【作者】 占志勇；

【导师】 周志春；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2013， 博士

【摘要】 油桐是原产我国的世界著名木本油料树种，从其种子中提炼而出的桐油是一种性质优良的干性油，广泛应用于工业中，具有极高的出口价值。利用经典蛋白质组学研究方法成功构建出油桐种仁在这一发育过程中的蛋白质表达谱，最终可运用蛋白质表达谱从蛋白质水平对油桐种仁中脂肪酸代谢过程进行阐述。发现油桐种仁中油脂含量与参与脂肪酸代谢的蛋白表达情况密切相关，油脂含量是由一系列参与脂肪酸代谢的蛋白（酶）共同决定的；此外，油体的动态发育过程更加形象化的体现了这一过程。论文为构建油桐种仁蛋白质表达谱从蛋白质水平理解其内油脂合成过程，对种仁蛋白质的提取、双向电泳体系优化、表达谱分析做了大量研究，得出研究结论如下：（1）分析了影响油桐种仁蛋白质提纯的关键因素，筛选确定TCA-丙酮结合酚抽法为有效提取油桐种仁蛋白质。实验结果表明种仁中的内含物对油桐种仁蛋白质提纯影响显著，采用TCA-丙酮结合酚抽法能针对油桐种仁中所富含的次生代谢物质如核酸、糖、酚类物质进行有效清除，故能制备出浓度高达8.1μg/μL的蛋白质样品；其次，SDS-PAGE实验表明，TCA-丙酮结合酚抽法对种仁中的高、低分子量蛋白质有较强的提取能力；再次， TCA-丙酮结合酚抽法所制备的蛋白质样品2-DE图谱分辨率高、背景清晰。（2）成功构建出具有高稳定性和高重复性的油桐种仁蛋白质双向电泳体系。具体针对能影响油桐种仁蛋白质双向电泳效果的蛋白质裂解液、上样量、等电聚焦时间以及SDS-PAGE电泳的运行条件等因素，进行优化对比实验，确定了各因素的最优条件。成功构建的最优油桐种仁蛋白质双向电泳体系为：以TCA-丙酮结合酚抽法制备样品后经过配方为7mol/L尿素、2mol/L硫脲、4%CHAPS、65mmol/L DTT和2%IPG buffer的裂解液裂解后，等电聚焦上样700μg进行聚焦70000Vh能够得到良好的分离效果。（3）研究发现油桐在花后130d~160d为其发育过程中油脂积累的关键时期。通过对油脂含量变化和种壳颜色变化将这一时期划分为3个不同的发育阶段S1（130d）、S2（145d）和S3（160d）。（4）在油桐种仁发育的3个不同阶段中共发现有144个蛋白质发生了差异表达，并通过MALDI-TOF/MS/MS成功鉴定出其中76个，将其分为能量代谢（25%）、脂肪酸代谢（15.79%）、抗性（14.47%）、蛋白酶（11.84%）等11个功能类别。（5）研究发现能量代谢一直是油桐种仁发育过程中除了脂肪酸代谢之外的最主要的生理代谢活动之一，TAC循环和糖酵解是其主要的代谢途径。S29（Malate dehydrogenase，苹果酸脱氢酶）的表达量随着种仁发育而逐渐增加，表明TAC循环中的草酰乙酸再生速度得到提升，而草酰乙酸的再生过程是维持TAC循环持续进行的关键。S62（3-phosphoglycerate kinase，3-磷酸甘油酸激酶）表达随着种仁发育而增加表明糖酵解代谢随着发育而得到增强。研究发现多数参与脂肪酸代谢的蛋白质在3个时期中的表达变化过程与种仁中油脂的积累速率变化相一致，均是抛物线的变化趋势，峰值出现在S2时期。这一发现有助于成功从蛋白质水平对种仁中油脂积累变化进行阐述。RT-PCR对参与能量代谢、脂肪酸代谢的10个蛋白质的研究结果表明，基因水平上的表达变化趋势与蛋白质水平的表达变化趋势具有较高的相似性，表明质谱鉴定结果具有较高的可靠性；同时，RT-PCR结果也可得出上述（5）（6）的结论。（6）在不同的发育阶段，油体有不同的细胞学特征。以此为基础可以将其发育过程分为发育初期、中期以及后期三个阶段。研究发现油体的数量与油脂含量表现出负相关关系；而油体大小与油脂含量则为正相关关系。油体动态发育过程与油桐种仁中脂肪酸积累、脂肪酸代谢相关蛋白表达变化具有密切联系。更多还原

【Abstract】 Tung trees are original from China and they are famous for tung oil in the world as an oilplant. Tung oils are widely used in industry as a good kind of dry oils.In the paper, the protein expression profiles were successfully constructed during thedevelopmental periods by the method of traditional and classical research on proteomics. Itcould help to explain the accumulation process of tung oil in tung seeds from the proteins level.The results indicated that there existed a close relationship between the oils accumulation andthe proteins expression especially for which related to fatty acids metabolism. The oilaccumulation was co-determined by a series of proteins expression and the development of oilbody visualization shows this process. In order to explain the oil biosynthesis from the proteinlevel, many research works were held on and the main results obtianed as follows:(1) TCA-acetone combined with phenol method was the most suitable for the protensextraction from tung seeds. This method can effective clear away the nucleic acids, sugars andphenols, thus the contribution of sample arrived at8.1μg/μL. It also found that it had a goodcapability in extracting high and low mass protiens after SDS-PAGE. In addition to, the resultsof2-DE pointed that the sample which prepared by TCA-acetone combined with phenol had aclear background and a good resolution.(2) It successfully constructed a2-DE platform for tung seeds with a high stability andrepeatability. Some factors can effects the2-DE process such as lysis solution, loading yield ofproteins, IEF and SDS-PAGE were compared. It indicated that the protein samples whichprepared by TCA-acetone combined with phenol were dissolved by7M urea,2M thiourea,4%CHAPS,65mM DTT and2%IPG buffer, then after IEF for70000Vh with the loadingyield of700μg, we could obtain a good2-DE patterns for tung tree seeds proteome.(3) It indicated that after flowering130d~160d was the key periods for the oilsaccumulation. This period can grouped into three stages by the change of oil content and thecolor change of seed coat. (4) A total of144proteins were determined as differential expression proteins, and76proteins were successfully identified by MALDI-TOF/MS/MS. They were classified into11classes including energy metabolism (25%), fatty acid metabolism (15.79%), defense-related(14.47%), protease (11.84%) and so on.(5) Energy metabolism (TAC cycle and glycolytic pathway) was the main change duringdevelopment besides fatty acids metabolism. The expression change of S29(malatedehydrogenase) and S62(3-phosphoglycerate kinase) during three stages indicated that TACcycle and glycolysis would be stronger along with development. The expression change ofmost proteins which related to fatty acids metabolism was similar to the speed of oilsaccumulation in seeds. It was parabola style, the highest value existed in S2. This result canhelpful for explain the oil accumulation from protein level. RT-PCR results indicated that theprotein which related to energy metabolism and fatty acids metabolism have a similar changeprocess with the MS results, it stands for the MS results owned a high reliability. We also canobtain a similar result as (5) and (6) by RT-PCR.(8) The oil bodies owned different cytological characteristics in the differentdevelopmental stages. According to this, the process of oil body development can divide intothree stages as the earlier stages, the middle and the last stages of development. There existed anegative correlation in the number of oil bodies in the endosperm cells and the oil content;existed a positive correlation in the diameter of oil bodies in cells and the oil content in tungseeds. The oil bodies development process owned a close relationship with the oilaccumulation in tun seeds and the proteins expression change which related to fatty acidsmetabolism.更多还原