【作者】 李文燕；

【导师】 王金发；

【作者基本信息】 中山大学 ， 遗传学， 2010， 博士

【摘要】 茉莉酸是植物体内广泛存在的一种植物激素,由亚麻酸经十八烷碳烯酸代谢途径合成的;它不仅作为内源生长物质,在植物的整个发育进程中起调节作用,而且还作为局部和系统的信号分子参与生物和非生物逆境胁迫的应答。12-氧-植物二烯酸还原酶(12-oxo-phytodienoic acid reductase,OPR)是茉莉酸生物合成即十八烷碳烯酸代谢途径中的一个关键酶,控制茉莉酸合成的最后步骤。在植物基因组中,OPR属于OYE家族,是黄素单核苷酸依赖的氧化还原酶,并且以多基因家族形式存在。目前,尽管对拟南芥及其他物种的少数几个OPR基因在生化和遗传方面进行研究并取得了一些重要结果,但是对于整个植物OPR基因家族的进化及生物学功能(生化和生理功能)并不很清楚。基于此,本研究在水稻第6染色体S5区基因注释和研究基础上,采取比较基因组学与现代分子生物学实验技术相结合的研究策略,首先从整体层面上对植物OPR基因家族进行系统发育分析,以探究植物OPR基因家族的起源、进化及潜在的功能分化;其次在植物OPR基因家族系统发育分析基础上,从局部层面上对水稻OPR家族基因进行分子生物学研究,以探究水稻OPR家族基因的蛋白结构、生化特性及可能的生理功能。1)在植物OPR基因家族系统发育方面:本研究采用综合生物信息学和系统发育的研究方法进行比较基因组学研究,以探究植物OPR基因家族的系统发育、结构进化及可能的功能分化,取得了以下结果:①利用公共数据库资源,在植物基因组中鉴定了105个OPR基因;其中,在6个不同谱系的11个代表性物种(藻类:莱茵衣藻和团藻;苔藓:小立碗藓;蕨类:卷柏;裸子植物:云杉;单子叶植物:水稻、玉米和高粱;双子叶植物:拟南芥、杨树和苜蓿)中鉴定了74个OPR基因,均以多基因家族形式存在。②通过系统发育分析发现,在植物基因组中,存在7个保守的OPR亚家族;其中,在水生植物中,仅存在一个OPR亚家族即sub.Ⅶ;而在陆生植物中,则存在六个OPR亚家族即subs.Ⅰ～Ⅵ。进一步分析提示,在水生和陆生植物分化后,谱系特异扩增尤其是串联重复是陆生植物OPR基因家族扩增与进化的主要机制,并导致多个OPR亚家族的产生。③通过exon-intron结构分析表明,不同谱系物种中的OPR基因内含子的数目和长度呈现多样性,而内含子的位置和相位却十分保守;这一研究结果并结合系统发育分析提示,在OPR家族基因结构进化过程中,不同谱系间及同一谱内的OPR基因发生持续内含子丢失,从而导致OPR基因结构的多样性。基于上述研究结果,提出了一个关于OPR家族基因结构演化的模型。④通过功能分化分析揭示,在进化过程中,由于受到局部正选择作用,不同OPR亚家族及其成员间发生了功能分化,并且导致功能分化的关键氨基酸位点主要分布于OPR蛋白三维结构的外侧,即α螺旋和底物结合区。2)在OsOPR家族基因分子生物学功能方面:本研究在植物OPR基因家族系统发育分析基础上,选择分别代表五个不同OPR亚家族的OsOPR基因即OsOPR04-1 (sub.Ⅰ)、OsOPR08-1 (sub.Ⅱ)、OsOPR06-1 (sub.Ⅲ)、OsOPR01-1 (sub.Ⅳ)和OsOPR02-1 (sub.Ⅴ),采取同源建模、原核表达、半定量RT-PCR及转基因方法研究水稻不同OPR亚家族基因的蛋白结构、生化及生理功能,取得了以下结果:①通过同源建模及蛋白结构比较分析显示,尽管不同亚家族OPR蛋白的三维结构的基本骨架高度保守即由8个α/β交替排列形成桶状结构,但仍存两个在蛋白三维构象上呈现明显差异的MVRⅰ和ⅱ区;位于MVRⅰ底物结合区决定底物与蛋白的特异结合,而MVRⅱ则影响底物进入由MVRⅰ形成的“口袋”。②通过蛋白体外表达及酶活分析表明,不同亚家族OPR蛋白酶活存在显著差异且呈现明显的底物偏好性;除OsOPR02-1外,其他四个OPR蛋白对反式-2-己烯醛、12-氧-植物二烯酸和顺丁烯二酸均呈现出强或中等程度的催化活性,提示这些亚家族OPR基因可能参与十八烷碳烯酸代谢的两个主要分支HPL和AOS途径,以及能量代谢TCA途径来调节植物的生长发育及防御应答。③通过基因表达谱分析显示,OsOPR家族基因的表达呈现明显的组织特异性且不同程度的响应多种非生物逆境胁迫诱导。OsOPR04-1 (sub.Ⅰ)和OsOPR08-1 (sub.Ⅱ)在各种组织及诱导条件下均呈现较强的表达,提示共存于单、双子叶植物中的subs.Ⅰ和Ⅱ基因可能在植物生长、发育及防御中起重要作用;而OsOPR06-1 (sub.Ⅲ)、OsOPR01-1 (sub.Ⅳ)和OsOPR02-1 (sub.Ⅴ)则在激素诱导下root中的表达明显高于shoot,提示仅存于单子叶植物中的subs.Ⅲ、Ⅳ和Ⅴ基因可能在根的生长过程中响应激素尤其是ABA和IAA信号起重要作用。④基于上述研究,提出一个关于OsOPR亚家族基因可能的生物学功能模型,从而为进一步通过反向遗传学(基因过表达和基因干涉)的方法深入研究OsOPR家族基因的生物学功能奠定基础。更多还原

【Abstract】 Jasmonic acid (JA) is a plant hormone and exists widely in plant, which is derived from linolenic acid via the octadecanoid pathway. Moreover, JA not only acts as plant growth regulator in various developmental processes, but also is local and systematic signaling molecule to response several of biotic and abiotic stresses. The 12-oxo-phytodienoic acid reductase (OPR) is one of the key enzymes in the octadecanoid pathway, which controls the last step of JA biosynthesis. In plants, the OPR genes, which belong to the old yellow enzyme (OYE) family, are flavin mononucleotide (FMN)-dependent oxidoreductases and form multigene families. Although discoveries about this family in Arabidopsis and other species have been reported in some studies, the evolution and biological functions (i.e. biochemical and physiological functions) of multiple OPRs in plants are not clearly understood. Therefore, based on the previous study of S5 on chromosme 6 of rice in our lab, we take the strategies of combining comparative genomics with the experimental methods and techniques of modern molecular biology, to investigate the phylogenetic evolution of OPR gene family in plants, and the protein structures, biochemical & physiological functions of OPR family genes in rice.Firstly, a comparative genomic analysis was performed by using a comprehensive bioinformatics & phylogenetic approaches to investigate the phylogenetic relationship, structural evolution and functional divergence among OPR paralogues in plants. The main results were as following:1) By using public database resources, 105 OPR genes were identified from plant genomes, including 74 OPR genes from 11 species representing the 6 major green plant lineages: green algae (Chlamydomonas reinhardtii and Volvox carteri), mosses (Physcomitrella patens), lycophytes (Selaginella moellendorffii), gymnosperms (Picea sitchensis), monocots (Oryza sativa, Sorghum bicolor and Zea mays) and dicots (Arabidopsis thaliana, Populus trichocarpa and Medicago truncatula).2) Phylogenetic analysis showed that seven well-conserved subfamilies exist in plants. All OPR genes from green algae were clustered into a single subfamily i.e. sub.Ⅶ, while those from land plants fell into other six subfamilies i.e. subs.Ⅰ~Ⅵ. Further analysis revealed that lineage-specific expansion, especially by tandem duplication, contributed to the current OPR subfamilies in land plants after divergence from aquatic plants.3) Interestingly, exon/intron structure analysis showed that the gene structures of OPR paralogues exhibits diversity in intron number and length, while the intron positions and phase were highly conserved across different lineage species. These observations together with the phylogenetic tree revealed that successive single intron loss, as well as indels within introns, occurred during the process of structural evolution of OPR paralogues. Then an evolution model was constructed to explain for the structural evolution from ancestral OPR to current OPR genes in plant species of different lineages.4) Functional divergence analysis revealed that altered functional constraints have occurred at specific amino acid positions after diversification of the paralogues, which leaded to significant functional divergence among all OPR subfamilies and their members. Strikingly, analysis of the site-specific profiles established by posterior probability revealed that the positive-selection sites and/or critical amino acid residues for functional divergence are mainly distributed inα-helices and substrate binding loop, indicating the functional importance of these regions for this protein family.Secondly, based on the phylogenetic analysis of OPR gene family in plant, five OsOPR genes (OsOPR04-1/08-1/06-1/01-1/02-1), representing five subfamilies (subs.Ⅰ~Ⅴ), were selected; then a comparative study was performed by using homology modeling, prokaryotic expression, semi-quantitative RT-PCR and transgenic techniques to provide insights into the five OsOPR protein structures, biochemical properties and physiological importance in rice. The main results were as following:1) Comparative analysis of the three-dimensional (3D) structure by homology modeling indicated all five OsOPR proteins contained a highly conserved backbone and consisted of (α/β)8-barrels. Notably, two middle variable regions (MVRⅰandⅱ) were also detected and defined. MVRⅰis comprised of substrate and protein, and MVRⅱaffects the substrate entrance into a "pocket" formed by MVRⅰ.2) Analysis of enzymatic characteristics revealed that all five OsOPR fusion proteins exhibit distinct substrate specificity. Different catalytic activity was observed using OPDA, trans-2-hexen-1-al and maleic acid as substrates, suggesting OsOPR family genes participate in two main branches of the octadecanoid pathway, including the allene oxide synthase (AOS) and hydroperoxide lyase (HPL) pathways, and the tricarboxylic acid cycle (TCA) energy metabolism pathway that regulates various developmental processes and/or defense responses.3) The transcript profiles of five OsOPR genes exhibited strong tissue-specific and inducible expression patterns under abiotic stress, hormones and plant wounding treatments. Furthermore, OsOPR04-1 and OsOPR08-1 (subs.ⅠandⅡ, respectively) transcripts were observed in all selected tissues and with all above-stress treatments, suggesting these two subfamilies play an important role during different developmental stages and in response to stresses. OsOPR06-1, OsOPR01-1 and OsOPR02-1 (subs.Ⅲ,ⅣandⅤ, respectively) expression were strongly up-regulated with hormone treatments in roots, which suggested these three subfamilies play an important role in responding to hormones especially ABA and IAA signals in roots.4) Based on the results from structure comparison, enzymatic characteristics and expression profiles, a model was proposed to elucidate the functions of OsOPR subfamilies. This study highlights important implications in the search for the true physiological role of OPR family members. Further experimental verification of these findings such as reverse genetics (gene over-expression or gene interference) may provide valuable information on the OPRs’physiological functions.更多还原