【作者】 张曦；

【导师】 冯辉；

【作者基本信息】 沈阳农业大学 ， 设施园艺学， 2013， 博士

【摘要】 植物表面的蜡质是覆盖在植物表皮细胞外的一层由亲脂性化合物构成的疏水层,主要成分包括脂肪酸、烷烃、伯醇、仲醇、醛类和酮类等,在十字花科蔬菜作物上一般呈灰白色霜状,因此称之为蜡粉。蜡粉的主要作用是减少非气孔的水分散失,保护植物免受外来的机械损伤、病虫害入侵、太阳辐射等。本研究以大白菜有蜡粉和无蜡粉的高代自交系为试材,利用扫描电子显微镜观察叶面蜡粉的结构,利用气相色谱-质谱联用技术检测蜡粉的化学成分。分析大白菜蜡粉性状的遗传特性,采用SSR分子标记技术对蜡粉基因进行精细定位,并利用荧光实时定量PCR技术对所预测的基因进行分析,验证了候选基因就是所要找的目的基因。主要研究结果如下：1.以有蜡粉和无蜡粉大白菜为试材,采用直接烘干法制备样品。经扫描电子显微镜放大5000倍后观察,大白菜表面蜡粉呈棒状结构,顶端略有乳头状突起；无蜡粉材料表面相对光滑。扫描电子显微镜下观察到的棒状结构,就是肉眼所见的白霜表型。无蜡粉大白菜表面没有蜡粉层结构。2.采用常温加热结合法用有机溶剂三氯甲烷将大白菜表皮蜡粉提取出来后,利用气相色谱-质谱联用技术分析大白菜表皮蜡粉成分,结果表明大白菜表皮蜡粉中含有7种偶数碳原子的饱和烷烃和9种醇类物质,其为蜡粉多种化合物中的主要成分。3.以大白菜有蜡粉自交系08A161和无蜡粉自交系08A235-2为亲本,配制F1、F2和BC1等世代材料,分析大白菜蜡粉性状的遗传特性。结果表明,大白菜表皮有、无蜡粉为质量性状,受一对核基因控制,有蜡粉为显性,无蜡粉为隐性。4.利用SSR技术对大白菜蜡粉基因进行定位。选取308株F2隐性纯合个体作为初步定位群体,通过筛选181对SSR引物,将蜡粉基因定位在A01连锁群上。在nia-m086a附近区域设计引物,得到两个与蜡粉基因紧密连锁的分子标记SSRzx45和SSRzx54,距蜡粉基因的遗传距离分别为2.3cM和0.8cM。5.将得到的这两个标记SSRzx45和SSRzx54在更大的F2隐性纯合个体间确定重组单株,确定蜡粉基因位于SSRzx79和SSRzx107两个标记之间,遗传距离分别为0.25cM和0.2cM。SSRzx79和SSRzx107之间的物理距离约为86.4kb。经与大白菜基因组序列信息比对,在这个区域范围内共有15个基因,其中Bra013809与拟南芥的CER2基因有类似的功能,已知CER2基因参与蜡粉的代谢,因此,预测Bra013809基因为与蜡粉代谢相关的一个基因。6.对Bra013809基因的序列特征进行分析,发现Bra013809蛋白可能为非分泌型蛋白,不存在N-端信号肽。该蛋白含有多种修饰性位点,包括N端豆蔻酰基化位点、cAMP和cGMP蛋白激酶磷酸化位点、酪氨酸磷酸化位点、酪蛋白激酶2磷酸化位点、N端糖基化位点和蛋白激酶磷酸化位点等等,在蛋白的5-286位为转移酶酶活结构域。因此,推测该蛋白具有转移酶的作用。聚类分析结果表明,Bra013809蛋白与拟南芥的CER2蛋白存在高度的同源性。7.为进一步探明Bra013809基因与蜡粉代谢相关,在转录水平上对Bra013809基因进行了表达模式分析。结果表明,Bra013809在有蜡粉植株中有高水平的表达,在无蜡粉植株中受到抑制,Bra013809在有蜡粉植株蜡粉分布的主要区域花茎和叶片上都有高水平的表达,说明Bra013809的确是一个与蜡粉代谢有关的基因。更多还原

【Abstract】 The cuticular wax covering the epidermal cells in plants which contains the lipophilic compounds is the hydrophobic layer. The main compounds include fatty acids, alkanes, primary alcohols, secondary alcohols, aldehydes, ketones, and so on. Cruciferous vegetable crops generally covered with white frosted. The function of the cuticular wax is to reduce non-stomatal water loss, protect the plant from external mechanical damage, pest invasion, and solar radiation. In this study, the inbred lines of waxy and glossy in Chinese cabbage were the test materials, using a scanning electron microscope to observe the epidermal wax structure and gas chromatography-mass spectrometry to detect chemical composition. Finding out the genetic regular of waxy trait, using SSR markers to locate the wax gene and analysis of the predicted genes using real-time PCR technology, further validation of candidate genes is the target gene. The main results are as follows:1. The material of waxy and glossy was dried directly and then using scanning electron microscopy to observe the samples. Scanning electron microscope found out that Chinese cabbage epidermal wax was the rodlike structure with top slightly papillae, and glossy in a magnification of5000times, compared with the waxy material, the performance of a relatively smooth. The scanning electron microscope observation results are consistent with the results with the naked eye observations, the rod-like structure that is observed under scanning electron microscope is the the white frosted phenotype seen to the naked eye, glossy has not such a structure of the wax layer.2. By the chloroform extraction, the Chinese cabbage epidermal wax was extracted and used gas chromatography-mass spectrometry to detect the wax composition. It contains saturated alkanes with an even number of carbon atoms and nine indeterminate alcohols, which is the main ingredient in the wax compounds.3.08A161and08A235-2inbred were selected as the parents. Genetic analysis was doing in the Fi, F2and BC1generation. The results showed that the waxy character was qualitative character, and the glossy trait is controlled by a single recessive genetic locus.4. Selecting308F2homozygous recessive individual as the wax gene for mapping population, screening181pairs of SSR primers and the wax gene located on A01linkage group, designing SSR primers in the nearby area of nia-m086a, two markers which linked wax genes closely were found: SSRzx45, and SSRzx54, genetic distances were2.3cM and0.8cM, respectively.5. These two markers will be join in the larger F2recessive individual to determine the recombinant, the analysis results showed that wax gene was located between SSRzx79and SSRzx107, genetic distance of0.25cM and0.2cM, respectively. The physical distance between SSRzx79SSRzx107about86.4kb and15candidate genes within this zone, Bra013809by comparison found Arabidopsis CER2has a similar function, and CER2involved in wax metabolic pathway, therefore predict that Bra013809is a gene associated with wax metabolism.6. The analysis of Bra013809protein showed that it may be a non-secreted protein and the protein does not exist in the N-terminal signal peptide for the gene sequence Bra013809characterized. The protein contained in a variety of modification sites including the N-myristoylation sites, cAMP-and cGMP-dependent protein kinase phosphorylation sites, and the tyrosine kinase phosphorylation sites, casein kinase2phosphorylation sites, N-glycosylation sites, protein kinase C phosphorylation sites in protein5-286transferase activity domain. Therefore we speculated that the protein was transferase enzyme. The cluster analysis showed that Bra013809protein had a high homology with Arabidopsis CER2.7. In order to proof Bra013809wax metabolism, we analysis the the Bra013809gene expression pattern at the transcriptional level. The results showed that a high level of expression in Bra013809waxy plants, glossy plants is suppressed and have higher levels of expression Bra013809on wax plant stems and leaves, which shows that Bra013809was a wax metabolism-related gene.更多还原