【作者】 高彩球；

【导师】 刘桂丰； 王玉成；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2007， 博士

【摘要】 刚毛柽柳（Tamarix hispid）为灌木或小乔木，具有强的抗旱、耐盐、耐水湿、抗沙埋能力，是优良的防风固沙、水土保持和盐碱地造林树种。而且能在含盐量1％的盐碱地上形成自然林，为盐碱土指示植物，是进行植物抗盐碱研究的理想材料。本文研究了刚毛柽柳在盐碱胁迫后不同时间点基因的表达，并克隆出抗逆相关基因。刚毛柽柳在0.4 mol／LNaHCO₃胁迫下，分别处理0 h（对照）、24 h和52 h，以叶片为材料，分别构建了3个全长cDNA文库。文库的滴度分别为7.5×10⁵ pfu、2.6×10⁶pfu、4.0×10⁶ pfu，插入片段平均长度分别为1.1、1.2和1.2 kb，平均重组率为97.5％。共获得9447条高质量的ESTs序列，其登录号为EH048198-EH057644。它们代表了3945条单一基因序列（Unigene），包括986个contigs和2959个singlets。3个文库具有的Unigene分别是1752、1558和1675。将9447条EST序列与GenBank的Nr数据库进行Blastx和Blastn分析，其中7365条ESTs（占77.96％）与已知基因序列相似性高(E-value＜10^-4)，其余2082（22.04％）条相似性低或没有相似性。与已知基因序列相似性高的7365条序列按功能分为12类，其中光合（photosynthesis）占20.31％，功能未知（functionunknown）占13.97％、细胞防御（Cell rescue，defense）占13.96％、能量（Energy）占7.59％、蛋白质合成（Protein synthesis）占7.58％、转运（Transport facilitation）占7.28％、转录（Transcription）占6.63％、细胞结构（Cell structure）占6.07％、代谢（Metabolism）占5.70％、蛋白质定位（Protein destination）占5.02％、信号转导（Signaltransduction）占3.86％、细胞生长／分裂（Cell growth，division）占2.04％。通过对3个cDNA文库分别进行EST分析和比较建立了NaHCO₃胁迫不同时间点的柽柳基因表达谱，鉴定了大量的盐碱胁迫响应基因。这些基因按功能可以分为：渗透调节、激素信号转导、活性氧清除、转录调控、蛋白合成和定位、离子平衡、核糖体蛋白、光合作用和代谢等。提示这些途径都可能参与了刚毛柽柳的耐盐碱胁迫。在文库中选择了9个表达明显差异的基因，利用实时定量（real time）RT-PCR技术，研究比较了这些基因在NaCl和NaHCO₃胁迫下基因表达模式，发现在NaCl或NaHCO₃胁迫24 h后这些基因表达趋势比较相似。但在胁迫52 h后，这些基因对NaCl和NaHCO₃胁迫响应表达趋势出现明显差异。文库中含有较多的抗逆相关全长基因，从中选择了液泡膜H⁺-ATPase c、c″亚基和2个GRP（glycine-rich RNA-binding protein）基因，对它们在NaHCO₃和NaCl胁迫下基因的表达进一步的分析。NaCl胁迫后ThVHA cl和ThVHA c″1表达量明显增加，ThGRP1和ThGRP2基因表达量下降；NaHCO₃胁迫后ThVHA cl和ThVHA c″1的表达量在24 h下降，52 h上升，而ThGRP1和ThGRP2为24 h上升，52 h下降。本研究发现刚毛柽柳对盐碱胁迫的响应涉及多个生理和代谢途径，并鉴定了一系列胁迫应答基因。这些结果可以作为木本植物耐盐的分子遗传研究的模式系统，使我们在分子水平上对木本植物耐盐机理有了进一步的认识。同时，丰富了林木基因工程育种理论，为抗逆基因工程育种提供一些优良的抗性基因。更多还原

【Abstract】 Tamarix hispida, a woody halophyte, thrives in saline or saline-alkali soil, having a high ability in resisting saline and saline-alkali stress. The expression profiling in response to salinity-alkali stress in T.hispida was studied by large-scale expressed sequence tags （EST） analysis, and some full-length cDNA sequence of stress resistance gens were cloned from T.hispida and further analyzed.Three cDNA libraries were constructed from leaf tissue of T. hispida plants that were well-watered （control） or exposed to 0.4 mol/L NaHCO3 for 24 and 52 h （the three libraries were signed as 0,24 and 52 h library, respectively）. The primary titer of the three libraries were 7.5x105 pfu, 2.6×106 pfu and 4.0×106 pfu,, with the average size of inserts of 1.1, 1.2 and 1.2 kb, respecitvly. The average rate of recombination in three libraries were all about 98%.A total of 9447 high quality expressed sequence tags （ESTs） were obtained from the three libraries. They were submitted to the GenBank databases with accession numbers from EH048198 to EH057644. The 9447 ESTs were assembled into 986 contigs and 2959 singlets. The numbers of Unigenes from the 0, 24 and 52 h library were 1752, 1558 and 1675, respectively.Among them, 7365 ESTs （77.96%） showed significant similarities (E-value＜10^-4) to gene sequences in the non-redundant （Nr） GenBank database, and the other 2082 （22.04%） had little, or no, similarity. The database-matched ESTs were further grouped into 12 funtion categories, i.e., photosynthesis （20.31%）, function unknown （13.97%）, cell rescue, defense （13.96%）, energy （7.59%）, protein synthesis （7.58%）, transport facilitation （7.28%）, transcription （6.63%）, cell structure （6.07%）, metabolism-related category （5.70%）, protein destination （5.02%）, signal transduction （3.86%）, and cell growth and division （2.04%）.EST analysis was performed to compare gene expression in the three libraries, and the transcripts responsive to NaHCO3 were identified. We found the transcripts encoding osmolyte biosynthesis, hormone signaling transduction, scavenging reactive oxygen species, transcriptional regulators, protein synthesis and destination, ion homeostasis, ribosomal proteins, photosynthesis and metabolism were significantly regulated under NaHCO3 stress.To investigate the differences in gene regulation between T. hispida after exposure to NaC1 or NaHCO3, nine transcripts, differentially regulated by NaHCO3 stress, were selected for further expression analysis using real time reverse transcription-polymerase chain reaction （RT-PCR）. Gene expression trends were similar after a 24-h exposure to either NaCl or NaHCO3, however, substantial variability was found after a 52-h exposure, indicating that short-term responses to either salt may not be significantly different. Bioinformation methods were conducted in cloning the full-length cDNA sequence genes from three libraries, and the full-length genes were obtained. Among these genes, the vacuolar-H⁺ ATPase subunit c,, vacuolar-H⁺-ATPase subunit c"and two unique glycine-rich RNA-binding protein genes were selected for further expression analysis using real-time RT-PCR. The expressions of ThVHA el and Th VHA c"l were significantly increased while the expressions of ThGRPl and ThGRP2 were continually decreased after exposure to NaC1. However, the Th VHA el and Th VHA c"l were both down-regulated after NaHCO3 treatment for 24 h and up- regulated for 52 h. The ThGRP1 and ThGRP2 genes were both up-regulated at 24 h and down- regulated at 52 h of NaHCO3 treatment.These sstudies showed that the response to saline-alkali stress in T. hispida is a complex one, involving multiple physiological and metabolic pathways. The results will enrich our knowledge of the stress tolerance of woody plants on a molecular level and provide new insight into saline-alkali tolerance in plants. In addition, the cloning and identification of saline-alkali tolerance genes and determination of their expression patterns under saline-alkali stress, may offer some attractive candidate genes and valuable information for improving saline-alkali tolerance of other woody plant species through genetic engineering.更多还原