【作者】 臧威；

【导师】 周志强； 庞秋颖；

【作者基本信息】 东北林业大学 ， 植物学， 2021， 博士

【摘要】 角果碱蓬(Suaeda corniculata)是天然分布在东北盐碱地的一种真盐生植物,对盐碱胁迫具有很强的耐受性,但目前对其适应盐碱胁迫的分子基础并不十分清楚。转录因子能够激活或抑制下游靶基因的表达,目前已被证实在植物抗逆应答过程中发挥重要作用。其中,热激转录因子(heat shock transcription factor,HSF)广泛参与植物生长发育及逆境响应过程,目前HSF基因在调节植物盐胁迫方面的功能还有待深入研究,盐生植物特别是角果碱蓬中HSF的功能研究尚未见报道。本研究以角果碱蓬为研究材料,利用生理分析及整合多种组学的研究方法,系统解析了角果碱蓬应答盐碱胁迫的代谢分子基础,并筛选到一个参与调控角果碱蓬耐盐性的基因ScHSFA1d。通过基因功能验证,初步认识了 ScHSFA1d转录因子正向调节植物耐盐性的功能,为深入理解ScHSFA1d的作用机制进行了理论探究。主要的研究结果如下:(1)角果碱蓬的盐碱胁迫生理分析相关结果表明,碱性盐胁迫对植物生长的影响比中性盐胁迫更严重。受到碱性盐影响,根的鲜重和相对含水量显著降低。两种胁迫下细胞内ROS水平均上升,清除ROS的抗氧化酶活性和非酶抗氧化物含量有不同程度的改变,根细胞的膜系统受到一定程度的损伤,细胞中积累了渗透调节物质(脯氨酸和甜菜碱)以应对盐碱胁迫。(2)基于1H NMR代谢组的分析结果表明,在NaCl处理的角果碱蓬根中检测到亮氨酸、异亮氨酸、缬氨酸、谷氨酰胺、苹果酸和2-酮戊二酸的含量显著上升。NaHCO3处理后的代谢特征并不完全相同,NaHCO3处理组中丙二酸、胆碱、甜菜碱三种代谢物含量上升,苹果酸、琥珀酸、蔗糖三种代谢物含量下降。在代谢组结果中,我们发现两种胁迫处理后甜菜碱含量远高于其他代谢物,而且在NaHCO3处理组中发现与甜菜碱合成代谢途径相关的代谢物磷酸胆碱和胆碱的含量也显著增加,甘氨酸甜菜碱的积累主要有助于胁迫过程中渗透稳态的重建。(3)蛋白质组研究结果表明,在中性盐和碱性盐胁迫后分别有65和32个蛋白质差异表达,许多生物学功能通路在盐和碱胁迫下出现不同的反应,如碳水化合物代谢、脂肪酸代谢、抗氧化系统和激素信号通路如茉莉酸信号途径:此外,积累萌发素类蛋白积极参与脂肪酸代谢和抗氧化过程,蛋白质折叠、加工和转运途径变化复杂,在角果碱蓬应对盐碱胁迫过程中发挥重要作用。同时,AsA-GSH循环和Prx/Trx通路在维持氧化还原稳态、清除过量的ROS和减轻氧化损伤等方面可能发挥了重要作用,从而帮助植物耐受盐碱胁迫。(4)转录组研究结果表明,角果碱蓬分别有997个和747个差异表达基因参与了盐碱胁迫应答。角果碱蓬中参与刺激响应、细胞生长、免疫过程和抗氧化剂活性等生物代谢过程的相关基因在盐碱应答过程中具有重要作用。大量的差异表达基因富集在内吞作用、植物激素信号转导、内质网蛋白质加工、光合固碳作用、酪氨酸代谢、维生素E和蛋氨酸代谢、精氨酸和脯氨酸代谢、苯丙烷类化合物、α-亚麻酸代谢、淀粉和蔗糖代谢、氨基酸生物分解和碳代谢等过程。对转录组数据中的转录因子进一步分析发现,48个转录因子在盐碱胁迫后差异表达,HSFs是本研究中差异表达数量最多的一类转录因子,多数HSFs在盐碱胁迫下被显著诱导。(5)在角果碱蓬转录组数据中筛选得到一个与盐碱应答密切相关的HSF转录因子,经过系统发育和蛋白结构分析最终命名为ScHSFA1d。通过qRT-PCR发现盐碱胁迫后ScHSFA1d在根和叶片中均上调表达。亚细胞定位结果表明ScHSFA1d定位于细胞核中,转录自激活实验结果说明ScHSFA1d的转录激活区域在氨基酸序列上的411aa到461aa处,包含一个AHA激活区。(6)通过在酵母和拟南芥中外源表达ScHSFA1d,发现其参与对耐盐碱性的调控。过表达ScHSFA1d拟南芥受胁迫损伤程度低于野生型拟南芥,ScHSFA1d能够提高拟南芥盐碱耐受能力。但拟南芥hsfa1d突变体与野生型拟南芥在生长表型和ROS积累等方面没有显著差异,并未使拟南芥表现出盐敏感表型,这说明拟南芥内源HSFA1d基因在调控拟南芥盐碱抗性过程中不具备重要作用。综合这些结果表明,甜土植物拟南芥和盐生植物角果碱蓬的HSFA1d同源基因在功能上存在差异,角果碱蓬的ScHSFA1d基因可能在植物盐碱抗性中起重要调控作用。更多还原

【Abstract】 Suaeda corniculata is a euhalophyte native to the saline-alkali soil of Northeast China which shows a higher alkali tolerance than other Suaeda plants.However,the molecular basis of the S.corniculata adaptation to salt and alkali stress is still not very clear.Transcription factors play an important role in plant stress resistance,through activating or inhibiting the expression of multiple target genes.Heat shock transcription factor(HSF)is widely involved in plant growth and development and stress response.At present,the function of HSF genes needs to be further studied such as regulating plant salt stress.And the function of HSF in halophytes has not been reported,especially in Suaeda.In this study,S.corniculata was used as the research material,and the metabolic molecular basis of S,corniculata in response to salt and alkali stress was systematically analyzed by using physiological and omics analysis.We screened a gene schsfald that may regulate the salt tolerance of S.corniculata.Through gene function verification,we preliminarily understand the function of ScHSFA1d in positively regulating plant salt tolerance.These studies laid a theoretical foundation for further exploring the mechanism of action of ScHSFA1d.The main results are as follows:(1)Physiological analysis of saline-alkali stress of S.corniculata showed that the effect of alkaline salt stress on plant growth was more serious than neutral salt stress.Affected by salt and alkali stress,the fresh weight and relative water content in roots were decreased significantly.The level of intracellular ROS increased under NaHCO3 and NaCl stress.Antioxidant enzyme activities and non-enzymatic antioxidant contents of ROS were changed to varying degrees.The membrane system of root cells was damaged to a certain extent.Osmoregulatory substances(proline and betaine)were accumulated in cells to cope with salt and alkali stress.(2)The results of 1H NMR metabolome analysis showed that the contents of leucine,isoleucine,valine,glutamine,malicacid and 2-oxoglutarate were significantly increased in the roots under NaCl stress.The metabolic characteristics after NaHCO3 treatment are not exactly the same.The contents of malonate,choline and betaine were increased under NaHCO3 stress,while the contents of malicacid,succinic acid and sucrose were decreased.In the metabolome results,we found that the content of betaine after two treatments was much higher than that of other metabolites.In addition,the contents of choline phosphate and choline,metabolites related to betaine anabolic pathway,were also significantly increased under NaHCO3 treatment.The accumulation of glycine betaine mainly contributes to the reconstruction of osmotic homeostasis during stress.(3)Proteomic studies showed that 65 and 32 proteins were differentially expressed under salt and alkali stress,respectively.Many biological functional pathways showed different responses under salt and alkali stress,such as carbohydrate metabolism,fatty acid metabolism,antioxidant system and hormone signal pathway,such as jasmonic acid signal pathway.In addition,the accumulation of germinating proteins actively participates in the process of fatty acid metabolism and antioxidation,and the changes of protein folding,processing and transport pathways are complex,which plays an important role in the response of S.corniculata to saline-alkali stress.At the same time,ASA-GSH cycle and PRX/TRX pathway may play an important role in maintaining redox homeostasis,removing excess ROS and reducing oxidative damage,to help plants tolerate salt and alkali stress.(4)Transcriptome analysis showed that 997 and 890 genes were involved in the response NaHCO3 and NaCl stress.The genes involved in the biological metabolic processes such as stimulation response,cell growth,immune process and antioxidant activity play an important role in the salt-alkali response.A large number of differentially expressed genes are enriched in endocytosis,plant hormone signal transduction,endoplasmic reticulum protein processing,carbon sequestration of photosynthetic organisms,tyrosine metabolism,vitamin E and methionine metabolism,arginine and proline metabolism,phenylpropane compounds αlinolenic acid metabolism,starch and sucrose metabolism,amino acid biodegradation and carbon metabolism.By analyzing the transcription factors in the transcriptome data,it was found that 48 transcription factors were differentially expressed under NaHCO3 and NaCl stress.HSFs were the most differentially expressed transcription factor in this study,and half of HSFs were significantly induced under NaHCO3 and NaCl stress.(5)A HSF transcription factor closely related to saline-alkali response was screened from the transcriptome data of S.corniculata.It was finally named schsfald after phylogenetic and protein structure analysis.Through qRT-PCR,it was found that schsfald was up-regulated in roots and leaves under NaHCO3 and NaCl stress.The results of subcellular localization showed that ScHSFA1d was located in the nucleus.The results of the transcriptional self-activation experiment showed that the transcriptional activation region of ScHSFA1d contained an AHA activation region from 411aa to 461aa on the amino acid sequence.(6)Overexpression of ScHSFA1d enhanced the tolerance of transgenic yeast and Arabidopsis to salt and alkali stress.The degree of stress damage of overexpressing ScHSFA1d Arabidopsis is lower than that of wild-type Arabidopsis.ScHSFA1d can improve the salinealkali tolerance of Arabidopsis.HSFA1d function deletion caused by Arabidopsis hsfa1d mutant,there is no significant difference in growth phenotype and ROS accumulation between hsfa1d mutant and wild-type Arabidopsis after stress,and the plant does not show salt-sensitive phenotype.These results show that endogenous HSFA1d gene in Arabidopsis does not play an important role in regulating salt and alkali resistance of Arabidopsis,The results show that there are functional differences between the two heat shock transcription factor HSFA1d homologous genes of sweet soil plant Arabidopsis and halophyte S.corniculata,which reflects the ScHSFA1d of S.corniculata.This genotype plays an important regulatory role in salt and alkali resistance.更多还原