【作者】 王凯；

【导师】 张猛；

【作者基本信息】 西北农林科技大学 ， 作物学， 2019， 硕士

【摘要】 中短链脂肪酸（Medium-chain fatty acids,MCFAs）包括辛酸（8:0）、癸酸（10:0）、月桂酸（12:0）、肉豆蔻酸（14:0）,是工业生产洗涤剂、肥皂、化妆品、表面活性剂和润滑剂重要原料。目前中短链脂肪酸的生产不能满足我们日益增长的需求。通过基因工程手段把其他植物尤其是萼距花属植物中调控中短链脂肪酸的关键酶转化到作物中在国外已经取得了一定的成果,然而在我国这些研究还处于起步阶段。我国许多樟科植物种子中都含有中短链脂肪酸,具有丰富的遗传资源。在我国樟科植物木姜子种仁中,富含约50%的月桂酸和20%的癸酸,是发掘提高中短链脂肪酸关键酶很好的材料。在脂肪酸从头合成过程中,FATB酰基-ACP硫酯酶FATB是控制脂肪酸碳链长短的关键酶。通过同源克隆的方法,我们从木姜子种仁中克隆得到了FATB。通过构建木姜子的FATB基因表达载体,转化拟南芥植株中,在拟南芥转化株系中对上述克隆的基因进行功能分析。为进一步探讨木姜子种子中月桂酸积累的机理,我们对发育中后期种胚的转录组进行了分析。本研究获得如下主要结果:1)从木姜子中克隆得到了一个FATB基因,基因的全长为1257bp。通过与已知物种的脂肪酸酰基ACP硫酯酶进行比对发现,该基因编码的蛋白聚类到FATB类群而不是FATA,并且我们的FATB蛋白的166Q、196W、198V、225G、226K、227N、228G位点,共同组成了硫酯酶活性部位4HBT。因此,将该基因命名为LpFATB。2)通过酶切连接的手段,将所得到的FATB基因构建到35S启动子启动的植物表达载体上。转化野生型拟南芥Col-0,通过对T₃代转化株系种子进行气相色谱分析,发现以Col-0为背景的LpFATB转化株系种子中,棕榈酸（16:0）的含量有明显提升,油酸（18:1）、二十碳烯酸（20:1）的含量都有一定程度的降低。棕榈酸较未转化的拟南芥野生型Col-0提高了2-3倍,最高达到了25.3%。这个结果表明,木姜子的LpFATB基因在拟南芥脂肪酸从头合成途径中对16:0-ACP的底物偏好性更强,而不是12:0-ACP。3)为了探究我们得到的FATB是否是木姜子种仁中唯一的一个,我们利用Illumina测序平台对木姜子发育中后期的种胚组织进行转录组测序,最终得到38897条高质量Unigenes序列。在获得的木姜子转录组数据中,通过将Unigenes与已知模式植物拟南芥基因序列进行比对分析,我们共发现247个Unigenes编码参与脂类代谢途径（包括脂肪酸合成、三酰甘油装配以及和油脂代谢相关的酶以及转录因子）。然而,我们并没有发现木姜子中存在与我们先前克隆的FATB不一样的拷贝。4)通过分析木姜子FATB与已经验证功能的可以在拟南芥中提高12:0的加州月桂FATB蛋白序列之间的差异,发现两个序列之间的N末端差异较大,可能是造成功能差异的原因,因此我们利用结构互换的方法把木姜子N端序列替换成加州月桂的N端序列,然而结果还是和木姜子FATB转化结果类似。这一结果表明,木姜子与加州月桂FATB之间在N端序列的差异,可能并不是造成底物差异的关键部位。并且,不同N端序列可能影响FATB的活性。更多还原

【Abstract】 Medium-chain fatty acids MCFAs,including octanoic acid（8:0）,decanoic acid（10:0）,lauric acid（12:0）,and myristic acid（14:0）,is an important material for industrial production of detergents,soaps,cosmetics,surfactants and lubricants.At present,the production of MCFAs can not meet our growing demand.The key enzymes from different plants especially in Cuphea which regulating MCFAs have been transformed into crops through genetic engineering in foreign countries.However,these studies are still in the initial stage in China,where many seeds contain MCFAs in Lauraceae.To make good use of these resourses,Litsea pungens,which rich in about 50%lauric acid and 20%decanoic acid in kernel,was a good material for discovering the key enzymes account for improving MCFAs in plant.During de novo fatty acid synthesis,FATB acyl-acp thioesterase FATB is the key enzyme to control the carbon chain length of fatty acid.One FATB was cloned from kernel of Litsea pungens by homologous cloning.Then,the LpFATB gene overexpression vector was constructed and transform into Arabidopsis thaliana wild type.Functional analysis of cloned gene was carried out in transformed lines.In order to further explore the mechanism of MCFAs accumulation,we analyzed the transcriptome of the kernel in the middle and late stage of development in Litsea pungens.The main results of this study are as follows:1)A FATB gene was cloned from Litsea pungens with a total length of 1257bp.Through comparison with the fatty acid-acyl ACP thioesterase of known species,it was found that the cloned genes were clustered into FATB group instead of FATA,and the amino acid 166Q,196W,198V,225G,226K,227N,228G in our FATB,together constitute the active site of thioesterase,called 4HBT.2)The LpFATB overexpression vector,which constructed by enzyme digestion connection technology was transformed into wild-type arabidopsis Col-0,and the fatty acid content of T₃ generation positive transformed seeds were analysed by gas chromatography,the content of palmitic acid（16:0）was obviously improved,oleic acid and eicosenoic acid were reduced in a certain degree.Compared with untransformed arabidopsis wild-type Col-0,the ratio of palmitic acid was increased by 2-3 times and the highest was 25.3%.This result indicates that the LpFATB gene is more favorable to the substrates of palmitate-ACP than that of laurate-ACP in the de novo fatty acid synthesis pathway.3)In order to explore whether the FATB obtained is the only one in the kernel,Illumina sequencing was used to sequence the transcriptome of the seed embryo tissue in the middle and later stages of the development of Litsea pungens,and 38897 high-quality Unigene sequences were finally obtained.By comparing and analyzing the gene sequences of Unigenes and the known model plant arabidopsis thaliana,we found that 247 Unigenes were involved in the lipid metabolism pathways（including fatty acid synthesis,triglyceride assembly,and enzymes and transcription factors related to lipid metabolism）.4)The protein sequences were analyzed between LpFATB and California bay UcFATB,we found that the N-terminal sequence of the protein between two sequences is bigger,which maybe the cause of functional differences.So we use structure swap methods to replace N-terminal sequence of Litsea pungens into California bay N-terminal sequence,but the result is similar with LpFATB.The results showed that the difference of N-terminal sequence between Litsea pungens and California bay may not be determining factor of substrate difference.Moreover,different N-terminal sequences may affect the activity of FATB.更多还原