designated CtFAD3, CtFAD7 and CtFAD8 have been isolated from safflower(Carthamus tinctorius L.) and submitted to GenBank. All of safflower deduced amino acid sequences showed the three histidine boxes (HDCGH, HXXXXXHRTHH and HVIHH) characteristic of all membrane-bound desaturases, and CtFAD7 and CtFAD8 possess a putative N-termianl signal peptide,6 and 27 aa respectively. Our Blast searches of the deduced aa sequences revealed that the deduced amino acid sequences of CtFAD7 and CtFAD8 showed higher similarities to other plant plastidialω-3 fatty acid desaturases (61-79%,63-78%, respectively), while OFAD3 showed higher similarity with other plant microsomalω-3 fatty acid desaturase (60-93%). In hydropathy and transmembrane analysis showed that the encoded polypeptides contain four putative hydropathy regions and transmenmbrane one to three times. Protein second-structure indicated that safflowerω-3 fatty acid desaturase were composed byα-helix andβ-sheet. Compared with the genome structures of safflower CtgFAD7 and CtgFAD8 and other plant plastidialω-3 desaturase genes, all the sequences contained 8 extons and 7 introns. The sizes of the internal 6 exons (from 2nd to 7th) were maintained in FAD7 and FAD8 from all the plant species.3. To investigate the regulatory mechanisms of the accumulation of fatty acids among the different safflower tissues, we studied the fatty acid composition and relative expression levels of theω-6 andω-3 fatty acid desaturase genes in roots, stems, petioles, leaves, flowers and developing seeds from safflower. Safflower vegetative tissues contained two main PUFAs,18:3 and 18:2, and two kinds of saturated fatty acids, palmitic acid (16:0) and stearic acid (18:0). Thus, safflower belongs to the group of so-called "18:3 plants". Difference between safflower and other 18:3 plant species was that no 18:1 presented in all the tested vegetative tissues. On contrary, large amount of 18:1 was detected in developing seeds while no 18:3 was found in this tissue. In roots, a new component, C18:3Δ9,12,15 alcohol, was detected in roots represented more than 20% when compared with fatty acids. The transcript analysis observed that all of theω-3 fatty acid desaturase genes were constitutively expressed. CtFAD3 was expressed in all the tissues except developing seeds, with highest mRNA accumulation in flower, followed by leaves, while CtFAD7 and CtFAD8 were mainly expressed in leaves. The relative percentages of 16:0 and 18:0 were decreased during seed development. The content of 18:2 decreased rapidly during the first 10 days of development, remaining steady afterwards till 15 DAF, and then it showed a fast and important increase of 18:2 in the later periods. CtFAD2-1, CtFAD2-3 and CtFAD2-8 are the main genes expressed in developing seeds and all of them show highest transcripts at the 10 day after flowering. Different from other plant, CtFAD3 did not express in developing seeds. At low temperature (5℃), both of 18:2 and 18:3 were increased in stems and petioles. In leaves, the percentages of 18:3 in leaves increased slightly (from 63.31 to 67.27%), with the compensation of 18:2, decreased from 12.73% to 8.70%. In roots, both of the percentages of 18:2 and 18:3 decreased, while interesting is the C18:3Δ9,12,15 alcohol was significantly increased from 22.41 to 32.13%. Express analysis indicated that the mechanism of temperature-dependent alterations of PUFAs composition in safflower membrane lipids is controlled at transcriptional and post-transcriptional level ofω-6 andω-3 fatty acid desaturase.4. Higher proportion of 18:2 in oil increases the chances of oxidation, which leads to unpleasant odors and tastes, thus limiting the storability of the oil. On the contrary, oils with high oleic acid (18:1) are less prone to oxidation and off-flavors and also extend the shelf life by delaying the development of rancidity. Hence recent research efforts are directed towards an improvement of oleic acid in oil crops. By RT-PCR method, the full-length cDNAs of CtFAD2-1 was isolated from safflower genotypes with normal and high ratio of oleic to linoleic acid, which were designated CtFAD2-1 and CtFAD2-1’, respectively. Sequence alignment of their coding regions revealed that a deletion of cytosine (C) exists at the position+603 bp of CtFAD2’ sequence of high oleic acid genotypes, which resulted in the shift of open reading frame (ORF) and truncated protein CtFAD2’, with the loss of the third box involved in metal ion complex required for the reduction of oxygen. Analysis of transcript level showed that the expression of CtFAD2’in high oleic acid genotype is significant lower than CtFAD2 in normal genotypes during seed development. CtFAD2-1 and CtFAD2-1’ were cloned into the expression vector, Pet30a and subsequently transformed into expression E.coli BL21 (DE3) pLysS. SDS-PAGE analysis showed that the 43 kDa target protein was visualized clearly in the cell membrane protein containing the CtFAD2-1, while the cells protein with CtFAD2-1’ did not showed this band. The enzyme activity experiment of yeast (Saccharomyces cerevisiae) cell transformed with CtFAD2-1 and CtFAD2-1’ proved that only CtFAD2-1 gene product showed significant microsomal oleate desaturase activity, partially convert 18:1 to 18:2. These results suggested that the change of CtFAD2’ gene sequence results in the deactivation and lower transcription of delta-12 fatty acid desaturase in high oleic safflower genotypes.5. The deduced amino acid sequences ofω-6 andω-3 fatty acid desaturase genes have been compared in order to infer their phylogentic relationships and functional diverge. All the deduced proteins shared three highly conserved histidine rich motifs suggesting a common origin. The histidine rich motifs in the sequences from higher plant were more conserve than that of from prokaryotes. All of the plastidialω-6 andω-3 fatty acid desaturase possess a putative N-termianl signal peptide with different amino acids. And we identified the functional region of the peptide with hydrophobic or neutral amino acids. Most of plant microsomalω-6 andω-3 fatty acid desaturase (FAD2 and FAD3) contained a KKXX-like motif at the C-terminal, while safflower CtFAD2-3, CtFAD2-4, CtFAD2-5, CtFAD2-6 and CtFAD2-7 did not contain this motif, instead an aromatic aa enriched signal (YKNK) was found at the C-terminus of these amino sequences and such signal peptide has been reported to be both necessary and sufficient for maintaining localization of the enzymes in the ER. The phylogenetic analysis revealed four distinct clusters within the membrane desaturases. One cluster consisted of Stearic-ACP desaturase, the second group included plant plastidialω-6 fatty acid desaturase, the third cluster comprised the Eukaryotes FAD2, and the fourth contained all of the plastidial and microsomalω-3 fatty acid desaturase. This arrangement of clusters suggested thatω-3 fatty acid desaturases originated in a prokaryotic lineage from aω-6 fatty acid desaturase gene. The diverging time of plastidial and microsomalω-3 fatty acid desaturase, seed type and housekeeping type FAD2 were after the formation of dicotyledonous and monocotyledonous plants. The statistical evidence of functional divergence between plastidial and microsomalω-3 fatty acid desaturase, seed type and housekeeping type FAD2 was found in this analysis. Further more, the site for functional divergence were identified and distributed near the HisboxⅠand HisboxⅡ. These results indicated that evidence of functional divergence in theω-6 andω-3 fatty acid desaturase gene family during the long evolutionary period.更多还原