【作者】 李军；

【导师】 王西平；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2013， 硕士

【摘要】 苹果（Malus×domestica Borkh.）是世界上最重要的水果之一，也是世界温带地区的主栽果树作物。苹果基因组序列的发布为在整个基因组水平上鉴定苹果的优良农艺性状、抗逆或抗病相关基因提供了重要资料，也为培育优良品质、抗逆或抗病的苹果新品种奠定了坚实的基础。在植物中，转录因子在许多重要的发育过程的调控网络中起着重要的作用。SQUAMOSA启动子结合蛋白（SBP）是一类植物特有的转录因子，在植物体的发育、激素信号转导和抗逆等方面起着重要作用。目前，多种植物中的SBP转录因子的功能已得到深入的阐述，而果树SBP转录因子的功能仍不清楚。本研究通过生物信息学分析方法从苹果基因组中鉴定得到苹果SBP转录因子基因家族的所有成员，然后对其进行结构域、miR156靶位点、系统进化、基因结构和基因同线等分析；研究了苹果SBP基因和miR156在各种组织器官中的表达模式以及苹果SBP基因对各种激素信号的响应情况，初步了解了苹果SBP转录因子的潜在的重要功能。获得的主要结果如下：1.苹果SBP家族基因的鉴定和命名。在苹果基因组和Genbank数据库中鉴定得到27个苹果SBP家族基因，并按照它们的染色体顺序对其进行命名。其中15个苹果SBP基因序列中含有miR156靶位点，可能是miR156的靶基因。2.植物SBP蛋白的系统进化分析。依据对SBP结构域蛋白的系统发生分析，将植物SBP蛋白分为8个亚族，苹果SBP蛋白分布于其中7个亚族。植物SBP蛋白在进化中是分歧的和多样化的。3.基因重复在苹果SBP基因家族的扩增中发挥了重要作用。基因结构、基因染色体位置及在苹果基因组范围内对MdSBP基因进行的同线分析表明串联重复和部分重复，以及整个基因组的重复，可能在苹果SBP基因家族的扩增和进化中发挥了重要作用，最终导致了MdSBP基因在数量上、结构上和功能上的多样性。4.苹果和拟南芥SBP基因的同线分析。对苹果和拟南芥进行的基因同线分析表明若干对同源的MdSBP及AtSPL基因分别位于两个物种的基因组同线区域内，表明这些基因对可能具有共同的祖先和相似的生物学功能。据此可以根据已知的AtSPL基因功能推测其同源MdSBP基因的功能。5.苹果SBP基因和miR156的组织特异性表达模式及其功能预测。MdSBP基因在苹果组织器官中表现出多样性的的时空表达模式。大部分MdmiR156靶向的MdSBP基因在茎、叶、顶芽及一些花器官中具有相对较高的转录水平，且与大部分的非MdmiR156靶向的MdSBP基因相比，它们表现出一个更具差异性的表达模式。而成熟MdmiR156大体上表为与其靶基因互补的表达模式，在幼果种子、成熟果种子、花蕾、根和雄蕊等中具有相对较高的表达水平，尤其是在幼果种子中，表明它可能在苹果种子发育中有着重要功能。根据不同物种的SBP基因和miR156的比较分析及其组织特异性表达模式，初步预测了MdSBP基因和MdmiR156在苹果中的功能。6.苹果SBP基因对不同植物激素有响应。对MdSBP基因在不同植物激素（乙烯、水杨酸、茉莉酸甲酯、脱落酸和赤霉素）处理下的叶片中进行的表达分析表明许多MdSBP基因对不同的植物激素有应答反应，表明MdSBP基因可能参与了胁迫或苹果发育中的不同植物激素信号转导的响应过程。更多还原

【Abstract】 Apple (Malus×domestica Borkh.) is economically one of the most important fruit cropsworldwide and the main fruit crop in the temperate regions of the world. The publication ofthe apple genome sequence provides important data for genome-wide identification of genesrelated with excellent agronomic traits, stress or pathogen resistance in apple, and is thefoundation of breeding novel apple cultivars that have excellent agronomic traits and arestress-or pathogen-resistant. Transcription factors play essential roles in the regulationnetworks of many significant developmental processes in plants. SQUAMOSA promoterbinding protein (SBP) is one type of plant-specific transcription factor and plays many crucialroles in development, hormone signaling and stress resistance in plants. To date, the roles ofSBP transcription factors have been substantially elucidated in many plants, whereas thefunction of SBP transcription factors in fruit crops remains unclear. In this study, all membersof apple SBP-box transcription factor family genes were identified in apple genome bybioinformatics approaches. Subsequently, domains, miR156target sites, phylogeny andevolution, gene structures and gene synteny of apple SBP-box genes were investigated.Moreover, expression patterns of apple SBP-box genes and miR156in various apple tissues ororgans and responses of apple SBP-box genes to various plant hormones were also analyzed,providing a basic understanding of the potential important roles of apple SBP transcriptionfactors. The major findings were as following:1. Genome-wide identification and annotation of SBP-box genes in apple.27putativeapple SBP-box genes were identified in apple genome and Genbank database, andnomenclature of them was carried out based on their chromosome orders.15of them hadmiR156target sites and were putative targets of miR156.2. Phylogenetic analysis of plant SBP proteins. According to the phylogenetic analysis ofSBP-domain proteins, plant SBP proteins were classified into eight groups, and apple SBPproteins were clustered into seven of the eight groups. The plant SBP proteins wereevolutionally divergent and diversified. 3. Gene duplications have played crutial roles in the expansion of apple SBP-box genefamily. Gene structure, gene chromosomal location and synteny analysis of MdSBP geneswithin the apple genome demonstrated that tandem and segmental duplications, as well aswhole genome duplications, have likely played critical roles in the expansion and evolution ofthe SBP-box gene family in apple, resulting in diversification of MdSBP genes quantitatively,structurally and functionally.4. Synteny of apple and Arabidopsis SBP-box genes. Synteny analysis between appleand Arabidopsis indicated that several paired homologs of MdSBP and AtSPL genes werelocated in syntenic genomic regions, indicating these syntenic orthologs likely share acommon ancestor and have similar biological roles. Thus we could confidently infer thefunctions of apple SBP-box genes based on their Arabidopsis homologs.5. Tissue-specific expression profiles of apple SBP-box genes and miR156, and theirfunction prediction. MdSBP genes exhibited diversified spatiotemporal expression patterns invarious apple tissues/organs. Most MdmiR156-targeted genes, which had relatively hightranscript levels in stems, leaves, apical buds and some floral organs, exhibited a moredifferential expression pattern than most MdmiR156-nontargeted genes. Whereas matureMdmiR156was generally expressed in a complementary fashion compared with their targetsand had relatively high expression levels in seeds of young fruit, seeds of mature fruit, flowerbuds, roots and stamens, especially in seeds of young fruit, suggesting its likely crucialfunction in seed development of apple. Based on comparative analysis of SBP-box genes andmiR156among different plant species and the tissue-specific expression profiles, wegenerally predicted the roles of MdSBP genes and MdmiR156in apple.6. Apple SBP-box genes were responsive to various plant hormones. Expression analysisof MdSBP genes in leaves upon different plant hormone (ethylene, salicylic acid, methyljasmonate, abscisic acid and gibberellin) treatments showed that many MdSBP genes wereresponsive to various plant hormones, indicating that MdSBP genes may be involved in theresponses to various plant hormone signaling in stress responses or in apple development.更多还原