【作者】 王保全；

【导师】 李国怀； 邓伯勋；

【作者基本信息】 华中农业大学 ， 果树学， 2012， 博士

【摘要】 桃树是全球广泛栽培的重要果树之一。新品种的选育和推广应用是促进桃产业健康持续发展的基础。基因工程技术由于目的性强、时间短等优点使其成为当前育种学家常用的手段之一,发掘植物体内的优良基因资源是应用基因工程技术创造植物新种质的必要前提。桃果实独特的成熟衰老进程,以及果实成熟期的生理生化物质代谢机制研究的深入,使桃树成为果树植物中研究成熟和基因组分析的模式植物。同时,桃树对土壤条件要求不太严格,栽培地域限制较小,为育种学家培育抵抗多重逆境胁迫的植物新品种提供了良好的种质资源。本研究从桃中成功克隆到PpERS1和PpADC基因及其启动子,并对它们进行了转基因功能验证,同时通过验证柑橘PtADC基因在转基因番茄抗旱过程中的功能,拓展了ADC基因在植物体内的分子生物学功能。主要结果如下：1.采用同源序列法从桃树叶片中克隆到乙烯受体基因PpERS1,基因序列包含1935bp的开放阅读框,编码644个氨基酸。其编码蛋白质分子量预测为72.4kDa,等电点为6.33。多序列比对和系统进化树分析表明PpERS1属于乙烯受体基因家族的ETR1亚家族,与蔷薇科植物的ERS1基因具有高度的同源性。生物信息学分析表明PpERS1基因开放阅读框含有5个外显子和4个内含子,此外,基因5’UTR序列中含有2个内含子,第1个内含子存在选择性剪切机制。基因表达分析表明PpERS1基因在桃树根、茎、功能叶片、花、幼果和成熟果实中均能表达,其中根组织中PpERS1基因的表达量最低,茎、功能叶片、花、幼果和成熟果实中PpERS1基因表达量分别是根组织的1.5、1.8、2.4、1.5和4.3倍。逆境胁迫条件下,PpERS1基因受到伤害、脱水、乙烯利、高盐和桃流胶病菌侵染的诱导表达,受到低温处理的抑制表达。2.应用染色体步移技术克隆到PpERS1基因启动子,其全长为2798bp.5’RACE结果表明转录起始位点为G碱基,位于翻译起始位点ATG上游-777bp处,生物信息学分析表明启动子序列含有典型的TATA box和CAAT box,并且含有乙烯、伤害和增强子响应元件,以及生长素、分裂素、脱落酸和赤霉素等激素响应元件,干旱、低温、病原菌等逆境胁迫响应元件。通过构建启动子全长和5’端系列缺失的GUS植物表达载体,并将其转化Micro-Tom番茄,GUS组织化学染色和荧光定量分析均表明启动子具有正常的启动子活性,并且启动子序列中内含子缺失不影响正常的启动子活性。不同组织器官和发育阶段GUS染色结果表明启动子在转基因番茄的根、茎、叶、花和果实中均能正常表达,在种子萌发过程中的胚根、上胚轴、子叶和真叶中也能正常表达,同时受到伤害处理诱导表达,GUS定量分析表明启动子受到伤害和乙烯利诱导表达,受到低温抑制表达,高盐处理不影响启动子的活性。3.采用同源序列法从桃中克隆到精氨酸脱羧酶基因PpADC,基因全长包含2178bp的开放阅读框,编码725个氨基酸。其编码蛋白质分子量预测为77.7kDa,等电点为5.19。多序列比对和系统进化树分析表明PpADC与苹果等蔷薇科植物的ADC基因具有高度的同源性。生物信息学分析表明PpADC基因没有内含子结构。逆境胁迫条件下,PpADC基因受到脱水、乙烯利、高盐和低温处理的诱导表达。将PpADC基因超量表达载体转化番茄Micro-Tom,转基因植株中PpADC基因超量表达,内源腐胺、亚精胺和精胺含量相对于野生型番茄明显提高,转基因植株生长发育迟缓、开花期延迟,外源施用赤霉素能有效恢复转基因植株生长势,基因表达分析表明转基因植株部分赤霉素合成相关基因表达明显受到抑制。4.应用染色体步移和电子克隆技术获得PpADC基因启动子,其全长为2043bp。生物信息学分析表明启动子的转录起始位点为A碱基,位于翻译起始位点ATG上游-453bp处,其序列含有典型的TATA box,并且含有干旱、低温、病原菌等逆境胁迫响应元件,以及生长素、伤害和生物钟调控响应元件。通过构建启动子全长和5’系列缺失的GUS植物表达载体,遗传转化Micro-Tom番茄,GUS组织化学染色分析表明PpADC启动子具有正常的启动子活性。5.PtADC转基因功能分析结果表明,转基因番茄中PtADC基因超量表达,转基因植株中内源腐胺和亚精胺含量相对于野生型番茄明显提高,但内源精胺含量没有明显变化。离体叶片脱水处理过程中转基因植株的失水率明显低于野生型番茄。处理结束时,形态学观察表明野生型番茄离体叶片相对于转基因植株明显萎蔫,转基因植株离体叶片的相对电导率明显低于野生型番茄,而叶绿素含量与相对电导率表现相反结果,并且NBT和DAB染色结果表明转基因植株ROS含量明显低于野生型番茄。干旱处理导致野生型番茄萎蔫程度明显高于转基因植株。更多还原

【Abstract】 Peach is one of the most important economic fruit trees among the world. The release and promotion of new cultivar underpin the healthy and sustainable development of peach industry. Genetic engineering is a feasible approach for cultivar breeding because of the sound technique foundation and high efficiency. But discovery of the desirable genes should be the prerequisite. Peach enjoys the unique characteristics of ripening and the accompanying biochemical mechanism has been studied intensively. Therefore, it has become the model for ripening study in fruit tree. In addition, peach is of no exception concerning stress restriction, which has provoked breeders throughout the world to create new cultivars with tolerance to multiple stresses in breeding program. In the current study, PpERS1and PpADC genes along with their respective promoters were cloned from Prunus persica and transferred into Micro-Tom tomato for functional analysis. Meanwhile, the transgenic tomato carrying PtADC were used to investigate its function under dehydration and drought stress. Main results were as follows:1. Based on the homogenous sequences, an ethylene receptor gene PpERSl was cloned from peach, containing an open reading frame (ORF) of1935bp which encodes a644amino acids polypeptide with a predicted molecular mass of72.4kDa and an isoelectric point of6.33. Analysis of the putative amino acid sequence suggests that PpERS1was clustered to into the ETR1subfamily, and showed high homology with those ethylene receptors from rosaceous plants. Bioinformatics analysis revealed that the coding region of PpERS1contained five exons interrupted by four introns, in addition, two introns located in the5’UTR of PpERSl gene, and alternative splicing occurred in the first intron of5’UTR. The expression analysis indicated that the PpERSl transcript was detected in all the tissues including root, stem, leaf, flower, fruitlet and ripening fruit. The expression of PpERSl was lowest in root and the expressions in stem, leaf, flower, fruitlet and ripening fruit were1.5,1.8,2.4,1.5and4.3fold higher to that of root, respectively. PpERSl expression was induced by injury, dehydration, ethephon, salt and peach tree gummosis, while low temperature down-regulated PpERSl transcription.2. A2798bp upstream region of PpERSl translation start codon (ATG) was cloned by genomic walking PCR.5’RACE result revealed that the transcription start site was G, located at-777bp upstream of ATG of PpERS1. In silico analysis of the PpERS1promoter revealed the presence of typical TATA box and CAAT box, ethylene response element, injury response element, enhancer element, hormone response elements including auxin, cytokinin, abscisic acid and gibberellin, and stress response elements including drought, low temperature and pathogen. We constructed vectors containing the PpERSl full-length and serial5’-deleted promoters with GUS reporter gene, and all the constructions were introduced into tomato (Lycopersicon esculentum cv. Micro-Tom) through Agrobacterium tumefaciens-mediated transformation. GUS activity analysis was carried out by histochemical staining and fluorescence detection demonstrated that the promoter activity was normal in transgenic plants and not affected by the introns contained in5’UTR. GUS activity analysis in tissue, organs and developmental stages indicated the PpERSl promoter was active in almost all tissues. Meanwhile, PpERS1promoter activity was detected in radicle, cotyledons, hypocotyls and euphylla during different development stages. Real Time PCR analysis suggested that promoter activity were induced by injury and ethephon treatment, suppressed by low temperature and immune to salt treatment.3. Based on the homogenous sequences, an arginine decarboxylase gene PpADC was cloned from peach, containing an open reading frame (ORF) of2178bp which encodes a725amino acids polypeptide with a predicted molecular mass of77.7kDa and an isoelectric point of5.19. Analysis of the putative amino acid sequence suggests that PpADC shared high homology with that from rosaceous plants. Bioinformatics analysis revealed that no intron was contained in the coding region of PpADC. The expression analysis indicated that the PpADC transcription were up-regulated by dehydration, ethephon, salt and low temperature treatment. The transgenic plants overexpressing PpADC contained higher Put, Spd and Spm level than that of the wild type (WT). Meanwhile, the transgenic plants showed dwarfism and late-flowering. However, the phenotype was rescued by gibberellin application. The expression analysis indicated that partial genes associated with gibberellin biosynthesis were suppressed in transgenic plant.4. A2043bp upstream region of PpADC translation start codon (ATG) was cloned by genomic walking and silicon cloning PCR. In silico analysis of the PpADC promoter revealed that the transcription start site was A, located at-453bp upstream of ATG of PpADC. The sequence of PpADC promoter contained the putative TATA box, stress response elements including drought, low temperature and pathogen, auxin response element, injury response element, and circadian regulation element. Vectors containing the PpADC full-length and serial5’-deleted promoters with GUS reporter gene were constructed and introduced into tomato(Lycopersicon esculentum cv. Micro-Tom) through Agrobacterium tumefaciens-mediated transformation. GUS activity analysis carried out by histochemical staining demonstrated that the promoter activity was normal in transgenic plants.5. The transgenic plants overexpressing PtADC contained higher Put and Spd level than the WT, while there was no difference in Spm level. Under dehydration, water loss of detached leaves was severer in the WT. Moreover, ion leakage of the transgenic lines was remarkably lower than that of the WT. However, chlorophyll content was higher in the WT. In addition, NBT and DAB histochemical staining revealed that WT accumulated more ROS than the transgenic lines in tomato. Therefore, drought tolerance of the transgenic lines was obviously improved.更多还原