【作者】 闫丽；

【导师】 夏光敏；

【作者基本信息】 山东大学 ， 细胞生物学， 2011， 博士

【摘要】 由于石油、天然气和煤炭等不可再生能源的常年开采而逐渐面临资源枯竭,全球正遭遇着越来越严重的能源危机。我国地域广阔、人口众多,资源丰富,但人均资源则相对匮乏,随着我国经济的高速发展,对能源需求量巨大,能源危机更为突出。因此,探索可再生、可持续、无污染、高效清洁的新能源已成为我国乃至全球科学家重点研究的目标之一。生物乙醇作为唯一一种能以液态形式存在的新能源,是我国现阶段最有可能实现产业化生产的主要生物能源产品之一,也是我国生物能源产业发展的重点。以粮食和油料作物为原料来生产乙醇可能会威胁国家粮油食品安全,而植物纤维素则是一种非常有潜力、前景广阔的生物乙醇的生产原料。我国有大量的盐碱地、荒地、次生退化土壤,因此结合我国国情,充分利用好这些宜耕边际性土地,筛选、培育适应性好、抗逆性强、具有较高生物量的能源作物高粱等,为生物乙醇生产提供大量优质原料具有重要意义。高梁具有C4植物的高光合效率,抗逆性强,生长期短,适应性广。即使在贫瘠、盐碱、干旱土地上种植也有较高产量和生物量。此外高粱基因组较小,有丰富的种质资源和生物信息学资源,又有大量EMS诱发的突变体,因此有必要选择出其中的耐盐碱种类进行深入研究、分析,培育出能在盐碱地、旱地正常生长发育的能源高粱新品系,对大量黄/褐中脉突变体的研究,有利于从分子机理上了解木质素代谢的调控,为进一步通过基因工程改良能源高粱创造条件。植物通过光合作用将绝大部分有机物转化为细胞壁成分,其主要成分为纤维素、半纤维素、木质素。木质素的水解产物影响纤维素酶对纤维素的水解效率,使纤维素转化为乙醇的成本提高。因此有必要系统认识高粱木质素合成及其基因表达调控的分子机制,分离木质素合成调控的关键基因。多种作物黄／褐色中脉(bmr)突变体的木质素含量明显降低,是研究木质素合成及调控的良好试验材料。但是至今为止,仅对少数bmr突变体与木质素合成的关系有所了解,大多数的突变体还有待于进一步研究。为了寻找耐盐碱的高粱品种,本论文对美国农业部收集的高梁种质资源进行筛选,获得抗盐碱种质材料,为在盐碱地发展能源植物提供了有用的实验材料；本论文同时通过抑制性差减(SSH)技术分别构建13种bmr突变体混合体与BTx623野生型对照的正、反向差减文库；从正反向文库中挑选cDNA片段制作基因芯片,分析其表达模式；根据基因的表达模式,从中筛选与木质素合成及调控相关的EST片段,并进一步克隆了部分差异表达基因；初步鉴定了这些基因的功能,为高粱及其它能源植物细胞壁成分的改良,培育低木质素的新种类提供了有益的线索。具体研究内容及结果包括：1.高粱耐盐性种质资源筛选根据美国农业部植物研究服务署(USDA-ARS)种植资源系统(National Plant Germplasm System)所收集管理的高粱信息,我们在其收集的1200多份甜高粱中选取锤度大于10,蔗糖含量高于4的甜高粱717份,及四万多份粒用高粱中高度大于2,5米,整齐度小于2.5的粒用高粱4222份,重新编号,进行大规模、高强度(150-200mmol/L NaCl)、长时间(25-40天)的耐盐性筛选。通过对甜高粱的两轮耐盐性筛选,共得到了耐盐株系83个,移栽成活65个株系,其中46个株系收到种子；对普通高粱进行耐盐碱筛选,并移种大田或花盆312个株系,大多数完成生活周期并获得了种子。在东营盐碱地中播种了31种筛选得到的耐盐甜高粱,结果表明碱地中甜高粱的出苗率略高于盐碱地。但其总生物量差异不大,盐碱地单株平均重量比碱地高。多数甜高粱在盐碱地中的锤度高于碱地。不同甜高粱的单株种子产量在盐碱地和碱地差异很大。盐碱地生长的甜高粱比同时播种在碱地的甜高粱提早成熟5-7天。2.利用SSH和cDNA芯片技术分析bmr突变体和野生型高粱BTx623的表达差异为了研究高粱bmr突变体细胞壁合成代谢及调控机制,我们用抑制性差减(SSH)文库和cDNA芯片联用的方式检验了bmr突变体和野生型高粱BTx623的差异表达基因。共得到153个差异表达基因,其中43个在bmr突变体中上调表达,110个基因在bmr突变体中表达受抑制。我们对其中12个差异表达基因进行半定量RT-PCR验证,结果证明了基因芯片数据是正确的。差异表达基因根据功能不同可分为11类：代谢、光合作用、遗传信息的加工、胁迫响应、蛋白命运、信号转导、转运、木质素合成、细胞过程和移动性、发育和调节及其它,其中代谢类的差异表达基因数量最多。光合作用类别的17个差异表达基因中,16个在bmr突变体中的表达都受到抑制,这一点和部分bmr突变体生物量减少的表型相一致。结果,我们从高粱bmr突变体中找到了一批差异表达基因,其中的CYP基因在bmr突变体中表达下调,这与bmr突变体中木质素含量降低一致；而木质素合成的苯基丙烷途径中的一个C4H基因,在bmr突变体中表达上调,该结果可能预示高粱的三个C4H在合成细胞壁的过程中,其功能有所分化；MYB.NAC.Lim等转录因子的表达在bmr突变体和野生型中没有明显的差异,但却发现bHLH转录因子在多个bmr突变体中明显上调表达。本文进一步对这3个基因进行了结构、表达分析及功能的初步鉴定。3.高粱木质素合成代谢相关基因的功能鉴定SbHLH的功能研究SbHLH是13种bmr突变株系与野生型SSH筛选和芯片杂交后得到的表达上调最高的一个转录因子,该基因信号在芯片杂交结果中重复出现达6次之多,表明其可能是多种bmr变体共有的一个调节信号。RT-PCR结果表明,7个bmr变体的叶片在7叶期该基因的表达也明显上调。SbHLh在根、茎、叶中有不同的转录活性,在叶中表达量最高,茎中的表达量最低,这与该器官的木质化程度有密切关系。在酵母菌中,全长SbHLH具有转录激活活性,表明它是一个转录因子；缺失N端低复杂性区时,即使HLH结构域完整存在也没有转录活性,说明该低复杂性区对HLH结构域及转录激活功能的重要性；C端缺失不能影响其转录激活能力。通过该蛋白偶联GFP,基因枪法转化洋葱表皮,结果表明该基因表达产物定位在细胞核。在拟南芥中过表达SbHLH后,转基因拟南芥的表型在幼苗期没有明显变化,也能正常完成的生长周期,但在后期由于茎伸长,过表达系比野生型容易发生倒伏。6周龄的冰冻切片染色结果及木质素含量测定表明,过表达拟南芥茎的木质素含量比野生型和空载体对照明显降低,木质素合成途径和类黄酮/花青素合成途径中部分相关基因的表达受到不同程度的抑制,如]DAL1,4CL,CCR1, HCT, COMT, CHI, UGT等基因。因此,SbHLH很可能是苯基丙烷代谢途径的转录抑制因子。SbCYP功能研究SbCYP基因在多个bmr突变体的7叶期中均呈现下调表达的趋势；与之相反,该基因在野生型高粱(BTx623)的5叶期到7叶期的表达呈上调趋势。在拟南芥中过表达SbCYP后,转基因拟南芥能完成正常的生长发育周期但在6周龄时的茎中,木质素含量和野生型拟南芥没有明显差异,不过在8周龄过表达的拟南芥株系茎中,木质素含量则明显升高；同时部分木质素合成相关基因(PAL1,4CL1, CCoAOMT, CCR1等基因)也被诱导表达,这些结果说明SbCYP参与了木质素的合成或调控。SbCYP的调控模式在bmr突变体与野生型高粱中明显不同,这可能是造成bmr突变体木质素含量降低的原因之一,也可能是受到其它上游基因(例如SbHLH)调控后出现的效应。SbC4H功能研究SbC4H基因在多个bmr突变体中都呈现出上调表达的趋势。将SbC4H生拟南芥中过表达后,在6周龄的转基因拟南芥,其茎中的木质素的含量较野生型明显降低,同时SbC4H过表达拟南芥中的部分木质素合成相关基因(例如4CL1,4CL, F5H1)的表达受到一定的抑制。总之,通过对高粱bmr突变体和野生型高粱中的差异表达基因SbHLH, SbCYP和SbC4H在木质素合成中的功能研究,发现他们在木质素合成及调控中起着重要作用,实验结果丰富了我们对bmr突变体以及木质素合成调控的认识,也为进一步的能源作物的细胞壁改良提供了理论基础和候选基因。更多还原

【Abstract】 We are facing more and more serious energy crisis due to exhaustion of fossil fuel reserves, such as coal, crude oil and natural gas。This situation is worse in China owing to the larger population, fewer resources per capita and more energy demand. Thus, it becomes increasingly necessary to the development of renewable, sustainable, clean and efficient energy sources. As the only renewable energy source of liquid fuel, ethanol is the most promising type of renewable energy sources in future and has received lots of attention in China. Increased exploitation of biomass energy from starch or oil crops will threaten food supplies through competition for land, which make lignocellulosic biomass a kind of ideal bioenergy resource. The presence of abundant of saline-alkali and marginal soils in China makes it very meaningful to exploit biomass resources not only produce sufficient biomass for converting to biofuels but also with stress-resistance which suitable for growing on the soils.As a C4plant, sorghum accumulates a significantly greater amount of carbon during photosynthesis than do C3plants. Most importantly, sorghum shows high tolerance to drought and low nutrition, which makes it possible for grown on lean soils and still achieves high yields. The availability of genome sequence informations and abundant number of germplasms together with a great deal of bmr mutants make sorghum not only a bioenergy crop which can be grown in salty and dry land, but also a model of lignin synthesis and regulation research.Plant cell walls comprise most of the dry body mass synthesized through photosynthesis, which mainly comprised of cellulose, semicellulose and lignin. The lignin in lignocellulosic biomass can lower the efficiency of converting cellulose to ethanol, thus, understanding the molecular mechanisms of lignin biosynthesis and regulation and identifying key genes related to lignin content is necessary for genetically improving the feedstock quality of bioenergy crops with lower lignin content. The brown-midrib (bmr) is a kind of genetic mutations found in a few species which often associated with reduced lignin biosynthesis. This genetic mutation may serve as a model of developing strategies for manipulating lignin content. Although the reduced lignin content in the bmr mutant plant is evidenced, at present very little is known concerning where the bmr mutations occur in the lignin biosynthesis pathway. In order to identify saline and alkali resistant sources in sorghum, we have completed a project on evaluation of the entire U.S. sorghum germplasm collections. Meanwhile, we have constructed forward and reverse subtracted cDNA library with bmr mutants and wild-type sorghum. Thousands of cDNA sequences have been selected from the SSH cDNA libraries and then were used to print cDNA microarrays for transcript analysis. According to the transcript profiling, we identified some differently expressed genes involved in lignin synthesis and regulation. Subsequent characterization and functional analysis of their role involved in cell wall biosynthesis will benefit for genetically improvement of cell wall components of sorghum and other bioenergy crops.The main results of this work are summarized as follows.1. Screening for salt-resistant germplasm line of sorghumWe have evaluated the salt resistance of717out of1200sweet sorghums and4222out of more than40,000grain sorghums in U.S. sorghum germplasm collections (treatment for25-40d under150-200mmol/L NaC1). For sweet sorghums, we obtained83salt-resistant lines,65of which survived after transplant in field or pots and46of them got seeds. For grain sorghums, we found312salt-resistant lines, and most of them can finish the life cycle.A total of31salt-resistant sweet sorghum lines were planted in the alkali field and saline-alkali land. The rate of germination in alkali field is higher than that in saline-alkali field. The overall biomass of sweet sorghums in the two kinds of field is almost the same, but the individual one of seedling in saline-alkali field is higher. The brix degree of most sweet sorghums in saline-alkali field is higher than that in alkali field. The seed yields of each sweet sorghum line in these two kinds of field are very different, with the mature time of sorghums in saline-alkali field is5-to-7-day earlier.2. Identification of differentially expressed genes in sorghum (Sorghum bicolor L.) brown midrib mutants using cDNA subtraction and microarray analysisFor dissecting genes involved in the cell wall metabolism that leading to the brown midrib phenotypes and their regulatory mechanisms in sorghum, suppression subtractive hybridization (SSH) combined with cDNA microarray profiling was performed to identify the differentially expressed genes in13sorghum bmr mutants. A total of153differentially expressed genes were identified, of which43genes showed up-regulated expression while the other110down-regulated in the bmr mutants. The expression pattern of12candidate genes through semi-quantitive RT-PCR confirmed the accuracy of the data from microarray analysis. All the differentially expressed cDNAs with significant protein homology could be classified into11functional groups: metabolism, photosynthesis, genetic information processing, stress responsive, protein fate, signal transduction, transport, lignin synthesis, cell process and mobility, development and regulation, and others. The most abundant group in the differentially expressed genes was metabolism. In the photosynthesis category,16of the17differentially expressed genes were down-regulated which coincident with the yield reduction phenotype of some bmr mutants. A few amount of differently expressed lignin synthesis related genes were identified, of which the expression of CYP was repressed in the bmr mutants which might responsible for the reduction of lignin content of the bmr mutants. However, the expression of another lignin biosynthetic gene cinnamic acid4-hydroxylase (C4H) was enhanced in the bmr mutants, which might indicated that monolignol biosynthesis from L-phenylalanine might occur by more than one route. No differentially expressed MYB, NAC and Lim transcription factors were found in the bmr mutants, however, the expression of a bHLH transcription factor was up-regulated in several bmr mutants. We then dissected the function of these three genes in lignin biosynthesis and regulation.3. Functional analysis of lignin biosynthesis related genes in sorghumFunctional analysis of SbHLHSbHLH was one of the most up-regulated transcription factors identified from SSH and microarray analysis and it showed obviously up-regulated expression in7of the13bmr mutants at7leaf stage. SbHLH appears six times in cDNA microarray analysis, which might indicated that SbHLH may be involved in lignin-biosynthesis regulation of several bmr mutants.The SbHLH showed different expression levels in roots, stems and leaves, with the highest level in leaves and lowest in roots, which correlated with the lignified degree of different organs. The full length SbHLH showed transcriptional activation activity in yeast which proved it a transcription factor. The deletion of the low complex N terminal region will lead to the lost of transcriptional activation activity even though the HLH domain is intact, which indicated the importance of this region to the activity of this transcription factor. Contrary, the deletion of the C terminal has no effect on the transcriptional activation activity of SbHLH. Transient expression of SbHLH-GFP fusion gene in onion epidermal cells indicated that SbHLH was located in the nucleus. Transgenic Arabidopsis overexpressing SbHLH showed no phenotypic alterations and could finish normal life cycle. However, the lignin content was reduced in the stem of transgenic plants. The overexpression of SbHLH down-regulated the expression of several genes of the lignin pathway and flavonoid/anthocyanin biosynthesis, such as PAL1,4CL, CCR1, HCT, COMT, CHI, UGT. Together, these results suggest that the sorghum SbHLH is a transcription factor repressing the phenylpropanoid biosynthetic pathway in Arabidopsis.Functional analysis of SbCYPThe expression of SbCYP was down-regulated in several bmr mutants at7-leaf-stage; however, it was up regulated in wild sorghum BTx623from5-leaf-stage to7-leaf-stage. Transgenic Arabidopsis plants overexpressing SbCYP could finish normal life cycle, with obviously increased lignin content in8-week stem although no obvious lignin content changes was observed in6-week stem. The overexpression of SbCYP induced the expression of several lignin biosynthesis related genes such as PAL1,4CL1, CCoAOMT and CCR1, which suggest that the sorghum SbCYP may contribute to the lignin biosynthesis and/or regulation. The differential expression of this gene between wild sorghum and bmr mutants would partly explain the lower lignin content of the bmr mutant, although this might due to the regulation of upper genes in the regulatory network (for example SbHLH).Functional analysis of SbC4H The expression of SbC4H was up-regulated in several bmr mutants. The involvement of thesorghum SbC4H in the lignin biosynthesis was investigated by overexpressing in Arabidopsis thaliana where the lignin content was reduced in6-week stem of transgenic plants when compared with wild type plants. The overexpression of SbC4H in Arabidopsis down-regulated the expression of several lignin biosynthesis genes such as4CL1,4CL and F5H1.In conclusion, the differently expressed genes in bmr mutants, SbHLH, SbCYP and SbC4H, may be part of the lignin biosynthesis and regulatory network according to the functional analysis. Furthermore, all the results have not only enriched our knowledge about the bmr mutant, lignin biosynthesis and regulation, but also provide new resources and theoretical basis for genetic improvement of lignocellulosic biomass with improved bioethanol production.更多还原