【作者】 袁红梅；

【导师】 姜静； 王玉成； 王柏臣；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 盐胁迫造成的盐害首先表现为低渗透势作用和离子毒害等。由于吸收了土壤环境中的大量盐分,植物水分亏缺,生理代谢发生紊乱,在这期间植物细胞积累一系列的蛋白质来保护细胞免受脱水伤害,其中LEA蛋白是最普遍的一种。虽然人们推测LEA蛋白可能在脱水胁迫中起重要作用,但对它们的生理生化功能及抗逆机理还不是十分清楚。本实验室通过农杆菌介导法将TaLEA基因(DQ663481)转化到小黑杨花粉植株中,获得了11个转TaLEA小黑杨独立转化株系,研究过程中发现了一株显著不同于其他转TaLEA小黑杨的矮化突变株(将此株系暂命名为dwf1),经过耐盐性分析发现，dwf1的耐盐能力与对照相比明显增强,同时dwf1还表现出叶型改变、叶片较早老化等突变性状。为深入研究dwf1的抗盐机理及突变表型的分子机理,本研究分别在DNA、mRNA以及蛋白三个水平对dwf1进行了研究。利用TAIL-PCR的方法,在dwf1中克隆获得了TaLEA基因的2个侧翼序列。通过生物信息学分析发现这2个序列分别位于一个反转录转座子区和一个未知基因(与葡萄的假定蛋白XM 002267539.1同源)的启动子区。利用RT-PCR的方法扩增该反转录转座子中的反转录酶,结果在dwfl和野生型对照WT中均没有扩增出目的条带,推测该转座子可能处于无活性状态。利用荧光定量PCR的方法检测该未知基因的表达情况,结果发现该基因在dwfl中上调表达,表达量是WT中表达量的2.22倍,推测TaLEA的插入可能影响了该基因的表达。目前,该基因功能未知。dwfl突变性状是否是由于该基因的上调表达引起的,还有待进一步的功能验证。在正常生长条件下，对dwf1小黑杨突变株与野生型对照进行了Affymetrix微阵列芯片的差异转录表达谱分析。利用SAM软件对芯片数据进行了分析,以q-value (%)<5, Fold Change> 2或<0.5为标准筛选出差异基因537个,其中280个基因上调表达,257个基因下调表达。通过实时荧光定量PCR技术验证,结果与芯片结果基本一致,证明芯片结果是可靠的。这些差异基因中除43个功能未知或未分类的基因外,其他基因分为11类,包括代谢、胁迫、蛋白合成及降解、激酶、细胞壁、激素、信号转导、转录调控、运输、细胞结构基因及细胞命运等。通过分析发现,与野生型对照相比,dwfl中很多胁迫诱导表达基因如Osmotin、RD22、chitinase、beta-1,3 glucanase、peroxidase、chalcone synthase、glutathione S-transferase、WRKY、bZIP、AP2/EREBP、NAC、serine/threonine kinase、stress-induced receptor-like kinase、lipid transfer protein等都上调表达。在正常生长条件,这些胁迫诱导表达基因的高量表达为dwfl的耐盐性提供了分子依据。同时,在dwfl中很多与激素合成及调控相关的基因,如Cullin-1、DWF5、BAK1、AIL5、JAZ10等的表达水平也发生了显著变化,推测这些基因的变化很可能影响了激素的水平,进而影响了植物的生长发育,从而导致了dwf1植株矮化等的表型。利用无标记定量分析(Label-free LC MS/MS)的方法对dwf1小黑杨突变株与野生型对照进行了差异蛋白表达谱分析。结果显示,以score值>200,Flod Change>1.3或<0.75为标准,去除冗余后共筛选出99个差异表达的蛋白，dwf1与野生型对照相比,其中上调表达蛋白32个,下调表达蛋白17个，dwf1中特异表达蛋白33个,野生型中特异表达蛋白17个。在99个差异蛋白中,除15个蛋白未能分类外,其它蛋白共分为8大类：代谢、胁迫、蛋白折叠、加工、降解、转录调控、细胞命运、运输、细胞壁及细胞骨架。将无标记定量分析的结果与Affymetrix微阵列芯片结果进行比较发现,在糖酵解、肌醇磷酸代谢、氮代谢等代谢通路中的差异基因,mRNA和蛋白质丰度差异基本吻合。而信号转导、转录因子、激素及核糖体蛋白、转录起始因子等相关基因的mRNA与蛋白质丰度差异显著。差异转录表达谱与差异蛋白表达谱的不一致性为今后对抗逆基因转录后调控机制的研究提供了重要线索。更多还原

【Abstract】 Primary symptoms of salt damage caused by salt stress comprise low osmotic potential effect and ion toxicity. Metabolic disorder of plant will occur due to lack of water when the plant absorbs too much salt from the soil. During that process, a series of proteins stored in plant cells will protect the cells from dehydration, among which the LEA protein is the most common one. Though it is believed that the LEA protein plays an important role in reducing dehydration stress, its physiological and biochemical functions and stress resistance mechanism are not clear yet. The TaLEA （DQ663481） gene was introduced into Populus simonii×P. nigra by Agrobacterium tumefaciens-mediated transformation. It was totally obtained 11 independent transgenic lines. Among them, a dwarf mutant （named dwfl） was found. Salt resistance analysis showed that the dwfl plant had stronger salt tolerance than the control group, and mutation symptoms like dwarf, change of leave shape, faster ageing of leave. To further research molecular mechanism causing mutant phenotypes of the dwfl plant and the salt resistance mechanism of the TalEA protein, we had studied the dwfl from the respective of DNA, mRNA and protein.Two flanking sequences of TaLEA gene were cloned from the dwfl by TAIL-PCR. Bioinformatics analysis showed that the two sequences were located in a retroposon and the promoter regions of an unknown gene （homologous with hypothetical protein of grape, XM₀02267539.1） respectively. After amplifying the reverse transcriptase of the retroposon by RT-PCR, the target band was not found in dwfl or wild type, so we supposed that the transposon was inactive. Fluorescence quantitative PCR was used for testing expression of the unknown gene and found that expression of the gene was up-regulated in the dwfl up to 2.22 times the expression of the control group. We presumed that the insertion may affect the expression of the gene. The function of the gene is still unknown. Whether the mutant characters of dwfl are related with the up-regulated expression of the gene shall be determined by further functional verification.Differential transcription expression profiling of the dwfl and wild type control group was carried out by Afrymetrix microarray in non-stress condition. Differential genes were screened by SAM software and 537 differential genes were selected on the basis of q-value （%）<5 and Fold Change>2 or<0.5.280 genes have up-regulated expression and others have down-regulated expression. Real-time fluorescence quantitative PCR verification showed the same results with the microarray chip analysis, thus the microarray chip analysis was reliable. Except 43 genes which have unknown functions or cannot be classified, the differential genes can be classified into 11 groups, including metabolism, stress, protein synthesis and degradation, kinase, cell wall, hormone, signal transduction, transcriptional regulation, transportation, cellular structure and cell fate. Analysis showed that most stress-induced genes had up-regulated expression, such as Osmotin, RD22, chitinase, beta-1,3 glucanase, peroxidase, chalcone synthase, glutathione S-transferase, WRKY, bZIP, AP2/EREBP, NAC, serine/threonine kinase, stress-induced receptor-like kinase, lipid transfer protein and so on. In normal growth condition, high level expression of these stress-induce genes provides the molecular basis for the salt tolerance of dwfl. Moreover, many genes, such as Cullin-1, DWF5, BAK1, AIL5, JAZ10 that were related to hormone synthesis and regulation in the dwfl also expressed significantly differently. These genes are very likely to influence the level of hormone. Hormone change may impact on growth of the plant, causing the mutant phenotypes of dwfl like dwarf.Differential protein expression profiling of the dwfl and wild type control group was carried out by Label-free LC MS/MS in non-stress condition. Differential proteins were screened and 99 differential non-redundant proteins were selected on the basis of Score value>200, q-value （%） <5 and Fold Change>1.3 or<0.75.32 proteins have up-regulated expression and 17 have down-regulated expression.33 proteins Specifically expressed in dwfl and 17 proteins Specifically expressed in WT. Except 43 proteins which have unknown functions or cannot be classified, the differential proteins can be classified into 8 groups, including metabolism, stress, protein synthesis and degradation, transcriptional regulation, cell fate, transportation, cell wall, and cytoskeleton. The results of Label-free LC MS/MS are compared with that of Affymetrix microarray. Analysis finds that mRNA and protein expression abundant of the genes in the glycosis, inositol phosphate metabolism, nitrogen metabolism pathway is similar. While mRNA and protein expression abundant of the genes that are related to signal transduction, transcription factor, hormone, ribosomal protein and translation initiation factor is significantly different. The inconsistency of the transcription and protein expression profiles may provide important clues to the research of post-transcriptional regulation of stress resistance genes.更多还原