【作者】 周祥明；

【导师】 张志毅； 苏晓华；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 美洲黑杨(Populus deltoides Marsh.)属于杨柳科(Salicaceae)杨属(Populus),分布范围广,而且优良杨树品种多数含有美洲黑杨成分,因此美洲黑杨在杨树遗传育种中占有主要地位。但由于杨树的许多性状受多基因调控,杂合度高,且生长周期长,使得利用常规育种进行林木遗传改良变得非常困难;此外,转基因植株可能对非目标生物和环境产生潜在的影响;另外,作为绿化树种和转基因受体材料,却又存在花粉过敏及基因逃逸等问题。为了解决杨树育种周期长及转基因安全性等问题,本研究拟以美洲黑杨雄性花芽为试材,以cDNA文库构建及其质量鉴定为切入点,采用大规模随机EST测序,进而分离得到与花发育相关的基因。在此基础上,开展与花发育相关基因的正反义植物表达载体的构建及其农杆菌介导遗传转化烟草等研究,并对所获得的转基因烟草植株进行分子检测及表型观察。通过上述研究得到以下主要结果:1.首次以美洲黑杨雄性花芽为植物材料,采用SMART技术,构建了第一个美洲黑杨雄性花芽全长cDNA文库。质量检测结果表明,美洲黑杨雄性花芽cDNA文库的滴度为6.8x106pfu·mL-1,文库扩增后的滴度为7×1012pfu.mL-1。经过蓝白斑计数,文库的重组率可达95%。平均插入片段大小为1 kb左右,说明所构建的文库完全符合目的基因分离筛选和表达建库的要求,可为进一步开展与杨树花发育相关基因的克隆及林木花发育分子机理的探讨等研究方面奠定重要的基础。2.随机挑取经PCR检测过的4 200个单克隆进行5’端测序,共获得3 092条原始序列。去除插入片段小于150 bp和污染序列后,获得了3 086条高质量的EST序列,通过聚类分析获得416个簇(clusters),并对每个簇进行单独拼接,进而得到451个重叠群(contigs)和1104个单一序列(singletons)。通过对1555个单一基因(uniquesequences)与NCBI数据库的比对,结果显示,34.7%(540个单一基因)与核酸和蛋白数据库无序列同源性,由此可推测,34.7%为新基因,且可能与花发育相关。同时获得了8个MADS-box基因,其中3个是杨树中未见报道的基因,且均含有全长的CDS。3.对3个未见报道的杨树新基因的其中两个进行核酸序列比对分析,结果表明,PdPI基因序列与荔枝、桦树、黄瓜以及苹果的MADS基因序列的同源性极高(均达80%以上),但PdAGL与核酸数据库中的基因序列同源性较低。通过对蛋白序列进一步比对分析后发现,PdPI和PdAGL均含有高度保守的MADS、中度保守的K区以及非保守的I区和C区,其中PdPI基因在C区还含有极保守的PI基序(motif)。系统进化树分析结果表明,PdPI和PdAGL分别属于MADS基因家族中的PI亚家族和DEFH 7亚家族。4.运用实时定量PCR(Real time PCR)方法对PdPI和PdAGL基因在花芽发育各时期表达量测定,结果发现,PdPI基因在花序出苞片的过程中表达量急剧上升,且在根中的表达量也较高,因此可推测该基因可能与花粉成熟以及根的生长发育有关。这说明拟南芥PI同系物不仅能在生殖器官中表达,而且在营养器官中也表达。另外,PdAGL基因不仅在花器官中表达,而且也在形成层和叶中表达。虽然PdAGL基因与金鱼草DEFH 7基因的亲缘关系最近,但其表达方式不同于DEFH 7基因。5.在PdPI和PdAGL基因的正反义植物表达载体的成功构建以及高效稳定烟草转化体系建立的基础上,采用农杆菌介导的叶盘法转化烟草,以NPTⅡ基因为扩增目的基因,并对所获得的具有卡那抗性的转基因烟草植株进行PCR检测。结果表明,目的基因已整合到转基因烟草植株的基因组中。此外,通过转基因烟草植株的表型观察,结果发现,转正义PdPI基因烟草植株存在提前开花现象,而转反义PdAGL基因部分植株出现叶片畸形、花期晚、花量少等特点。更多还原

【Abstract】 Populus deltoides Marsh., belonging to the family of Salicaceae, is distributed worldwide and plays an important role in the genetic improvement for the Populus. But the fact that many of the traits of poplars are controlled by multiple genes, pulsed high heterozygosity, and a long life cycle makes conventional breeding very difficult, moreover the risks associated with the use of engineered plants include the potential effects on non-target organisms and environment, and causing allergic reaction to human due to the escape of transgenes through pollen dispersion. In order to shorten the breeding cycle and to reduce the pollution derived from pollens, a cDNA library for the male buds of P. deltoides was constructed and several genes involved in flower development were isolated based on EST sequences in databases in this study. In addition, many transgenic tobaccos with sense and anti-sense genes were produced via Agrobacterium tumefaciens-mediated transformation. The results are as follows:1. A full-length cDNA library for the male floral buds of P. deltoides was constructed by using SMATR （the Switch Mechanism At the 5’ end of RNA Templates） technique. The quality detection indicated that the library had a starting titer of 6.8×10⁶ pfu·mL^-1 and a final titer of 7×10¹²pfu·mL^-1 after amplification. It also had a high ratio of recombination （up to 95%） with the inserts at an average size of approximately 1 kb. These results showed that this library was highly quality, which is suitable for the isolation and expression of target genes and provide an important foundation for the studies on the molecular mechanisms involved in the flower development of forest trees.2. By large-scale ESTs sequencing of 4 200 randomly selected clones from the constructed library of male floral buds of P. deltoides, 3 092 raw sequences were obtained. About 3 086 valid ESTs were generated after cleaning of inserts that is shorter than 150 bp, and the contaminants were phylogenetically classified into 416 clusters. An assembly of sequences within independent clusters finally 451 contigs and 1 104 singletons were identified. BLAST analyses within these 1 555 unique sequences against NCBI database demonstrated that 540 unique sequences （34.7%） did not have homologous proteins or nucleotide sequences in the database, suggesting that they might be new genes involved in the floral development. In addition, eight MADS-box genes, including three full-length genes that have not been reported, were obtained.3. Multiple alignment analyses showed that PdPI gene of male floral buds of P. deltoides shared high similarity （over 80%） with the MADS genes from Litchi chinensis, Betula pendula, Cucumis sativus and Malus domestica, while PdAGL gene did not have homologous nucleotide sequences in the database. Further analyses indicated that there were a highly conserved MADS domain, a middle level of conserved K domain and the variable I and C domains present in both PdPI and PdAGL genes. In addition, a highly conserved PI motif was found in the C domain of PdPI gene. The results of phylogenetic analyses showed that PdPI and PdAGL genes were belonging to the subfamilies PI and DEFH 7 respectively.4. The expression pattern of PdPI and PdAGL was analyzed by using quantitative real-time PCR analyses. A dramatic increase in the expression of the PdPI gene was detected in a reproductive organ, including the catkins and roots, It can be suggested that PdPI gene might be involved in the processes of pollen maturation and root development of P. deltoides. The results showed that the PI homo log of Arabidopsis was expressed not only in reproductive organs, but also in vegetative organs Although PdAGL was closely related to the DEFH 7 from Antirrhinum, the expression pattern of PdAGL gene observed in P. deltoides was significant difference from that of DEFH 7.5. A sense and an anti-sense plant expression vectors for PdPl and PdAGL were constructed and were transferred into tobacco via Agrobacterium tumefaciens-mediated transformation based on the establishment of a high efficient genetic transformation system. PCR analysis with NPT II specific primers confirmed the integration of foreign genes into the genome of transgenic tobaccos. The observation of phenotype revealed the pre-mature transgenic tobaccos with sense gene and anti-sense-gene-containing tobaccos with abnormal leaf, delayed flowering and reduced amount of flowers.更多还原