【作者】 袁晓伟；

【导师】 亓宝秀； 李新征；

【作者基本信息】 山东农业大学 ， 细胞生物学， 2013， 博士

【摘要】 植物的固着性使它整个生命过程中不可避免面临各种不利的外部环境（如干旱胁迫、盐胁迫、涝害、高温、低温、病原菌侵袭等）。在长期的进化过程中，植物形成了一套响应胁迫和提高抗性的机制。因此，理解植物应答逆境胁迫伤害、适应性变化等诸多生理过程，对提高农作物的产量和品质有重要意义。内源二十碳二烯酸和二十碳三烯酸对拟南芥生理影响VLCPUFA（svery long chain polyunsaturated fatty acids）对人体营养和健康非常重要。虽然人体自身能够合成VLCP（UFAs，但其效率极低。目前，该类脂肪酸的主要来源是深海鱼油，其主要有效成分是二十碳五烯酸（EPA）和二十二碳六烯酸（DHA）。但由于过度捕捞、环境污染等诸多原因，导致鱼油的供给已远不能满足市场需求。随着植物转基因技术的发展，许多生产超长链多不饱和脂肪酸的转基因油料作物已诞生，如油菜，大豆和亚麻等。因此，研究这些异源脂肪酸在植物生理和对非生物胁迫的耐受性具有可行性。该研究将为转基因植物中商业化生产超长连多不饱和脂肪酸奠定理论基础。我们以內源合成二十碳二烯酸（EDA）和二十碳三烯酸（ETrA）的转IgASE1基因的拟南芥作为材料，结合外源添加实验，研究EDA和ETrA对拟南芥的生理表型及干旱胁迫的耐受性的影响。主要研究结果总结如下：（1）RT-PCR分析表明，IgASE1基因在转基因拟南芥中表达；气相色谱分析其叶片脂肪酸组成，结果表明：內源合成的EDA和ETrA占总脂肪酸含量的25.6mol%；叶片膜脂组成分析表明，EDA和ETrA参与了生物膜脂的构成，从而将膜脂内不饱和脂肪酸的含量由野生型的59.6mol%，增加到转基因植株中73.7mol%。（2）连续多代拟南芥生长表型调查结果表明，与野生型拟南芥相比，转基因拟南芥表型上有差异：苗期生长缓慢，花较小且花瓣较薄，种子更饱满，后熟时间缩短。（3）外源添加ABA实验结果显示，与野生型拟南芥相比转基因拟南芥在种子萌发，苗期生长及气孔的张开方面对ABA的敏感性增强。（4）选用萌发后3天的幼苗和萌发后3周的苗子分别进行模拟干旱和断水处理实验，结果表明转基因拟南芥比野生型拟南芥对干旱的耐受能力强。利用qRT-PCR分析依赖于ABA途径的关键基因（RD29B、 RD26和CBF3）和非依赖于ABA途径的关键基因（COR15A、ADH1和CBF2）的表达情况，结果表明转基因拟南芥对干旱的强耐受力来源于依赖ABA途径和非依赖ABA途径的共同作用。（5）外源添加EDA和ETrA可以在野生型中模拟转基因拟南芥对ABA敏感和对干旱耐受的表型，即EDA和ETrA在拟南芥对ABA和干旱的响应过程中作用。植物XB3-like蛋白家族功能研究具有同源性结构域及序列，进化上相关的蛋白质归在同一个蛋白家族，对蛋白家族的分析研究有利于对该蛋白家族的功能及进化进行系统研究。随着基因组测序的飞速发展，至今已有五十多种植物完成或正在进行全基因组测序，这为在多个物种中分析比对一个蛋白家族提供可行性。水稻XB3，是XA21丝氨酸/苏氨酸激酶的底物，在XA21介导的抗病途径中起到重要作用。在拟南芥中同源基因有5个，分别命名为XBAT31、XBAT32、XBAT33、XBAT34和XBAT35。对XBAT32和XBAT35的研究发现，它们在植物的生长发育及抗逆过程中起到重要作用。经过我们对这些蛋白结构分析发现，它们都包括锚定重复序列（ANK）和保守的C3HC4型指环结构。因此，我们将同时含有ANK和C3HC4指环结构的蛋白归为一个蛋白家族，并命名为XB3-like蛋白家族。本研究利用生物信息学的方法分析比对数据库提供的所有物种序列（29种），对该蛋白家族的功能及进化进行系统研究，以期找到具有合适功能的基因用于生产实践。（1）我们利用生物信息学方法从已公布的29种全基因组测序的物种中搜索到187个同时具有ANK和C3HC4型指环结构的蛋白。因其与XB3结构上相似，我们命名为XB3-like蛋白家族，编码这组蛋白的基因命名为XB3-like基因家族。对29个物种中XB3-like基因的数量统计后发现，两种藻中无XB3-like基因，结果说明该蛋白家族在植物进化过程中可能是由蕨类开始出现的。（2）根据XB3-like蛋白间的同源性关系构建进化树，发现该家族蛋白可分为三组，且三组蛋白的指环结构域之间存在明显差异。另外，我们还发现多数植物中XB3-like蛋白分别属于上述三组蛋白，而蓖麻和苜蓿中仅有2个XB3-like蛋白且都属于第Ⅰ组。（3）对模式植物拟南芥和三种作物（水稻、玉米和大豆）的芯片分析结果显示，该基因家族在植物的整个生命周期和不同的组织中均有广泛表达和偏好性表达，这个结果说明该家族基因可能在植物不同发育阶段和不同组织发挥重要的生物学功能。（4）我们利用荧光定量的方法检测到拟南芥和玉米中多个XB3-like基因的表达受激素和非生物胁迫信号的影响，预示着它们可能在植物生长发育以及胁迫响应过程中扮演了重要角色。（5）拟南芥XBAT31表达受多种激素和非生物胁迫信号的诱导。于是我们对XBAT31基因的功能产生兴趣。我们将基因上游2Kb启动子序列与GUS报告基因连接后表达，发现XBAT31在不同组织均有表达，其中茎、叶及花序中表达量较高。（6）XBAT31基因低量表达突变体在萌发期和苗生长早期对盐胁迫耐受性较野生型拟南芥低。其中，突变体内非依赖ABA途径内的基因表达量明显降低，可能是使突变体在萌发期和苗期对盐胁迫的耐受性降低的原因。因此，我们推测XBAT31在盐胁迫信号的转导过程中可能起正向作用。更多还原

【Abstract】 Plants are sessile organisms hence they cannot escape unfavourable environmentalconditions within their life cycle, such as high salinity, drought, waterlog, high or lowtemperature, pathogen attack and mechanical agitation. For long-term evolution, plants haveevolved finely tuned stress signaling and resistance mechanisms. Understanding themechnisms of plant stress responses and adaptation to enrionmental cues is very important inthe improvement of yield and quality of crops.Effect of heterologous eicosadienoic acid and eicosatrienoic acid on physiologicalchanges in ArabidopsisThe very long chain (≥C20) polyunsaturated fatty acids (VLCPUFAs), such aseicosapentaenoic acid (EPA) and docosapentaenoic acid (DHA), are essential for humanhealth and nutrition. They can be synthetized in human, but the productivity is very low. Oilyfish is good source for these fatty acids. However, the depletion of marine fish resources andenvironmental pollution result in severe shortage of fish oil. Therefore, various attempts havebeen carried out to engineer oilseed crops, such as canola, soybean and flax, to produce thesevery long chain polyunsaturated fatty acids with some success. However, before introducingthese crop plants to agriculture, the roles of VLCPUFAs in transgenic plants such asphysiological changes and tolerance to abiotic stressed have to be evaluated. This will providevital information for the commercialization of VLCPUFA-producing transgenic plants.Homozygous single-copy transgenic Arabidopsis plants expressing the IgASE1, in whichEDA and ETrA was produced, were used for this study. We compared the difference betweentransgenic Arabidopsis and wild type in their morphology and responses to abiotic stress andbelow are our findings: (1) We carried out RT-PCR to detect the transcript level of IgASE1from rosette leavesand the result showed that IgASE1was indeed expressed in these transgenic plants. We nextmeasured the total fatty acid content in the leaf tissue by gas chromatography and the resultshowed that the transgenic plants contained two additional fatty acids conresponding to EDAand ETrA compared to the wild type. The result of the total fatty acid content in the leaf tissueshowed that these two fatty acids accumulated to25.6mol%of total fatty acids. We measuredthe total fatty acids in membranes and showed that these two fatty acids were found inmembrane of transgenic plant and the accumulation of unsaturated fatty acids was up to73.7mol%of the total fatty acids and compared to59.6mol%in the wild type plant.(2) Compared to the wild type Arabidopsis, we found the transgenic plant growed slowerduring seedling stage and the leaves were smaller. The flowers of the transgenics were smallerwith smaller petals that appear to be translucent. Seeds of the transgenic plants were largerand less dormant than the wild-type.(3) To test the sensitiveness of transgenic Arabidopsis to ABA, given the importance ofABA in the abiotic stress adaptation, the germination, early seedling growth and stomatalopening of wild-type and transgenic Arabidopsis were compared. The result showed that thetransgenic seedlings were hypersensitive to ABA during germination, early seedlingdevelopment and stomatal closing.(4) We mimicked the drought condition by treating three-day-old seedlings of both thewild-type and the transgenics with glycerol or mannitol for7days. Three weeks old plantswere subjected to drought stress by withholding water for22days. These results indicate thatthe transgenic plant exhibited enhanced drought tolerance than the wild-type Arabidopsis. Todetermine whether the drought tolerance of35S:IgASE1transgenics is due to ABA, wecompared the expression profiles by quantitative qRT-PCR of some drought-inducible genesin ABA-dependent and ABA-independent pathways. The result showed that both theABA-dependent and the ABA-independent drought-inducible genes displayed increasedtranscript levels in the transgenic plant during drought stress, implying that the adaption todrought stress in the EDA and ETrA producing transgenics involves both the ABA-dependentand ABA-independent pathways.（5）Exogenous application of EDA and ETrA can mimick the ABA and drought responses in wild type plants similar to that found in transgenic plants. These results indicatedthe involvement of these eicosapolyenoic acids in the regulation of ABA mediated seedgermination, early seedling development as well as in responses to drought of the matureplants, at least partially.Global analysis of XB3-like protein family in plantsProteins in the same family share homology domains or sequences and are relatives inevolution. Therefore, global analysis of a protein family is conduceivable in the study of theirfunction and evolution. With the rapid development of DNA sequencing technology, the fullor partial genome sequences of more than50plant species have been completed or ongoing.Therefore, it is feasible to analyze a protein family in a large number of plants.In Oryza sativa，XB3is a substrate for the XA21serine and threonine kinase and isnecessary for Xa21-mediated immunity. In Arabidopsis, five homologos genes have beennamed XBAT31, XBAT32, XBAT33, XBAT34, XBAT35, respectively. So far, XBAT32andXBAT35have been reported played roles in plant growth and development and stresstolerance. We analysed the domains of the proteins’ sequences using tools and found that allthe proteins contained ankyrin repeats (ANK) and a conserved C3HC4RF domain. Then wenamed the proteins contained ankyrin repeats (ANK) and conserved C3HC4RF domain theXB3-like proteins. We used bioinformatics methods to gather extensive information regardingthis family in the29plant genomes that have been completely sequenced. Then the functionand evolution of these proteins were studied, and the interesting gene will be found and used.(1) Using bioinformatics methods, we identified187proteins which contain ANK and aconserved C3HC4RF domain, from29species with complete genomes and named theseproteins the XB3-like proteins because they are structurally related to the rice (Oryza sativa)XB3and the encoding genes the XB3-like gene. In this study, we observed that the XB3-likegene family originated from the ferns and were identified in27species of land plants (noXB3-like gene in Chlamydomonas reinhardtii or Volvox carteri).（2）To clarify the phylogenetic relationship among the XB3-like genes and to infer theevolutionary history of the gene family, the full-length protein sequences of the XB3-likefamily members in plants were used to construct a joint unrooted phylogenetic tree, from which it can be observed that the proteins fell into three major groups (group I to group III)with well-supported bootstrap values. Interestingly, we found that the C3HC4-type RFdomain was conserved in each group. In addition, most plants contain3groups of XB3-likegenes, except for Ricinus communis and Medicago truncatula, which only contains group IXB3-like genes.（3）To investigate the expression profile of the XB3-like gene family in plants, we usedbioinformatics methods to gather extensive microarray information regarding this family inthe model plant Arabidopsis and in other crops (Oryza sative, Zea mays and Glycine max).These results demonstrated that the accumulation of XB3-like gene transcripts wasdemonstrated during different developmental stages and in different tissues. It is suggestedthat the XB3-like proteins may be play roles in during different development stages and indifferent tissues.(4) To investigate the potential function of the XB3-like gene family in plant, wesurveyed the responses of XB3-like genes to phytohormones and in Arabidopsis and Zea maysusing real-time PCR. The results demonstrated that phytohormones and mimic abiotic stressesaffect the expression of XB3-like genes and suggest that these proteins may play roles ingrowth and development as well as responses to abiotic stresses in plants.(5) Then we are interested in XBAT31, because the expression of XBAT31was inducedby all phytohormones and mimic abiotic stresses tested. Upstream of the gene2Kb sequencepromoter was used to promote the expression of GUS reporter gene. And the result showedthat XBAT31were expressed in different tissues, especially high expression in stems, leavesand inflorescence.(6) We compared the responses to salt stress between wild type and RNAi mutant, inwhich the expression of XBAT31was less than in wild type, during germination and seedlinggrowth. The result showed that the mutants couldn’t tolerate the salt stress. To determinewhether the less tolerance of mutants was dependent ABA, we compared the expressionprofiles by quantitative qRT-PCR of some of abiotic-induced genes in ABA-dependentpathway and ABA-independent pathway. The result showed that the transcript level ofABA-independent genes was reduced in mutants, and that will be part of reason for lesstolerance in mutants than wild type Arabidopsis. Therefore, we hypothesized that the XBAT31may play a positive role in response to salt stress signal.更多还原