【作者】 赵玲玲；

【导师】 郝玉金； 翟衡；

【作者基本信息】 山东农业大学 ， 果树学， 2007， 博士

【摘要】 非生物胁迫严重影响植物的生长和发育,利用基因工程对作物进行遗传改良提高其抗逆性,是保障作物高产优质安全生产的有效途径,具有广阔的应用前景。多胺在植物细胞内参与诸多生命活动,并参与植物的抗逆性形成,SAMDC是多胺生物合成的关键酶,苹果MdSAMDC2基因的表达能够被低温、干旱和盐等逆境诱导上调,而其在植物体内的生物学功能还没有验证。为了确定MdSAMDC2的表达模式,本研究通过TAIL-PCR技术分离了该基因的启动子,初步研究了该启动子的启动活性;通过Gateway技术构建了MdSAMDC2的植物表达载体,并导入烟草使该基因过量表达,进一步研究了逆境条件下该基因的表达水平,测定了各项抗性生理指标,表明过量表达MdSAMDC2能够提高转基因烟草的抗逆性。ICE1(Inducer of CBF expression 1)是CBF3的正调节因子,已有研究表明,过量表达ICE1能够明显提高转基因拟南芥的耐寒性,说明ICE1在植物耐寒性形成中起重要作用。本研究从苹果中克隆到MdICE1基因,并对其表达和功能进行了初步研究,转基因研究表明MdICE1过量表达可以提高烟草的耐寒能力。主要结果和结论如下:1、根据MdSAMDC2基因的cDNA序列,设计了该基因编码区的3′和5′端引物并进行克隆,利用Gateway技术构建了植物表达载体,通过农杆菌浸染法转入烟草,PCR检测证明MdSAMDC2基因已经整合到烟草基因组中,RT-PCR表明MdSAMDC2在烟草中得到表达,所有转基因植株生长正常,能够完成生活史。用HPLC测定了转基因植株的多胺水平,发现转基因植株的多胺含量明显上升,并且不同多胺的比例发生了变化,Spd比例相对提高,Put和Spm的比例相对下降。分别用低温、20%PEG模拟干旱和NaCl等逆境胁迫进行处理,分析了转基因植株的抗逆生理生化变化,发现在逆境条件下,过量表达MdSAMDC2可以提高Spd和Spm的积累,降低Put含量,同时提高了脯氨酸含量以及SOD和CAT的酶活性,降低了MDA含量,表明转基因烟草抗逆性得到了提高。2、根据MdSAMDC2的5′端序列设计3条反向引物和4条随机兼并引物,以苹果基因组DNA为模板,利用改进TAIL-PCR法克隆到了长度为680bp的MdSAMDC2启动子,序列分析表明,该启动子区包含多种逆境诱导启动子的特异序列元件,如ACGTA序列元件、ASF1元件、MYC识别位点、MYB1和MYB2识别位点等。构建了该启动子和GUS基因的重组表达载体,将重组质粒导入农杆菌LBA4404,用农杆菌介导法转入烟草,GUS染色显示转基因烟草的叶片为蓝色,表明该启动子具有启动活性。3、为了验证不同抗性水平苹果砧木在逆境下的多胺反应,以山定子(M. baccata Borkh)、新疆野苹果(M. sieversii(Ledeb)Roem)、烟台沙果(M. prunifolia(Willd)Borkh)和莱芜难咽(M. micromalus Makino)等4种苹果砧木为试材,测定了低温胁迫下不同砧木叶片的多胺含量变化及MDA、抗氧化酶的变化等生理指标。结果表明,低温胁迫能明显诱导多胺总量(PAs)、腐胺(Put)、亚精胺(Spd)和精胺(Spm)的发生,耐寒性强的山定子和新疆野苹果PAs、Put和Spd增加显著,相关分析表明,叶片MDA相对增加量与PAs、Put和Spd的增加量呈极显著负相关,与Put/PAs比值呈显著负相关,而与(Spd+Spm)/Put、Spd/PAs、Spm/PAs呈显著正相关,表明当低温胁迫下叶片PAs、Put、Spd增加量较大时,苹果砧木耐低温胁迫的能力比较强。4、以苹果为材料,利用同源序列法和RACE技术克隆苹果MdICE1的全长cDNA序列(GenBank登录号为EF495202),该cDNA长度为1890bp,编码531个氨基酸,保守性分析表明,MdICE1蛋白质序列上包含由60个氨基酸残基组成的bHLH(basic Helix-Loop-Helix)功能域,同源性比较表明,不同bHLH蛋白功能域的同源性高达90%以上。将该蛋白氨基酸序列与拟南芥等植物的bHLH蛋白进行在线多重序列比较,并构建系统树,发现MdICE1与拟南芥ICE1(AAP14668)同源性最高,系统树将这两个蛋白聚为一类,因此可以确定MdICE1是拟南芥ICE1在苹果中的一个同源基因,编码bHLH类蛋白。表达和功能分析结果如下:①构建了MdICE1的原核表达载体pET-30(a)+MdICE1,转化BL21,筛选重组菌株。经IPTG诱导后,SDS-PAGE发现大小为64.5kDa的特异蛋白产物。②将MdICE1-GFP融合蛋白在洋葱表皮细胞内瞬时表达,只在细胞核中观察到绿色荧光,表明MdICE1定位于细胞核,Northern杂交结果表明MdICE1表达受低温和盐胁迫诱导。③将MdICE1插入pBI121构建表达载体,利用农杆菌浸染法将35S::MdICE1转入烟草。PCR检测表明MdICE1已经整合到烟草基因组中,RT-PCR表明MdICE1在转基因烟草中能够表达。用低温胁迫处理转基因株系,分析了该基因表达模式和转基因植株的抗逆生理生化变化,表明过量表达MdICE1的转基因烟草的抗寒性明显提高。更多还原

【Abstract】 Abiotic stresses often adversely affect both plant growth and crop productivity. To cope with abiotic stresses, breeding new varieties which hava modified stress tolerance is one of the most important approaches. Polyamines are involved in many life processes in plant cells. Increasing evidences indicate that polyamine accumulation confers stress tolerance on plants. SAMDC is a step-limiting enzyme for polyamine biosynthesis. MdSAMDC2 encoding a SAMDC in apple is induced by low temperature, dehydration and salinity. But its biological function in plants is yet to be identified. A modified TAIL-PCR strategy was used to isolate the promoter region of MdSAMDC2 from apple, and then the ability of MdSAMDC2 promoter to drive the downstream ORF was valuated.In order to characterize the in vivo biological function of MdSAMDC2 in plant, transgenic tobaccos over-expressing MdSAMDC2 were obtained. The enhanced tolerance of transgenic lines to abiotic stresses suggests that MdSAMDC2 is a candidate gene for genetic engineering to improve plant tolerance to stresses. In addition to MdSAMDC2, we also isolated MdICE1 from apple. Functional analysis demonstrated its crucial role the responses of plant to cold stress. Over-expressing MdICE1 remarkably improved the tolerance of transgenic tobacco to low temperature. The main results of this study were summarized as follows.1) Specific primers were designed based on the cDNA sequence of MdSAMDC2. Its ORF was cloned and inserted into expression vector pMDC32 with Gateway recombination. Then the resultant construct was introduced into tobacco with Agrobacterium-mediated transformation. PCR verified the integration of MdSAMDC2 into tobacco genome. Furthermore, RT-PCR detected a high level of MdSAMDC2 transcript in transgenic tobacco. All transgenic tobaccos normally grew and developed in appearance. Polyamine titers in transgenic plants were determined with HPLC. The results showed that the titers of three polyamines increased remarkably. After treated with low temperature, 20% PEG and salt respectively, transgenic plants accumulated more Spd and Spm but less Put than non-transgenic control. Moreover, proline content and the activity of SOD and CAT increased while MDA content decreased. So over-expression of MdSAMDC2 in tobacco enhanced the tolerance of transgenic plants to stresses.2) To investigate the expression pattern of MdSAMDC2, the promoter region of MdSAMDC2 was isolated from apple with a modified TAIL-PCR approach. The promoter is 643 bp in size and contains several basic elements for promoter such as TATA-box, CAAT-box, stress-induced elements and so on. To examine its ability to drive a downstream ORF, the promoter was inserted into a plant expression vector to replace CaMV 35S promoter driving GUS gene. Transient expression of GUS in transgenic tissues was detected with histochemical GUS staining. As a result, GUS activity was detected in transgenic plants. GUS activity can be induced by low temperature and high salt.3) MDA and polyamine contents in leaves of four apple rootstocks were exmined to explore the changes of polyamines in apple during low temperature stress. The results showed that MDA content could indicate the cold tolerance of four rootstocks at time point 6h during low temperature stress. The titers of total polyamines, put, spd and spm were significantly induced by low temperature. MDA content was negatively correlated with polyamines and the changes of Spd and put content while changes of relative content of spm(Spm/PAs)and spd content(Spd/PAs)are positively correlated with (Spd+Spm)/Put. The changes in polyamines content, Put content and Spd content under low temperatures were significant and could be used as indicator of cold tolerance in apple rootstocks.4) RT-PCR and RACEs were used to isolate MdICE1 (Inducer of CBF Expression 1) from apple. The full length cDNA of MdICE1 was registered in Genebank with an accession number EF495202. MdICE1 is 1890 bp in size and encodes 531 amino acid residues. Its full-length cDNA was inserted into vector PET-30a (+) and then transferred into E. coli strain BL21. As a result, a 64.5 kDa protein was specifically induced by IPTG treatment visualized by SDS-PAGE. BLAST analysis indicates that MdICE1 is a homolog of Arabidopsis ICE1. To investigate the subcellular localization of MdICE1 in cells, a construct producing MdICE-GFP fusion protein was introduced into onion epidermal cells. GFP florescence was observed only in the nucleus, suggesting that MdICE1 was localized to nucleus.MdICE1 expression patterns under different stresses in apple were detected with Northern blotting. The result showed that the expression of MdICE1 was induced by low temperature and salt stresses. To determine the function of MdICE1, transgenic tobacco plants were created with ectopic expression of MdICE1 driven by cauliflower mosaic virus 35S promoter. After treated with low temperature and salt stresses, transgenic plants produced increased proline and activated SOD and CAT enzymes, while decreased MDA content. All evidences indicated that ectopic expression of MdICE1 in tobacco confers cold tolerance on transgenic plants.更多还原