【作者】 周淑梅；

【导师】 王玮；

【作者基本信息】 山东农业大学 ， 植物学， 2013， 博士

【摘要】 小麦是禾本科小麦属的重要栽培作物，也是我国第二大粮食作物。全世界约有35%-40%的人口以小麦作为主要粮食。小麦生长过程中经常遭受干旱胁迫的危害而导致减产。干旱胁迫导致植物体内蛋白质损伤，细胞内无功能蛋白的积累增多，多种酶受到影响造成一系列生理生化代谢过程紊乱，甚至导致细胞、组织、器官或者植株的死亡。因此，胁迫条件下无功能蛋白质的及时清除成为植物抵抗逆境的重要机制，而泛素-26S蛋白酶体系统在清除这些无功能蛋白过程中起着至关重要的作用。泛素连接酶E3是泛素-26S蛋白酶体系统的关键酶，其中F-box蛋白是多亚基泛素连接酶E3的关键亚基，也是识别底物蛋白的特异组分，在泛素-26S蛋白酶体系统中居于关键部位。前人研究发现F-box蛋白在植物抗逆性中起着重要作用。本研究从抗旱小麦品种山农16中获得了泛素连接酶SCF复合体F-box亚基基因TaFBA1及其启动子区域，对其序列特征、表达模式进行了分析，构建了组成型和胁迫诱导型表达载体，转化烟草和拟南芥获得了转基因植株，对转基因植株的抗逆性及其生理机制进行了研究。主要结果如下：（1）利用同源克隆从小麦中分离到F-box基因TaFBA1（JN038382）。该基因cDNA全长为1334bp，基因的开放阅读框编码325个氨基酸，分子量约为36.6kDa，具有两个保守区F-box结构域和AMN1（Antagonist of mitotic exit network protein1）结构域。（2）利用Tail-PCR分离了TaFBA1基因的启动子区域（JQ065654），长度为1104bp；PlantCARE功能软件预测表明，该启动子区域含有茉莉酸甲酯、水杨酸、乙烯缺氧胁迫、胚乳表达、伤口、植物防御与抗性等响应元件以及参与干旱诱导的MYB结合位点。（3）将p35S-TaFBA1-GFP融合蛋白在洋葱表皮中瞬时表达。通过荧光显微镜观察到TaFBA1-GFP融合蛋白在细胞膜、细胞质和细胞核中都有分布。（4）qRT-PCR分析表明，TaFBA1基因在根、茎、叶、小穗中均有表达，其中叶片中表达最丰富，茎中最低。PEG、NaCl、MV等逆境胁迫条件均能诱导该基因的上调表达，同时外施ABA、MeJA、SA、ETH等激素也可诱导TaFBA1基因的上调表达。（5）构建了基因的原核表达载体pET-TaFBA1，并在大肠杆菌BL21中表达了融合蛋白。将诱导蛋白分离纯化并溶于PBS中获得抗原，免疫小白鼠制备了抗体。检测抗血清效价为1:1500。Western杂交表明，干旱诱导小麦叶片中TaFBA1蛋白的表达丰度提高。（6）将获得的TaFBA1基因编码序列与含有35S启动子的pBI121载体重组，构建了过表达载体pBI121-35S-TaFBA1。利用农杆菌介导的叶盘法转化烟草，获得过表达TaFBA1的转基因烟草植株。利用农杆菌介导的花序侵染法转化拟南芥，获得了过表达TaFBA1的拟南芥转基因植株。利用PCR对带卡那抗性的转基因烟草和拟南芥植株进行检测，结果证明成功地获得了过表达TaFBA1烟草和拟南芥植株。Western blot同样证实转基因烟草株系中TaFBA1蛋白的表达明显。（7）选择3个过量表达TaFBA1的转基因株系用于基因抗逆性功能研究。结果表明，干旱胁迫下过表达TaFBA1烟草植株比野生型植株萌发率较高，萌发较快；过表达TaFBA1烟草幼苗比野生型叶片数较多，侧根数较多，生长量较高，说明过表达TaFBA1提高了烟草的抗旱能力，维持了较好的生长势。干旱胁迫下，过表达TaFBA1烟草比野生型植株积累较少的ROS，相对电导率较低，丙二醛含量较少，说明过量表达TaFBA1基因在一定程度上缓解了干旱胁迫导致的活性氧胁迫，细胞受伤害程度较低，细胞膜完整性较好，膜透性受影响较小。干旱胁迫下过表达TaFBA1烟草比野生型植株相对含水量较高，气孔开度较大，光合速率较强，说明转基因植株根系发达，吸水能力强，维持较好的水分状况，从而能够维持较高的光合速率，增强了叶片的耐干旱能力。（8）检测了转基因植株的耐氧化胁迫能力。MV处理条件下，过表达烟草种子比野生型具有较高的萌发率；转基因幼苗失绿、根系受抑制程度较轻，生长情况较好；表明过表达TaFBA1的转基因烟草抗氧化胁迫能力较强。用MV处理叶圆片后发现，转基因株系相比野生型植株叶绿素含量较高，相对电导率较低、丙二醛含量较少，抗氧化酶SOD、POD、APX、CAT活性高且较稳定，积累ROS较野生型少，细胞受伤害程度较小，说明转基因烟草的抗氧化胁迫能力较强。进一步检测氧化胁迫相关基因表达发现，在正常情况和MV处理条件下，过表达TaFBA1均影响了氧化胁迫相关基因的表达。（9）观察了转基因株系整个生育期的生长发育表型。与野生型烟草相比，过表达TaFBA1转基因烟草叶片畸形率较高。同时发现，过表达TaFBA1转基因拟南芥比野生型生长减缓，植株矮小，叶片呈现明显皱缩，缺刻增多。（10）为了减轻过表达TaFBA1基因对植株表型造成的影响，同时保持TaFBA1基因的耐逆性功能，我们用胁迫诱导启动子RD29A代替35S启动子，构建了胁迫诱导表达载体pBI121-RD29A-TaFBA1，利用农杆菌介导的叶盘法转化烟草，获得胁迫诱导表达TaFBA1烟草植株。对诱导型转基因植株的初步观察研究发现，干旱和氧化胁迫条件下，胁迫诱导表达TaFBA1转基因烟草植株比野生型烟草的耐干旱和耐氧化胁迫能力较强，表型与野生型差异不明显。以上研究表明，TaFBA1基因不仅在植物抵抗干旱胁迫和氧化胁迫时具有重要作用，而且还可能与植物的生长发育相关。更多还原

【Abstract】 Wheat is a monocots and an important crop from gramineae family, and the secondleading cereal crop in china. More than one third population in the world take wheat as themain food. Drought stress restrains wheat growth and yield. Drought stress may causevarious adverse effects on plant growth and development, such as dwarf plant, shorter rootlength, smaller leaf area and decreased biomass. Besides, proline content in leaf andelectrolyte permeation were significantly increased, associated with photosynthesis decreased,sometimes the whole plant is killed badly. The accumulation of waste proteins in cells wereincreased under stress conditions. Ubiquitination of proteins through ubiquitin/26Sproteasome system (UPS) is an important regulative manner in response to various abioticstresses in plants. Ubiquitin ligase E3, as a main determinant of substrate specificity in UPSwas involved in plant stress adaptability. F-box protein characterized by an F-box motif is asubunit of SCF complex, which works as determinant in substrate recognition. It is veryimpotant to investigate the relationship between F-box and stress tolerance in plants.In the present study, we isolated and characterized an F-box gene, TaFBA1, fromTriticum aestivum L. cv. Shannong16with stronger drought resistance. Sequence comparisonand expression analysis were analyzed. The full-length TaFBA1cDNA was subcloned intothe expression vector pBI121downstream of the CaMV35S promoter or RD29A promoter toform pBI121-35S-TaFBA1or pBI121-RD29A-TaFBA1constructs. The constructs weretransferred into arabidopsis plants and tobacco plants mediated by Agrobacteriumtumefaciens. And further functional analysis on the transgenic plants were analyzed in thiswork. The main results are listed as follows:(1) We isolated an F-box gene from wheat, named TaFBA1(GenBank ID: JN038382). TheTaFBA1contains a978bp open reading frame (ORF), and encodes a325amino acidspolypeptide of molecular weight36.6kDa. There are two conserved regions, an F-box motif and an AMN1（Antagonist of mitotic exit network protein1）motif in this protein.(2) We also isolated the promoter region of TaFBA1(GenBank ID: JN065654). The promoterfragment length is1104bp. Promoter sequence analyzed by PlantCARE showed that it hasTATA-box, CAAT-box and some cis-acting elements such as MYB binding site, cis-actingregulatory elements involved in MeJA, SA response, some light-induced responsive elementsand other transcription factor-binding sites.(3) p35S-TaFBA1-GFP fusion protein was constructed and transiently expressed in onionepidermal cells. It was observed with fluorescence microscope that the TaFBA1-GFP fusionprotein was found in the membrane, cytoplasm and nucleus.(4) qRT-PCR showed that TaFBA1mRNA was expressed in the leaves, stems, roots andspikes, and the transcript level in the leaves was higher than that in the roots and spikes, andin the stems, its expression level was minimal. NaCl, PEG and MV stresses led to asignificant increase of TaFBA1transcript level in wheat leaves. And exogenous application ofABA, SA, MeJA and ETH also increased the TaFBA1mRNA in wheat leaves.(5) A recombinant of prokaryotic expression vector pET-TaFBA1was constructed andtransformed to E.Coli. BL21. The strong induced fusion protein bands were collected intophosphate buffer (PBS) solution and were used to immunize mice to obtain antiserum. Thevalue of antibody reaches1:1500. Western blot analysis revealed the presence of the strongpositive protein signals corresponding to TaFBA1in transgenic plants. The results showedthat the expression of TaFBA1at protein level was induced by drought stresses and changedwith the treatment.(6) The full-length TaFBA1cDNA was subcloned into the expression vector pBI121downstream of the CaMV35S promoter to form pBI121-35S-TaFBA1constructs. Theconstructs were first introduced into Agrobacterium tumefaciens LBA4404and GV3101bythe freezing transformation method under the kanamycin selection pressure. The transgenicplants were verified by PCR and qRT-PCR. It was indicated that the TaFBA1gene had beenrecombined into tobacco and arabidopsis genome and the transgenic plants were obtained.(7) Compared to WT plants, overexpression of TaFBA1transgenic plants showed higherresistance to drought stress by increasing germination rate, leaf number, lateral number, rootlength and growth vigor. Under drought stress, the MDA content, ROS accumulation, relative electrical conductivity of transgenic plants were obviously lower than those of WT plants.These results showed that overexpression of TaFBA1alleviated the permeability ofmembrane, cell damage degree and oxidative damage under drought stress. Furthermore, netphotosynthetic rate (Pn) and relative water content (RWC) in WT and transgenic plantsdecreased markedly under drought stress, the decrease of Pn and RWC was more slower intransgenic plants compare to WT. The results indicated that overexpression of TaFBA1maintained high photosynthetic rate and strengthened the stress resistence of plants.(8) Overexpression of TaFBA1in tobacco conferred resistance to methyl viologen (MV)induced oxidative stress. Under MV treatments, the growth of TaFBA1-overexpressingtobacco plants was better than the WT plants, which including more green cotyledons andlonger root length. In addition, TaFBA1-overexpressing tobacco plants showed less ROSaccumulation and higher activities of antioxidant enzymes (SOD, APX, CAT and POD)compared to WT plants under MV treatments. Furthermore, the expression levels ofROS-related genes were changed by overexpressed TaFBA1.(9) Compared to WT plants, overexpression of TaFBA1transgenic plants showed highproportion of abnormal leaves. At the same time, transgenic arabidopsis plants exhibiteddecelerated growth and abnormal leaves compared to WT.(10) For further research, The expression vector pBI121-RD29A-TaFBA1was constructed,and the constructs was transferred into tobacco plants mediated by Agrobacteriumtumefaciens LBA4404under the kanamycin selection pressure. The transgenic plants wereverified by PCR. It was indicated that the TaFBA1gene had been recombined into tobaccogenome and the stress-induced expression of TaFBA1transgenic plants were obtained. Wefound that stress-induced expression of TaFBA1transgenic plants showed higher resistance todrought stress and oxidative stress compare to WT plants.In conclusion, TaFBA1plays an important role in the biological processes related toplant growth and development, adaptation to drought stress and oxidative stress.更多还原