【作者】 盖英萍；

【导师】 郑成超；

【作者基本信息】 山东农业大学 ， 生物化学与分子生物学， 2008， 博士

【摘要】 植物在长期的进化过程中具备了各种不同的生理生化机制,以抵抗、适应各种逆境。在众多非生物胁迫中,低温是限制许多重要作物产量和分布的重要环境因素。植物通过改变形态、生理和生化过程,使胁迫应答基因表达,合成特殊的蛋白质来响应低温胁迫。到目前为止,关于低温胁迫对植物生长发育以及各种代谢活动影响产生的机理还不清楚。本研究利用差异蛋白质组学策略首次对低温胁迫下棉花幼苗、烟草悬浮细胞蛋白质组变化以及外源转录因子CBF1A表达对烟草悬浮细胞蛋白质组的影响进行了研究,以期为揭示植物对低温胁迫响应的分子机制提供依据。1利用差异蛋白质组学策略,采用三氯乙酸-丙酮沉淀法( TCA/acetone precipitation)提取棉花幼苗总蛋白质,建立了棉花幼苗蛋白质双向电泳体系;双向电泳凝胶经银染和考马斯亮蓝R-350热染,扫描获取电泳图片,利用ImageMaster 2D Platium Software分析比较低温胁迫处理幼苗与未低温处理幼苗的蛋白质组差异;发现银染的凝胶中有54个差异表达蛋白点,其中29个蛋白点在低温处理后表达量升高,25个蛋白点表达量降低;利用质谱技术成功鉴定出其中的8个差异表达蛋白点,其中的7个蛋白点被鉴定为同一种蛋白β-球蛋白,另一种蛋白鉴定为Rubisco大亚基;通过对棉花子叶生理生化指标的测定发现,低温胁迫会引起棉花幼苗子叶肽链内切酶活性的增强;子叶净光合速率和羧化效率降低;棉花子叶内活性氧产生速率升高,脂质过氧化加速。基于上述结果,我们认为低温胁迫会加速β-球蛋白和Rubisco大亚基的降解,并对蛋白的降解模式进行了分析。本文为从分子水平上揭示棉花幼苗低温响应机制研究奠定了基础,为深入了解低温胁迫下蛋白的降解机制提供了依据。2利用差异蛋白质组学策略,采用三氯乙酸-丙酮沉淀法提取烟草悬浮细胞总蛋白质,建立了烟草悬浮细胞蛋白质双向电泳体系;分析比较了烟草悬浮细胞在低温胁迫处理后蛋白质组的变化,通过对差异表达蛋白点的鉴定,发现LEA–类似蛋白NtLEA7-3的丰度在低温处理后上调,为低温诱导表达升高蛋白。通过荧光定量PCR分析发现低温胁迫下该蛋白mRNA水平变化与蛋白表达水平变化情况基本一致。利用RACE技术得到NtLEA7-3基因全长cDNA (GenBank登录号:EF532409)。NtLEA7-3 cDNA全长为1 267 bp,该序列含有一个969 bp的完整开放读码框,编码322个氨基酸,蛋白质理论分子量约为35.7 kDa,等电点为4.86。对NtLEA7-3编码的蛋白质进行结构预测分析表明,该蛋白富含无规卷曲(Coil),高达65.31%,其次是α-螺旋(Helix),为19.69 %,而β-折叠(Strand)只有15.00%。将NtLEA7-3编码区插入原核表达载体pET30a (+),并转化到大肠杆菌菌株BL21中,经IPTG诱导,NtLEA7-3融合蛋白在BL21菌株中成功表达。将得到的NtLEA7-3编码区插入植物表达载体pBI121中,构建了NtLEA7-3植物表达载体pBI121-LEA;成功地将其转入到烟草和悬浮细胞中,获得了转基因烟草植株及悬浮细胞,通过对转基因植株及悬浮细胞的表型和低温胁迫下生理生化特征分析表明,NtLEA7-3成功地表达可以提高转基因植株和悬浮细胞的耐低温胁迫能力。将NtLEA7-3编码区插入含有GFP基因的植物表达载体pBI121中,构建了NtLEA7-3-GFP的植物表达载体pBI121-LEA-GFP,成功地将NtLEA7-3-GFP融合基因转入拟南芥和烟草悬浮细胞,发现NtLEA7-3定位于细胞核中。本文不仅为研究植物的抗逆机理提供了参考,为植物抗性育种提供了有效的候选基因,同时为深入研究NtLEA7-3的结构、功能和分子调控机制奠定了基础。3成功地将棉花低温胁迫相关转录因子CBF1A基因转入了烟草悬浮细胞中,利用差异蛋白质组学策略分析了转CBF1A基因烟草悬浮细胞和野生型烟草悬浮细胞的蛋白质组差异,发现外源CBF1A基因的表达会引起烟草悬浮细胞蛋白质组的变化,有7个蛋白点表达差异显著,其中4个蛋白点表达升高,3个蛋白点表达降低。利用质谱技术成功鉴定出其中6个蛋白点,代表了5种不同的蛋白,其中3个是功能已知蛋白,即推测的叶绿体半胱氨酸合成酶前体、苹果酸脱氢酶类似蛋白、短链脱氢酶,另2个蛋白为功能未知蛋白。本研究为揭示CBF1A对植物的调控机制提供了参考。更多还原

【Abstract】 During the long-term evolution, plants have developed sophisticated biochemical and physiological mechanisms to adapt and resistance to various environmental stresses. Among different stress, cold is a major environmental limitation to crop productivity and to the distribution of wild species. Plants acquire resistance to stress environment by reprogramming metabolism and gene expression, gaining a new equilibrium between growth, development and survival. But the mechanism of low temperature stress on growth and the accompanying metabolic changes remain to be elucidated in detail. In this study, we carried out comparative proteomic analyses to investigate the proteomic response of cotton (Gossypium hirsutum L.) and Nicotiana tabacum cell suspension culture under chilling stress and the proteomic changes of total proteins in Nicotiana tabacum cell suspension culture when the cotton CBF1A were expressed in them. The aim was to provide a better understanding about the underlying molecular mechanisms for plant response to low temperature.To gain a better understanding of chilling stress responses in cotton (Gossypium hirsutum L.), we carried out a comparative proteomic analysis. Cotyledon proteins of 1-week-old cotton seedlings treated with or without chilling treatment at 4℃were extracted, separated by a two-dimensional gel electrophoresis and compared. Among 1500 protein spots reproducibly detected on each gel, 25 protein spots were down-regulated and 29 were up-regulated. Seven cotton proteins were identified by mass spectrometry analysis as beta-globulin A precursor fragments. One was identified as a Rubisco large subunit fragment, providing evidence of both seed storage protein and photosynthetic protein destruction by chilling stress. The related physiological and biochemical analysis showed that there was a significant increase in endopeptidase activity and activated oxygen generation rate, and an obvious decrease in carboxylation efficiency and maximum net photosynthetic rate of cotton seedlings under chilling stress. Based on the above results, the degradation mechanisms of beta-globulin and Rubisco under chilling stress were discussed. In conclusion, our study provides new insights into chilling-stress responses in cotton and better understanding of the degradation mechanisms of seed storage proteins and photosynthetic proteins under chilling stress.In this study, differential proteomic analysis was conducted to characterize the proteins in Nicotiana tabacum cell suspension culture that were differently expressed in responsive to chilling stress with 2-DE and MS/MS. The gels were analyzed by ImageMasterTM 2D Platinum software. About five hundred reproducible protein spots were detected, among which one protein spot was up-regulated in the chilling stressed samples. MALDI-TOF-TOF MS analysis followed by database searching helped to identify the spot as late embryogenesis abundant (LEA)–like protein. To investigate the change of gene expression at the mRNA level, we performed qPCR analysis. The results showed that the mRNA level is correlated well with protein level. A full-length cDNA encoding the protein (designated as NtLEA7-3, GenBank accession No. EF532409) was cloned from Nicotiana tabacum cell suspension culture by rapid amplification of cDNA ends. The cDNA was 1 267 bp containing a 969 bp open reading frame which was deduced to encode a peptide of 322 amino acids whose predicted molecular mass was 35.7 kDa and isoelectric point was 4.86. It was predicted that the structure of NtLEA7-3 was rich in coils, and was poor in helix and strands. The coding region of the NtLEA7-3 was inserted into an expression vector, pET30a (+), and transformed into Escherichia coli BL2l. The fusion protein was successfully expressed with IPTG induction. The plant expression vector pBI121-LEA with this fragment under the control of 35S promoter was constructed and transformed into Nicotiana tabacum plants and cell suspension culture. Northern blot and Western blot analysis indicated that the NtLEA7-3 was expressed successfully in them. The related physiological and biochemical analysis showed that the NtLEA7-3 was a very efficient gene for improving chilling tolerance of Nicotiana tabacum. The coding region of the NtLEA7-3 was inserted into an expression vector pBI121 containin GFP and transformed into Arabidopsis thaliana plants and Nicotiana tabacum cell suspension culture. Then the transgenic Arabidopsis thaliana plant roots and cell suspension culture were observed through fluorescence microscopy and the results showed the NtLEA7-3 were expressed in the nucleus. This research not only provides a better understanding for the molecular mechanisms of plant response to low temperature, but also a useful candidate gene for resistance breeding. Furthermore, this study may be helpful to study the structure, function and regulation of the NtLEA7-3.The cotton CBF1A gene under the control of 35S promoter was transformed into Nicotiana tabacum cell suspension culture successfully. A comparative proteomic analysis was carried out to gain a better understanding of the proteins differently expressed in responsive to CBF1A expression in Nicotiana tabacum cell suspension culture. Among the protein spots detected, 7 protein spots displayed differential expression. There were 4 up-regulated and 3 down-regulated protein spots in the transgenic cell suspension culture. MS analysis followed by database searching helped to identify 6 spots representing 5 different proteins. The identified proteins include putative chloroplast cysteine synthase 1 precursor, malate dehydrogenase like-protein, short chain dehydrogenase and two unknown protein. This research provides a better understanding for the molecular regulation mechanisms of CBF1A.更多还原