【作者】 萧凤回；

【导师】 翟虎渠；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 博士

【摘要】 干旱和缺水是农业生产面临全球性难题,选育和利用抗旱耐旱品种是节水农业和减缓旱灾最经济的途径。传统育种方法选育抗旱品种受到诸多因素的限制,干旱胁迫分子机制研究和基因工程科技术为品种抗旱性改良提供了新途径。研究干旱胁迫可诱导基因启动子对提高转基因分子育种的效率至关重要,尤其对启动子的评价、选筛、改造和利用子是不可缺少的环节。本研究应用分子克隆和基因瞬间表达等技术,开展了大麦和水稻干旱可诱导LEA基因启动子的分离、克隆和功能分析研究,得到如下结果：1.采用PCR技术,从大麦品种"Sahara"和水稻品种"Jarrah"中克隆到5个LEA基因启动子HVA1s、Dhn4s、Dhn8s、rab16Bj、wsi18j。序列分析表明,5个启动子与原报道的从大麦品种"Himalaya"和"Dicktoo"克隆的HVA1、Dhn4和Dhn8和从水稻品种"Toride"和‘"Josaeng Tongil"克隆的rab16B和wsi18启动子比较,有95.3%～99.8%序列同一性,不同来源的启动子均在序列差异。5个启动子均含数量和类型不同胁迫应答顺式作用。2.为了检测不同启动子及其突变体的瞬时表达水平,构建了15个表达载体。其中GFP表达载体5个,GUS表达载体5个,HVAls启动子ABRE序列碱基替换突变载体3个和DRE/CRT碱基替换突变载体1个,wsi18js启动子ABRE序列碱基替换突变载体1个。3.以大麦幼苗、愈伤组织、部分器官为基因枪转化受体进行基因瞬间表达最佳材料筛选,开展了启动子表达质卡粒基因DNA浓度、表达诱导因子及处理水平等研究,建立了快速检测启动r诱导表达水平和功能分析的定性检测和定量检测方法体系。4.采用干燥、ABA、NaCl、低温等诱导处理,检测对不同启动子瞬间表达的影响。结果显示：①Dhn8s启动子在干燥和ABA诱导及对照处理中,均呈现表达的非特异性,并且表达强度最高,表现为组成型的非特异性启动子。而Dhn4s、HVAls、rab16Bj和wsi18j启动子为诱导型启动子,但诱导表达的强度存在差异,ABA诱导表达强度为Dhn4s> HVA1s> wsi18j> rab16Bj,干燥诱导表达强度为Dhn4s>HVA1s> wsi18j> rab16Bj。②Dhn4s、HVA1s、rab16Bj和wsi18j启动子对低温和NaCl处理诱导的反应均较弱,但存在一定差异。其中HVAls最强,wsi18j较弱,Dhn4s反应弱,rab16Bj没有反应。5. HVAls启动子DRE/CRT序列碱基件换突变研究表明,DRE/CRT序列碱基替换后对ABA和干燥处理诱导的表达水平略有下降(分别降低7.7%和10.9%),而对低温处理诱导的表达水平下降明显,只相当于野生型的37.9%。说明DRE/CRT主要负责对低温胁迫的应答,但也被ABA和干旱所诱导。6. HVAls启动子中以ACGT为核心序列的ABA应答元件ABRE1、ABRE2和ABRE3序列逐步碱基替换突变研究表明,ABRE3突变后HVAls对ABA和干燥诱导表达水平下降40%-50%；ABRE2和ABRE3同时突变后表达水平下降89%～92%；ABRE1、ABRE2和ABRE3同时突变后表达水平基本同ABRE2和ABRE3同时突变的水平。证实HVAls中的3个ABRE元件中,只有ABRE2和ABRE3具有高效的ABA和干旱胁迫诱导功能,ABRE1不是真正的ABRE顺式调控元件。7. wsi18j启动子ABRE元件碱基替换突变研究显示,在对照中,野生型和突变型的质粒均不表达。经ABA处理后,两者都表达,但突变型启动子表达强度显著减弱,不到野生型的1/4。证实了Wsi18j启动子CE3-ABRE复合体对ABA的应答表达起主要作用。wsi18j与wsi18启动子应答元件分析农明,2个启动子含有的干旱、低温和ABA应答顺式元件的类型和数量完全相同,但对ABA诱导应答有本质的不同。由于碱基的缺失导致wsi18j与wsi18二个启动子的元件间距离改变。碱基差数占相应元件间最大趴离的百分比有明显的差异。CE3与ABRE间相对碱基差最大,为16.67%。Wsi18启动子对ABA反应不敏感,与其ABRE核心基序两侧缺失各1个C碱基并改变CE3-ABRE间的距离有关。8.本研究的创新和价值有：1)拓宽了瞬间表达快速检测干旱可诱导启动子特性方法的应用空间,建立的检测方法一是可应于转基因分子育种的高效启动子筛选；二是可应用于环境胁迫基因调控体系和路径中转录因子的关系和交互作用研究是；三是可应用于启动子顺式作用元件的功能和互作研究；四足可应用于生物信息学预测启动子功能推测的检验。2)在相同背景下客观地比较了LEA基因启动子特性和差异,并对其利用价值进行评价,为利用这些启动子提供了新的试验依据。3)发现LEA基因启动子存在属于非诱导型启动子Dhn8s。更多还原

【Abstract】 Drought and water deficit are a globle problem for agricultural production. To breed and utilize crop varieties resistant or tolerant to drought is the most economical approach for water-saving farming and buffering drought disaster. It is very important and necessary to investigate the gene promoters drought-inducible for enhancing efficiency of molecular breeding through transgenic technology, especially in evaluation, screening, modification and application of promoter. In this thesis, the isolation, cloning and functional analyses of promoters belonging to LEA (late embryogensis abundant protein) gene have been carried out from barley (Horcleum vulgare L.) and rice (Orizy sativa L.) by using molecular cloning and transient expression technique. The results are as following:1. Five LEA gene promoters have been cloned from barley variety Sahara and rice variety Jarrah using PCR techonolgy, including HVAls、Dhn4s、Dhn8s、rabl6Bj、wsil8j. Sequencing analysis showed that there are95.3%～99.8%consensus identities between these five and those of HVA1、Dhn4and Dhn8gene from barley varieties "Himalaya" and "Dicktoo" as well as from rice varieties "Toride"and "Josaeng Tongil" reported previously. Sequence differences were found between those promoters from different varieties. These five promoters are various in their number and type of cis-acting elements stress-responsive.2. In order to determine the expressional levels of the cloned promoters and their mutants,15expression vector constructs have been constructed,5for GFP reporter,5for GUS reporter;3for ABRE sequence base-substitution mutation and reporter as well as1for DRE/CRT sequence base-substitution mutation and reporter of HVA1promoter;1for ABRE sequence base-substitution mutation and reporter of wsi18js promoter.3. Barley seedlings, cultured barley calli and some organs were used for gene-gun transformation receptor so as to select the best candidate material for transient experiment. Studies on DNA concentration of the promoter expression vector for transient experiment, factors and treatments for inducing expression have also been conducted. A methodology has been established for rapid test and determination of expression level of the promoters stress-inducible, based on qualitative and quantitative analyses.4. Dry, ABA, NaCl, cold as treatments for inducing the different promoter’s transient expression and their effects have been investigated, the results indicated:1) Dhn8s showed non-specific and highest expression under dry, ABA and control treatments, which means it is a constitution promoter without specific requirement. Dhn4s、HVAls、rab16Bj and wsi18j promoters were induce-dependent and differed in their expression levels. Under ABA induction, their expression levels were in such an order: Dhn4s> HVAls> wsi18j> rabl6Bj. Response to dry treatment, they were in Dhn4s> HVAls> wsi18j> rab16Bj.2) HVA1s、Dhn4s、rab16Bj and wsi18j promoter being weak in response to cold and NaCl, they were different from each other. Comparatively, HVAls was strongest, wsi18j and Dhn4s weaker, and rabl6Bj without response to cold..5. DRE/CRT sequence base-substitution mutation of HVAls promoter showed that it was reduced in expression level (7.7%和10.9%down) under ABA and dry treatments. While under cold treatment, the mutant promoter expression level dropped to37.9%in comparison with the wild one, indicating the responsibility of DRE/CRT mainly for cold stress response. But DRE/CRT was still related to ABA and dry response.6. Base-substitution mutations on the responsive elements ABRE1、ABRE2和ABRE3sequences containing ACGT core motif in HVA1promoter showed that:the expression level of ABRE3mutant dropped by40%～50%under ABA and dry treatments; ABRE2+ABRE3mutant decreased by89%～92%; ABRE1+ABRE2+ABRE3mutant was the same as ABRE2+ABRE3mutant. These results proved that just ABRE2and/or ABRE3were high-efficient elements among the above three, ABRE1was not a real ABRE cis-acting element.7. Base-substitution mutation on the responsive element in wsi18j promoter showed that the wild and the mutant did not expressed under control treatment, while after ABA induction both of them expressed in way that the mutant was only1/4of the wild in expression level. This result proved that CE3-ABRE complex in Wsi18j played a major role in responsive expression. Analysis on responsive elements in wsi18j and wsi18promoters revealed that both have the same number and type of elements related to dry, cold and ABA response. Base deletion and distance change between CE3与ABRE elements in wsi18promoter may be the reason for why they are completely different in response to ABA. There is respectively one C base deletion flanking the ABRE sequence of CE3-ABRE complex.8. The innovation and usefulness on this research:1) The room of the methodology has been broadened for rapid test and determination of expression level of the promoters stress-inducible, which could be used for the efficient selection of promoters for transgenic molecular breeding. The method can be applied to study the relation and crosstalk between pathways in the transcription factor regulatory networks. It can also be utilized for investigate the function of and the interaction between sic-acting elements. Finally, it would be useful for confirmation of bioinformatic prediction on a promoter’s function.2) The characteristics and differences of LEA gene promoters have objectively been compared under the same backgroud.3) It was found that there be promoter like Dhn8s not inducible by stress but constitutively expressional in LEA gene family.更多还原