【作者】 宋敏；

【导师】 马正强；

【作者基本信息】 南京农业大学 ， 生物信息学， 2013， 博士

【摘要】 植物对于不良环境缺乏躲避能力,因此进化出在一定程度上适应高盐、干旱、贫瘠、冷热、病害、虫害等诸多不利环境的能力。在基因组水平上,植物进化出一系列与逆境适应相关的基因,比如抗病基因、胁迫响应转录因子和凝集素基因等。对这些基因的研究将加深对植物适应环境的进化机制和功能机制的了解,有利于农作物的遗传改良。JRL(Jacalin-Related Lectin)是植物凝集素中的一个家族,该类蛋白至少具有一个jacalin结构域。为了了解JRL基因及其编码蛋白的特征以及JRL基因与植物适应性的关系,我们首先通过全基因组搜索鉴定了小麦、玉米、高梁和短柄草基因组中的JRL基因。虽然这些物种的基因组大小不同,但每个基因组所含有的JRL基因数都在20个左右。小麦有三个亚基因组,其JRL基因数也最多,达67个。同一物种内及不同物种间的JRL蛋白序列长度和序列一致性变异范围较大。通过与蛋白结构已被解析的Heltuba和Benlec比较,在小麦JRL蛋白中发现了9个新的保守位点,其中p4中保守残基与先前报道的不同。接近一半的小麦jacalin结构域具有两个糖结合位点。从JRL蛋白的结构域组成上看,禾本科植物的JRL蛋白具有的jacalin结构域数目不超过3个,且嵌合型JRL比例要高于拟南芥。拟南芥更容易出现多个jacalin结构域重复的类型。87.2%的拟南芥JRL蛋白只有jacalin结构域,其中21.3%的DIR蛋白(Dirigent Protein)有4个jacalin结构域。针对包含dirigent结构域和jacalin结构域的DJRL基因,在目前已完成和基本完成测序的植物基因组中进行搜索,结果表明DJRL基因是禾本科植物特有的基因。这一类基因也是小麦、水稻、玉米、高粱和短柄草中主要的嵌合型JRL基因。协同进化分析表明DJRL基因的dirigent结构域和jacalin结构域存在显著的协同进化关系。在系统发生分析中,我们将小麦、水稻、玉米、高粱、短柄草和拟南芥的JRL蛋白可分成8个类群。大部分拟南芥JRL蛋白聚集在同一个群,与禾本科JRL蛋白区分明显。结合拟南芥和禾本科植物JRL蛋白的结构域组成特征,推测拟南芥和禾本科植物分化后可能采用了不同的进化方式。其中一个群包括了来自小麦、水稻、玉米、高粱、短柄草和拟南芥的JRL蛋白,这些蛋白都具有WDDG基序,该基序在拟南芥中具有85%以上的保守性,而在禾本科植物中保守性很低。我们认为这一分支的蛋白可能代表了更古老的JRL蛋白类型。EST数据分析表明,小麦JRL基因主要在根茎叶等营养组织表达,极少在种子中表达。在这些基因中,50个有来自处理组织的EST,其中36个被显著诱导,占小麦JRL基因总数的53.7%。对30个小麦DJRL基因进行的半定量RT-PCR分析表明,除了EST分析检测到的11个响应生物或非生物胁迫的DJRL基因外,还有13个基因响应至少一种非生物胁迫,9个响应一种或多种生物胁迫,23个基因受MeJA、SA或ABA等一种或多种植物激素的诱导。这一结果说明,大多数小麦JRL基因可能与胁迫响应有关。DIR是另一类与JRL,基因在进化和多样性上类似的基因,参与木质素和木脂素的合成。对小麦、水稻、拟南芥和杨树的DIR基因进行了全基因组鉴定,获得了135个小麦DIR基因和44个杨树DIR基因。此外还新鉴定到8个水稻DIR基因和1个拟南芥DIR基因。比较发现,同一物种内DIR蛋白序列一致性变异范围较大。杨树和拟南芥的DIR基因结构简单,多数无内含子；三分之一的水稻DIR基因具有1-5个内含子。大多DIR基因只包含一个dirigent结构域。水稻和拟南芥DIR基因以串联复制为主要扩张方式,杨树DIR基因以串联复制和片段复制为主要扩张方式。EST分析表明小麦DIR基因可在根、叶、花和种子等多个组织中表达。30%的小麦DIR基因受胁迫诱导。更多还原

【Abstract】 Plants are sessile and must adapt to various environmental conditions and stresses, such as high salinity, water and nutrient deficiencies, low and high temperatures, infection by diseases, and attacks by insect pests. At the genome level, this type of adaptive evolution is exemplified by the rapid amplification and functional diversification of the gene families involved in stress responses. Both jacalin-related lectins (JRLs) and dirigent (DIR) family genes have been reported particitation in plant respond to biotic and abiotic challenges. An investigation of these genes would shed light on the evolutionary and functional mechanisms by which plants respond to variable growth environments and lead to the identification of genes critical for crop improvement.JRLs are carbohydrate-binding proteins and involve in various biological processes. In the presence of homeologous subgenomes, the67genes may encompass orthologs from unidentified homoeologous loci. Grouping using95%identity yielded56unique genes, with approximately18genes per sub-genome. We therefore concluded that at the subgenome level, wheat is similar to Brachypodium, sorghum, and maize in JRL gene number but has fewer genes than Arabdopsis (47) and rice (31). JRL proteins are variability in sequence length and domain arhitectures. Compared with the structure of JRL Heltuba and Banlec, thirty-three highly conserved residues showing at least85%similarity. Of the12key residues necessary for the integrity of the β-prism fold of Heltuba,11were conserved in wheat JRLs and one was conserved when75%chemical similarity was used as the cutoff, implying most TaJRL jacalin domains adopt the β-prism structure. Nine new conserved residues were identified in the wheat jacalin domains, including three in133, two in β4, one in β5, one within β5-β6loop, one in β8, and one in β12. Nearly half of the wheat jacalin domains had two carbohydrate-bingidng sites.Domain architectures of JRL proteins are diverse. Our data showed percentage of holo-JRL in Arabidopsis higher than grass plants and percentage of chimeric JRL lower than grass plants. The domain content and organizations are very similar among grass members but different with Arabidopsis in this family. This finding suggests that the domain organization of JRL family was established after the Arabidopsis-grass split and before grass speciation. DJRL, which contains an N-terminal dirigent domain and a C-terminal jacalin domain, are very likely specific to grass plants. The correlation analysis revealed that the divergent evolution of the N termini from one another is highly positive correlated with the divergent evolution of the C termini from one another.To infer the evolutionary relationships of JRL proteins in different plant species, a phylogenetic tree was constructed using the conserved jacalin domains in the rice, Arabdopsis, sorghum, maize, and Brachypodium jacalin proteins. This tree revealed eight clades. Most of AURL members fell into its own subclass, separating from monocot plants. Members from five grass species intended to be clustered together, exhibiting lineage-specific expansion. The clade Ⅱ cereal JRLs, like A. thaliana JRLs, had a conserved WDDG motif, although this region is poorly conserved in cereal JRLs on the whole. It could therefore be speculated that this clade represents the more ancient JRLs.Digital expression data showed that most of the wheat JRL genes were predominately expressed in vegetative tissues (including root, leaf, stem, shoot and crown) but rare in seed. Fifty of the identified TaJRL genes had ESTs from treated tissues, and36showed significant induction by biotic and/or abiotic treatments, accounting for53.7%of the identified TaJRL genes. Indition, another13JRL genes were responsive to at least one of the abiotic treatments and9genes were induced by at least one of the biotic treatments via sqPCR.23JRL genes showed up regulation by at least one of three stress hormones. These data imply most wheat JRL genes play important roles in stresses adaptation to wheat.Dirigent proteins (DIRs) are critically involved in the formation of lignans, a diverse and widely distributed class of secondary plant metabolites exhibiting interesting pharmacological activities and implicated in natural plant defense. In this study, a total of135and44DIR genes were identified in wheat and poplar. While8and1new DIR genes were identified in rice and Arabdopsis genome, respectively. DIR proteins are variability in sequence. Most PtDIR and AtDIR genes have no intron and1/3OsDIR genes have1-5introns. Most DIR proteins contain single dirigent domain. A large number of DIR genes were expanded via gene duplication,29PtDIR,45OsDIR and10AtDIR genes were tandemly duplicated, respectively. There are19and4genes were segmentally duplicated in poplar and rice genomes, respectively. Digital expression data showed that wheat DIR genes were expressed in different tissues.30%TaDIR genes showed significant induction by biotic and/or abiotic treatments. This study will facilitate further studies on DIR family and provide useful clues for functional validation of DIR genes in higher plants.更多还原