【作者】 师静；

【导师】 尹伟伦； 卢存福；

【作者基本信息】 北京林业大学 ， 植物学， 2012， 博士

【摘要】 沙冬青属豆科沙冬青属,是古老的第三纪残遗种,为亚洲中部荒漠地区特有的常绿阔叶灌木,具有极强的抵御低温、干旱、盐、高温胁迫的能力,是一种理想的抗逆基因资源植物。但目前对沙冬青的研究主要停留在生理及植被保护方面,对其抗性分子生物学机理方面的研究较少。据此,我们以沙冬青为材料,对其非生物胁迫相关基因的功能进行了较为系统的研究,探讨沙冬青抗逆机理,从而为抗逆品种选育提供数据和技术支持,同时也期望为通过基因工程技术培育抗逆植物新品种提供新的基因资源。本文的主要研究结果如下：1、沙冬青KS型类脱水素基因AmCIP及其基因家族功能分析及鉴定1.1沙冬青KS型类脱水素AmCIP的低温保护功能鉴定AmCIP编码的脱水素中含一个富K区域(HKEGLVDKIKDKVHG),与典型的脱水素K区(EEKKGIMDKIKELPG)有所不同。AmCIP还含有一个S区,但是不含有Y区,因此我们将其命名为KS型类脱水素。本研究的结果显示：转AmCIP烟草的抗寒性获得提高；在E. coli ER2566中超表达AmCIP能增强宿主菌抵御冻融伤害的能力；纯化得到的重组AmCIP是水溶性的蛋白,并具有热稳定性；重组AmCIP能在冻融胁迫下保护乳酸脱氢酶的活性；YFP/AmCIP融合蛋白定位在洋葱内表皮细胞的细胞质和细胞核中。因此推测沙冬青KS型类脱水素AmCIP可能在沙冬青细胞质和细胞核中发挥作用,并与沙冬青低温防御机制密切相关。1.2沙冬青KS型类脱水素基因家族的克隆与序列和功能分析根据AmCIP序列设计引物,从低温、干旱、盐、高温胁迫处理的沙冬青中扩增得到一组与AmCIP同源性很高的基因。它们编码的蛋白长106至290个氨基酸残基,这些蛋白与AmCIP的同源性也很高,加上AmCIP共计为126个。我们对这些蛋白采用AmP+氨基酸残基数+字母的命名方式。经过分析,这些蛋白的分子量从11.661 kDa到30.688kDa不等。每个蛋白中所含的甘氨酸、谷氨酸、天冬酰胺、谷氨酰胺以及组氨酸占氨基酸总数的60%以上,其中甘氨酸的含量最高。除AmP200P为碱性蛋白外,其余蛋白均为酸性蛋白。这些蛋白具有33个氨基酸残基长的保守N端和71或73个氨基酸残基长的保守C段,在这两段保守序列之间有0-8个22或24或26个氨基酸残基长的重复片段,这些重复片段之间也具有较高的同源性。这些蛋白在保守的C端区域中都含有一个15个氨基酸残基长的富含K的,与典型脱水素K片段类似的区域,并且这些蛋白还都具有一个S片段,但是不存在Y片段,因此我们称这个家族为沙冬青KS型类脱水素家族。这些蛋白均为亲水性蛋白,并含有3-11个酪蛋白激酶Ⅱ磷酸化位点和1-16个N-肉豆蔻酰化位点,推测这些类脱水素可能在沙冬青细胞中参与细胞信号转导活动。另外,某些蛋白还含有RGD细胞附着序列。这些蛋白都有较高比例的柔性区域。这些蛋白内部亲缘关系很近,与4种低温响应的脱水素亲缘关系最近,包括鬼箭锦鸡儿(Caragana jubata)AEJ20970.1,棘豆(Oxytropis campestris subsp.johannensis) AEV59620.1和AEV59619.1,以及棘豆(Oxytropis arctobia)AEV59613.1。用纯化的重组AmCIP溶液制备抗血清,用此抗血清对低温、干旱、盐、热胁迫处理的沙冬青进行免疫印迹分析。结果显示,沙冬青KS型类脱水素家族在正常生长条件下的沙冬青中的表达水平很低,但是受低温、干旱、盐、热胁迫信号的诱导,并随着胁迫处理时间的延长表达量也逐渐升高。根据以上结果推测,此沙冬青KS型类脱水素家族可能参与了沙冬青的非生物胁迫防御机制。在此之前尚未有研究发现具有类似特点的大型脱水素家族,沙冬青KS型类脱水素家族的发现可以为脱水素的分子进化以及沙冬青独特的抗逆机理研究提供宝贵的理论基础。2、沙冬青防御素基因AmDF的功能鉴定沙冬青防御素基因AmDF全长414 bp,含有一个207 bp编码68个氨基酸的开放阅读框,推测编码的蛋白分子量为7.66 kDa,等电点为8.92,包含8个保守的半胱氨酸残基可以形成4对二硫键。蛋白内含有一个Knot1结构,这是具有抗菌活性的γ-硫堇(γ-thionin)和Knottin中常见的结构。此编码蛋白与大车前(Plantago major)防御素CAH58740.1的亲缘关系最近。荧光定量PCR结果显示,AmDF在正常生长条件下的沙冬青中表达水平较低,然而其表达量在低温、干旱、盐以及热胁迫条件下表达量都出现上调,并且主要在低温胁迫后期富集。YFP/AmDF融合蛋白定位在洋葱内表皮细胞的细胞质和细胞核中。然而转AmDF烟草的抗低温、干旱、盐胁迫能力未获得提高。因此推测沙冬青防御素AmDF可能参与低温胁迫下对外界环境中病原体的抵御活动,从而提高沙冬青在低温胁迫下的存活率,不过这还需要进一步实验进行验证。3、沙冬青非生物胁迫抗性相关功能基因表达分析3.1沙冬青非生物胁迫下荧光定量内参基因选择植物对逆境条件的反应不取决于某一单个因子,而是大量基因协同作用的结果。对某一个基因、或某一类基因的功能进行分析,难以完整解释性状的表现机理,而对表达的基因进行综合的研究则有利于揭示植物性状的复杂分子本质。为了探索基因某方面的功能,首先就要了解其在植物体内的表达。实时荧光定量PCR具有准确、灵敏、重复性好、通量高等优点,而被研究者广泛应用于基因的表达分析。为了去除不同样本在RNA产量、质量和反转录效率上可能存在的差别,以得到可靠的目标基因的表达水平和表达模式,选取合适的内参基因显得尤为重要。本研究第一次对沙冬青低温、干旱、盐、高温胁迫下荧光定量内参基因的选择进行了系统的分析。利用geNorm和NormFinder两个软件对14个内参候选基因在22个沙冬青样品(包括对照1个,低温、干旱、盐胁迫各5个,热胁迫6个)的表达稳定性进行分析后发现,eIF1和eIF3在22个沙冬青样品中表达稳定性最高,Tub2、Abc1和EF1的表达稳定性最低。通过对AmHsp90的表达进行分析,证明了geNorm和NormFinder两个软件给出的候选基因的排名都是可靠的。3.2沙冬青非生物胁迫相关功能基因在低温、干旱、盐以及热胁迫下的表达分析采用qPCR分析方法,用eIF1和eIF3作为内参,对沙冬青中55个胁迫相关的功能基因,包括AmSOD01～04、AmCAT01、AmAPX01、AmAPX02、AmGPX01、AmPrx01、AmPrx02、AmTrx01～06、AmFNR01、AmFd01～04、AmGrx01～05、AmHsp70-1～3、AmHsp90-1、AmDnaJ01、AmDnaJ02、AmLEA01、AmLEA14、AmLOX01、AmLOX02、Am1433-01～03、AmCAB01～07、AmFBP01～02、AmFBA01、AmEno01、AmPGK01～02、AmMT01～03、AmFer01在低温、干旱、盐以及热胁迫处理后的沙冬青中的表达情况进行分析。表达结果显示这些基因在正常生长条件下的沙冬青中都有表达,说明它们可能为沙冬青在正常生长条件下维持生理代谢所需要的基因。然而它们的表达大多受胁迫信号调节,在表达水平和表达模式上具有各自的特点。抗氧化相关基因在低温、干旱、盐以及热胁迫的各阶段都出现不同程度的上调,说明沙冬青对氧化胁迫有较强的控制力。光合作用相关的AmCAB01～07以及糖异生和糖酵解相关的AmFBP01～02、AmFBA01、AmPGK01～02和AmEno01在非生物胁迫的后期都出现降低,说明沙冬青为了适应长期的恶劣环境,可能在胁迫后期的降低自身对能量和同化物的需求。其余基因也在在胁迫处理的不同阶段出现了上调或下调的现象。推测这些基因可能对沙冬青胁迫防御机制的建立起到积极的作用。这些基因表达模式的多样性,也说明了植物逆境胁迫响应机制的复杂性。总之,经过长期的地质历史时期的适应性进化,沙冬青获得了在沙漠极端恶劣条件下生存的策略,进化得到一些自身特殊的分子防御机制。本研究的成果对沙冬青强抗逆机制的研究提供了重要的实验和理论基础,同时也为农作物和树木抗逆品种的选育提供了许多可供选择的胁迫基因。更多还原

【Abstract】 Ammopiptanthus mongolicus is the only evergreen broadleaf shrub endemic to the Alashan desert, northwest sand area of China, where the climate is arid, the site is desertification and salinization and the temperature is blow-30℃in winter, up to 40℃in summer. Situating long in adverse environments, A. mongolicus has gradually evolved typical superxeromorphic structures and molecular metabolic characteristics to adapt the adversity, such as drought, salinization, cold, heat and other stresses. Tolerance to various adversities makes A. mongolicus an excellent model of woody plants for the study of molecular mechanisms underlying abiotic stresses tolerance. However, few studies were focused on molecular biology of A. mongolicus. Hereby, we carried out a systematic study on the functions of some A. mongolicus abiotic stress-related genes to explore molecular mechanism of stress tolerance, and to provide data and technical support for stress tolerance and resistance breeding of crops. The main findings in this study are as follows:1. Identification of A. mongolicus a KS-type dehydrin-like protein AmCIP and a super KS-type dehydrin-like protein family.1.1 Identification of protection role of a KS-type dehydrin-like protein AmCIP from A. mongolicus under cold stress.Herein firstly, we report function analysis of a dehydrin-like protein AmCIP unified with a distinct K-segment of HKEGLVDKIKDKVHG similar to the 15-mer consensus sequence EEKKGIMDKIKELPG of typical dehydrin. Expression of AmCIP in transgenic tobacco leaded to enhanced tolerance to cold stress for both geminating seeds and seedlings. Over-expression of AmCIP in transgenic E. coli significantly improved freeze-thawing tolerance of host E. coli cells. Moreover, the recombinant AmCIP kept soluble pre-and post-10 min boiling treatment, and conferred stabilization of the LDH under freeze-thawing stress, consistent with the characteristic of dehydrins. YFP-AmCIP fusion protein was subcellular localized in the cytoplasm and nucleus in onion inner epidermal cells, suggesting that AmCIP might function in the cytoplasm and nucleus in cells. The combined results indicated that AmCIP with a distinct K-segment was a hydrophilic dehydrin-like protein and closely associated with resistance to cold stress. This research offered the valuable information for molecular mechanism of dehydrins and stress responses in A. mongolicus.1.2 Cloning, sequencing and functional analysis of a super KS-type dehydrin-like gene family in A. mongolicus.A super AmCIP homologous gene family was obtained through PCR by primers pairs according to DNA sequence of AmCIP. The total present members of this AmCIP homologous gene family account for 126 plus AmCIP. The coded proteins of these genes range from 106 to 290 amino acid (aa) residues, which share high degree of homology with AmClP and between each other, while share low degree of homology with other typical dehydrins. And this dehydrin family has not been reported before. These proteins are named through AmP+protein length+different English letters to present different members, such as AmP106A, AmP106B, AmP106C, et al. The calculated molecular mass of these proteins ranged from 11.661 KDa to 30.688 KDa. Glycine, glutamate, asparagines, glutamine and histidine in each of these protein accounted for more than 60% of the total number of amino acid residues, in which the content of glycine is the highest. As predicted, almost all the members of this A. mongolicus KS-type dehydrin-like protein family are acidic proteins, except for AmP200P which is a basic one.These proteins have a 33 aa conserved N-terminal region, and a 71 or 73 aa conserved C-terminal region. Between these two conserved regions, there are 0～8 conserved repeated amino acid fragments, the length of which is 22 or 24 or 26 aa. And these repeated amino acid fragments also have high degree of homology. Each of these proteins also has a distinct K-segment similar to the 15-mer consensus sequence EEKKGIMDKIKELPG of typical dehydrin, and an S-segment in the conserved C-terminal region, but has no Y-segment. So this group of proteins was called KS-type dehydrin-like protein family of A. mongolicus. These proteins are all hydrophilic and flexible proteins with 3-11 casein kinaseⅡphosphorylation sites,1-16 N-myristoylation sites, suggesting these proteins may be involved in signal transduction activities during abiotic stress. Some members of this family also have cell attachment sequence. Phylogenetic analysis indicated that the members of the KS-type dehydrin-like protein family of A. mongolicus are closely related to each other, and this group is most closely to AEJ20970.1 of Caragana jubata, AEV59620.1 and AEV59619.1 of Oxytropis campestris subsp Johannensis, AEV59613.1 of Oxytropis arctobia which are all cold responsive dehydrins.Expression of this A. mongolicus KS-type dehydrin-like protein was analysed through western blot by using antiserum agains AmCIP. Results showed that this A. mongolicus KS-type dehydrin-like protein family was constitutively expressed with low level, and accumulated at the late stage of cold, drought, salt and heat stresses.The combined results indicate that this A. mongolicus KS-type dehydrin-like protein family plays important roles in abiotic stress responses. Prior to this, no such big dehydrin family has been reported, and the discovery of them is valuable for future researched on evolution of dehydrin and anti-stress mechanism of A. mongolicus.2 Functional identification of A. mongolicus defensin gene AmDFAmDF was obtained through solid-phase subtractive hybridization from cold stressed A. mongolicus. Sequence analysis showed that the overall length of AmDF gene was 414 bp, and the cDNA of AmDF contained a 207 bp ORF encoding a polypeptide of 68 amino acids with a calculated molecular mass of 7.66 kDa and a theoretical pI of 8.92. AmDF contained eight conserved cysteine residues which could form four disulfide bonds, and a Knotl structure which is common in y-thionin and knottin with antimicrobial ability. Phylogenetic analysis indicated that AmDF was most closely related to Plantago major defensin CAH58740.1. Real-time quantitative PCR analysis showed that AmDF was constitutively expressed, up-regulated by cold, drought, salt and heat stress, and specifically accumulated at the late stage of cold treatment. YFP-AmDF fusion protein was subcellular localized in the cytoplasm and nucleus in onion inner epidermal cells, suggesting that AmDF might function in the cytoplasm and nucleus in cells. However, the cold, drought and salt tolerance of AmDF transgenic tobacco was not improved. The combined results indicate that AmDF protein plays some roles in anti-microbial activities under cold stress to elevate the survival rate of A. mongolicus under cold stress, which need to be confirmed by further study.3. Expression analysis of abiotic stress-related functional genes in,4. mongolicus3.1 Reference gene selection for A. mongolicus under cold, drought, salt and heat stressAbiotic stress tolerance of plant is not determined by single gene, but resulted from products of great many genes. The mechanism of stress tolerance could be hardly explained by analysis of one or a class of genes. However, a comprehensive research on expressed genes is advantageous in revealing the complex stress tolerance mechanism, which is more and more based on gene expression. qPCR is more widely used for its multiple advantages of accuracy, sensitivity, reproducibility, and high throughput. Nevertheless, the accuracy of qPCR was significantly influenced by the reference genes used. By now, no report has so far described the selection of reference genes to get stringent normalization for qPCR in A. mongolicus.We identified reliable reference genes for normalization of qPCR data in A. mongolicus under abiotic stresses from 14 reference gene candidates (UBQ, Tub1, Tub2, Abel, Ubc1, Ubc2, Ubc4, Ubc5, elF1, elF2, eIF3, eIF4, EF1, EF2). We set a series of 22 experimental samples covering the control and different time points under cold, dry, salt, and heat stresses. According to geNorm and NormFinder, the combination of elF1 and eIF3 was more sufficient to confer an accurate normalization across all the treatments, which was confirmed by normalizing qPCR data of AmHsp90. In contrast, Tub1, Abel, and EF1 were ranked poorly and should be excluded as reference genes.3.2 Expression analysis of abiotic stress-related functional genes in A mongolicus.55 anti-abiotic stress genes were chosen from an EST data of cold-and drought-stressed A. mongolicus, including AmSOD01～04, AmCAT01, AmAPX01, AmAPX02, AmGPX01, AmPrx01, AmPrx02, AmTrx01～06, AmFd01～04, AmFNR01, AmGrx01～05, AmHsp70-1～3, AmHsp90-1, AmDnaJ01, AmDnaJ02, AmLEA01, AmLEA14, AmLOX01, AmLOX02, Am1433-01～03, AmCAB01～07, AmFBP01～02, AmFBA01, AmPGK01～02, AmEno01, AmMT01～03, AmFerOl, which are known to be involved in anti-stress mechanism. The expression of these genes was analysed in the same above 22 samples by qPCR through normalizing against elF1 and elF3. Results showed that these genes are all constitutively expressed in A. mongolicus, suggesting that they had basic functions under normal growth conditions. The balance and coordination of them provides highly efficient machinery to maintain normal growth and metabolism. The 55 anti-stress genes exhibited diverse expression levels and differentially up-or down-regulated expression patterns under abiotic stresses, indicating different anti-stress protection roles of them under abiotic stresses. The anti-oxidative genes are up-regulated at various stages of cold, drought, salt and heat stresses, suggest a powerful control for generating oxidative damage under abiotic stress. Photosynthesis associated genes AmCAB01～07, and gluconeogenesis and glycolysis associated genes AmFBP01～02、AmFBA01、AmPGK01～02 and AmEno01 were down-regulated at the late stages of abiotic stresses, indicating that A. mongolicus may reduce its demand for energy and assimilates at the late stages of abiotic stresses to survive the long-term hard environment. The combined results suggested that these genes may play positive roles in mechanism of A. mongolicus abiotic stress tolerance. The diversity of expression patterns of these genes under abiotic stresses provides a preliminary impression of the complicated stress tolerance mechanisms in A. mongolicus. These results are valuable for future research on gene expression and abiotic stress tolerance in A. mongolicus.In conclusion, A. mongolicus has evolved a complete set of defense mechanism to survive in the extreme environment of desert. The findings in this study confer a better understanding of the special anti-stress system of A. mongolicus, and provide important guidance for future researches on plant anti-stress activities.更多还原