【作者】 周磊；

【导师】 郝东云；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 土壤盐碱化的危害日益严重,因此,研究植物耐盐胁迫机理具有重要意义。本文选择了中度耐盐碱的短芒大麦为实验材料,比较分析了野生型和耐盐型短芒大麦耐盐机制的差异。首先使用不同浓度梯度的NaHCO3分别处理野生型短芒大麦,最终确定了最适的处理浓度:100mM NaHCO3.在后续的实验中,运用AFLP技术分析出野生型和耐盐型短芒大麦基因序列的差异,得到4条与非生物胁迫相关的基因片段;说明通过AFLP技术是可以筛选到野生型和耐盐型短芒大麦中与盐碱相关的差异基因片段的;同时利用cDNA-AFLP以及Blast软件等技术分析,找到了在野生型和耐盐型短芒大麦有差异的7个转录片段,下一步,可将此片段连接到表达载体,通过转基因等手段在植物中过表达来验证植物的耐盐性是否有所提高。通过本研究,我们分别在基因组水平和基因转录水平找到了再野生型和耐盐型短芒大麦中有差异的盐碱相关片段,为盐碱调控网络的完善奠定了基础,同时为盐碱胁迫条件下植物分子育种提供了可以利用的基因资源。实验过程中采用了生理实验确定植物合适的处理浓度,反转录半定量PCR、AFLP、巢式PCR、cDNA-AFLP等实验技术和手段解析出野生型和耐盐型短芒大麦在盐碱胁迫条件下响应机制的异同,同时筛选得到了在盐碱胁迫条件下有明显应答反应的基因序列,为今后植物耐盐碱机理研究和耐盐碱植物育种工作提供了理论依据。更多还原

【Abstract】 The soil salinization is the most urgent problem for agriculture. It can influence most plants’normal growth. But we also find some plants can grow and breed for community under this extreme environment. Hordeum brevisubulatium (Trin.) link is a kind of perennial forage grass belonging to gramineous type barley genus, which salt-tolerance capacity is strong. The mechanism of adapting salinity environment made shortsubulate barley as an ideal plant for researching of anti-adversity.In order to know the salt-tolerance mechanism and utilize key salt-tolerance gene, this paper chose wild type and salt-tolerance type barley as material, analyzed the physiological response to NaHCO3 stress, determined the suitable concentration to deal with the plants. At the same time, we analyzed the difference of gene expression by RT-PCR and cDNA-AFLP technonlgies, then acquired the relative important genes by nested PCR. The results were as follows:Through physiological experiment to determine the suitable treeting concentration for the plants: 100mM.Using RT-PCR to compare DREB1 and CIPK gene that expressed differently between wild type and salt-tolerance type barley at different time. We chose a-tubulin as the internal parameters. The results showed that in the plants which treeting with alkaline salt, its DREB1 expressed highest after 6hrs, and while treeting with neutral salt, DREB1 expressed highest at 12hrs later. Anyway CIPK expressed highest after 24hrs. Alkaline condition has little influence on CIPK, but under NaHCO3 stress, the response of wild type and salt-tolerance type barley are consistent.Under alkaline salt stress, Wild type and salt-tolerance type Barley have obvious difference in phenotype. So this study analyzed sequence difference by the AFLP technology. The result shows the difference is 1% when stating 1400 sequences.Using nested-PCR obtained the gene sequence of CIPK、NHX3、DREB1 The experiment respectively cloned the cDNA sequence of CIPK、NHX3、DREB1 from the normal type and the anti type of barley. At the same time, BLAST aligned the sequence. Analyzed the difference between the two type found the proteins has no difference. So we can not deduced, transcription has correlation with anti-salt.Analyse the expression difference by cDNA-AFLP. The normal type and the anti type of barley displayed different expression profile. By BLAST aligning, we chose one TDF which is a relative gene about salt stress. Then we designed primers for the sequence amplification. The sequence of the gene is the same in both wild type and tolerance type. But the expression level has difference in the two tyoe barley. Therefore the next step we may prove the tolerance of the gene through transgenic technology.更多还原