【作者】 王雷；

【导师】 姜廷波；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2010， 博士

【摘要】 为研究小黑杨在不同盐浓度和不同胁迫时间的生理响应,本实验测定0、75、150、250 mM NaCl胁迫下,小黑杨第6小时和第1、2、4、6、8天的SOD活性、POD活性、MDA含量及叶绿素相对含量,结果表明：随着胁迫时间的延长和盐浓度的增加,小黑杨叶绿素相对含量逐渐降低,MDA含量升高。POD和SOD的活性则呈现逐渐增强后减弱的趋势。通过对小黑杨叶部形态观察发现,250 mM NaCl胁迫第二天,叶片失水,叶柄变软；随着胁迫的持续进行,叶片萎蔫变黄直至最后变成褐色,卷曲变脆。以上结果表明小黑杨并非强耐盐植物,对低浓度盐有一定耐受性,但高浓度盐则会对其造成伤害。为进一步研究盐胁迫下小黑杨基因应答机理,以正常生长和盐胁迫下的小黑杨叶片为试材,进行cDNA-AFLP分析。64对引物组合共获得4407条转录基因片段,其中差异片段2027条(其中上调表达996条,下调表达1031条)。回收161条差异条带,成功再扩增与克隆121条差异片段,测序得到107条原始序列,经聚类分析后得到86条唯一序列,Genbank登录号为GW672587-GW672672,其功能涉及转录调控、应激反应、光合作用、信号转导、代谢等10个功能类别。其中功能未知的基因占最高比例,约为18.6%；其次为参与代谢的基因,约占17.44%；参与转录调控的基因约为12.79%,信号转导的基因占11.63%；,与细胞合成相关的基因占10.47%,转运占4.65%,细胞降解占4.65%,参与应激反应的基因占5.81%、发育相关基因占6.98%、光合作用与氧化还原的基因均占6.98%。环锌指蛋白基因是小黑杨盐胁迫后诱导表达的基因,应用RACE技术克隆出具有完整开放读码框的小黑杨环锌指蛋白(PsnRZF)基因,该基因全长1061 bp,其中5’非翻译区为184 bp,3’非翻译区为82 bp,开放读码框为795 bp,编码264个氨基酸,预测蛋白的分子量为30.25 kDa,理论等电点为8.04。实时定量PCR检测的结果显示：小黑杨PsnRZF基因在不同器官中表达量存在差异,该基因在叶中的表达量最高,其次为茎,在根中的表达量最低。随着胁迫时间的延长,PsnRZF基因在叶和茎中的表达量变化均呈现逐渐升高后降低的趋势,在叶中该基因表达量峰值出现在胁迫后的第6天,在茎中该基因表达量峰值出现在胁迫后的第4天；而在根中,该基因表达量的变化不显著。为验证小黑杨环锌指蛋白(PsnRZF)基因的功能,构建植物表达载体,利用农杆菌介导法将小黑杨环锌指蛋白基因导入烟草中。通过对正常生长条件下和盐胁迫条件下,转基因烟草生理指标的检测发现,PsnRZF基因在不同的生长条件下,可能发挥不同的功能。在正常生长条件下,PsnRZF基因的过量表达对植物体来说是有害的；但在盐胁迫条件下,PsnRZF基因的过量表达可以缓解膜脂的过氧化作用。更多还原

【Abstract】 To study physiological response of Populus simonii×P.nigra to salt stress, SOD activity, POD activity, MDA content and relative content of chlorophyll were measured at 6 h,1 d,2 d, 4d,6d,8d under 0,75,150,250 mM NaCl stress. As the salt concentration increased and the stress time continued, POD and SOD activity increased gradually and then decreased. MDA content was increased and chlorophyll content was decreased gradually. By Morphological observation of Populus simonii×P.nigra leaf, it was found that water loss and petiole softening was serious under NaCl sress at the second day. Under continued stress condition, leaf was wilting and yellow, brown, crimping and brittle. This also showed that Populus simonii×P.nigra was not salt-tolerant plant. It had a certain tolerance to low concentrations of salt but high concentration of salt was harmful.To identify genes involved in salt stress responses, we carried out cDNA-AFLP analysis on leaves under normal growth and NaCl stress. Selective amplifications with 64 primer combinations allowed the visualization of about 4407 transcript-derived fragments (TDFs). 2027 of TDFs were differentially expressed.996 TDFs were up-regulated and 1031 TDFs were down-regulated.161 differentially expressed TDFs were excised from gel, eluted and re-amplified.121 TDFs were re-amplified and 107 TDFs sequenced successfully, yielding 86 unique sequence. These TDFs were submitted to the Genbank as accession number of GW672587-GW672672. They were further classified into 10 functional categories based on their putative functions, i.e., unknown protein(18.6%), metabolism(17.44%), regulation of transcription(12.79%), signal transduction(11.63%), cellular biosynthesis(10.47%), transport (4.65%), cellular catabolism(4.65%), response to stress(5.81%), photosynthesis and redox (6.98%), development process(6.98%).PsnRZF gene was induced in Populus simonii×P.nigra under NaCl stress. The 1061 bp full length cDNA of ring zinc-finger gene was isolated by rapid amplification of cDNA ends (RACE), including a 184 bp 5’untranslated region, an 82 bp 3’untranslated region and a 795 bp open reading frame encoding 264 amino acid residues. The molecular weight of deduced protein was 30.25 kDa with a theoretical pI of 8.04. Real-time PCR revealed that PsnRZF gene was expressed differentially in roots, stems and leaves. The expression level of PsnRZF gene in leaves and stems was increased gradually. The peak value was appeared at the 6th day in leaves and 4th day in stems under NaCl stress. The expression level of PsnRZF gene in roots was not changed significantly under NaCl stress.To verify PsnRZF gene function, the plant expression vector with PsnRZF gene was constructed, and transformed tobacco was performed by agrobacterium-mediated method. Physiological analysis showed that PsnRZF gene may play different roles under different growth conditions. Over expression of PsnRZF gene is harmful to plant under normal condition. But over expression of PsnRZF gene under salt stress can relieve the damage of membrane lipid oxidation.更多还原