【作者】 孙立平；

【导师】 杨兴洪； 李德全；

【作者基本信息】 山东农业大学 ， 植物学， 2012， 博士

【摘要】 高温、低温和干旱等非生物胁迫是影响植物生长发育的重要环境因子，植物受到这些胁迫信号刺激后，会产生一系列响应蛋白来保护细胞代谢。热激蛋白（HSP）是植物对外界胁迫所做的应答，是一类重要的胁迫诱导蛋白，在逆境胁迫下的表达增强可提高植物抵御各种逆境因子胁迫的能力，在植物与环境长期协同进化过程中起到非常重要的作用。小分子量热激蛋白（sHSP）是热激蛋白家族中一类分子量约在15-30kDa的热激蛋白，许多体内及体外的研究表明，sHSP在细胞中起分子伴侣作用。作为“分子伴侣”，sHSP可以与正在合成的多肽结合，使其正确折叠；能够引导新生肽穿过细胞器膜结构，使蛋白定位于细胞的不同部位。在高温胁迫下，sHSP可以阻止热变性蛋白的聚集或阻止不可逆的蛋白变性，或有利于蛋白质在高温胁迫变性之后的复性，在植物耐热性中起重要作用。本研究从热激处理的玉米（郑单958）叶片中分离到一个细胞质classⅠsHSP16.9的关键基因，命名为ZmHSP16.9，对其序列特征、表达模式，以及过表达转基因烟草的抗逆生理功能进行了初步研究。研究结果对进一步了解sHSP的功能以及利用基因工程技术改良玉米的综合抗逆性，具有重要的理论价值和实践意义。主要结果如下：（1）ZmHSP16.9基因的克隆及序列生物信息学分析提取热激处理的玉米叶片总RNA，通过RT-PCR的方法分离到sHSP基因ZmHSP16.9，该基因开放阅读框编码一个152个氨基酸的蛋白。将该基因同其他小分子量热激蛋白同源序列比对，结果表明该基因与细胞质calssⅠsHSPs家族聚类在一起，氨基酸序列分析表明该基因同其他植物的细胞质classⅠsHSPs高度同源。通过ProtComp v.9.0database（http://linux1.softberry.com/berry.phtmL?topic=protcomppl&group=programs&subgroup=proloc）数据库对ZmHSP16.9进行亚细胞定位预测，结果显示其定位于细胞质中。构建pBI121-ZmHSP16.9-GFP表达载体，并在洋葱表皮细胞中瞬时表达，在荧光显微镜下观察到细胞质内有GFP激发的绿色荧光。通过以上结果可以推测ZmHSP16.9基因为细胞质classⅠsHSP基因。（2）ZmHSP16.9组织特异性和表达模式分析对玉米幼苗进行干旱、高温、低温、H2O2及ABA处理，提取叶片总RNA进行Northern杂交，结果表明，正常生长条件下玉米叶片中未检测到ZmHSP16.9基因的转录产物，ZmHSP16.9在热激的玉米根、茎、叶中均有表达，但在叶中表达量最高。高温和外施H2O2均能诱导ZmHSP16.9基因的表达，表明ZmHSP16.9为热激和氧化胁迫诱导特异表达的基因。（3）ZmHSP16.9原核表达载体的构建、抗体制备和Western blot分析构建了原核表达载体pET-ZmHSP16.9，并在大肠杆菌BL21中表达融合蛋白，诱导、纯化蛋白，免疫小白鼠，制备抗体。Western杂交结果表明，ZmHSP16.9蛋白在玉米幼苗叶片中受高温和氧化胁迫诱导。（4）过表达ZmHSP16.9转基因烟草的鉴定构建pBI-ZmHSP16.9正义表达载体，成功转化烟草。Northern blot和Western blot结果均表明，ZmHSP16.9基因已经在转基因植株中稳定表达。（5）高温条件下过表达ZmHSP16.9基因烟草种子萌发率和幼苗长势良好高温胁迫条件下，过表达ZmHSP16.9基因的转基因烟草种子比野生型烟草种子萌发率高，过表达ZmHSP16.9转基因烟草幼苗比野生型幼苗长势更好。（6）过表达ZmHSP16.9转基因烟草在高温胁迫条件下的生理变化为了检测过表达ZmHSP16.9转基因烟草在高温胁迫条件下的生理变化，测定了一系列与植物逆境胁迫有密切关系的生理指标，包括H2O2和O2的积累量、抗氧化酶的活性、MDA含量和膜透性。结果表明，未处理的转基因和野生型烟草的这些抗逆指标没有明显区别。但是高温胁迫处理后，不管是野生型还是转基因植株烟草体内H2O2，O2的积累量明显增加，但野生型烟草增加的更明显；高温胁迫下过表达ZmHSP16.9转基因烟草通过维持较高的抗氧化酶活性增强活性氧清除能力，减轻活性氧对细胞的伤害；高温条件下，转ZmHSP16.9基因烟草的丙二醛含量、相对电导率比野生型烟草低。这些结果表明，过表达ZmHSP16.9基因提高了转基因烟草对高温胁迫的抗性。（7）过表达ZmHSP16.9基因提高了转基因烟草对氧化胁迫的抗性氧化胁迫条件下，过表达ZmHSP16.9基因的烟草种子比野生型烟草种子萌发率高；过表达ZmHSP16.9基因烟草幼苗比野生型幼苗长势好。利用不同浓度的H2O2模拟氧化胁迫，发现转基因各株系烟草叶圆片在较高或较低浓度的H2O2溶液中，均保持较高的叶绿素含量。这些结果表明，过表达ZmHSP16.9的转基因烟草耐受氧化胁迫的能力较强。更多还原

【Abstract】 Abiotic stresses such as high temperature, low temperature and drought, influence plantgrowth and development. These stress signals can stimulate plant which can produce a seriesof responsive protein to protect the cell metabolism. Heat Shock Protein (HSP) is one kind ofimportant stress-induced proteins, producing when the plants are in response to externalstresses. Under stress, increasing expression can strengthen the ability of the plants to resistvarious stress factors, which plays a very important role in the long-term co-evolution processof plants and the environment, and has an important significance in taking it as a study ofstress physiology. The small HSP (sHSP) is a kind of heat shock protein family that range insizes from approximately15to30kDa. Many studies in vivo and in vitro indicate that sHSPact as molecular chaperones in cells. As molecular chaperones, sHSP can bind to and facilitatethe synthesizing polypeptides to correct folding; leading new-synthesized polypeptides totransverse the organelle membranes and locating them in different position. Under hightemperature stress, sHSP can prevent the heat-denatured proteins assembling, preventunreversible proteins denaturalization, or be propitious to the renaturation of heat-denaturedproteins. They play very important roles in plant’s thermotolerance.In the present study, we isolated and characterized a novel sHSP gene, ZmHSP16.9, frommaize leaves under heat stress. Sequence comparison, expression analysis and furtherfunctional studies on the over-expressing ZmHSP16.9plants were analyzed in this work.These data have the important theoretical and practical significance in further understandingthe functions of sHSP and using genetic engineering to improve corn comprehensiveresistance. The main results are as follows:(1) Molecular cloning and bioinformatics analysis of ZmHSP16.9geneTotal RNA was isolated from maize leaves under heat stress, we isolated the sHSP genesZmHSP16.9by RT-PCR, and the ZmHSP16.9encodes a protein of152amino acids.Comparing the gene with other small heat shock protein homologous sequence alignment, the results showed that the gene and the cytoplasmic class I sHSPs family clustered together.From the amino acid sequence, we can draw the conclusion that the gene with other plantcytosolic class I sHSPs is highly homologous. The amino acid sequence of ZmHSP16.9wasanalyzed in the ProtComp v.9.0database(http://linux1.softberry.com/berry.phtmL?topic=protcomppl&group=programs&subgroup=proloc). ZmHSP16.9was predicted to be localized in the cytosolic. The fusion protein pBI121-ZmHSP16.9-GFP was expressed transiently in onion epidermis, and the GFP greenfluorescence could be detected in the cytoplasm with the fluorescence microscope. The resultsclearly indicate that ZmHSP16.9is mainly localized in the cytoplasm. From these data, wespeculate that the gene belongs to cytosolic class I sHSP genes.(2) Tissue specificity and expression analysis of ZmHSP16.9in Zea maysThe young seedlings of maize were treated by drought, high temperature, lowtemperature, H2O2and ABA, and total RNAs were extracted and Northern blot was carriedout. The results showed that maize leaves under normal growth condition could not detectZmHSP16.9gene transcripts, but ZmHSP16.9mRNA were expressed in the leaves, stems androots of maize under heat-shock treatment, and the level in the leaves was highest. Hightemperature and H2O2treatments led to a significant increase of ZmHSP16.9transcript level.These results suggest that the ZmHSP16.9gene is involved in response to heat temperatureand oxidative stresses.(3) Recombinant of prokaryotic expression vector pET-ZmHSP16.9, antibody productionand Western blotting analysisA recombinant of prokaryotic expression vector pET-ZmHSP16.9was constructed andexpressed in E.Coli. BL21. The strong induced fusion protein was purified and used toimmunize white mice to obtain antiserum. Western hybridization revealed ZmHSP16.9inleaves of maize seeding was induced by heat temperature and oxidative stresses.(4) Identification of the transgenic tobaccoThe coding region of ZmHSP16.9was introduced into the vector pBI121under thecontrol of the CaMV35S promoter and then transformed into WT tobacco (NC89). Thetransgenic lines showed expression of ZmHSP16.9at both mRNA and protein levels byNorthern blot and Western blot. (5) The over-expression of ZmHSP16.9increased seed germination and seeding growthof tobacco under high temperatureCompared to WT plants, the over-expressing ZmHSP16.9transgenic tobacco showed asignificantly higher germination rate, and the seedling growth was better than WT plantsunder high temperature stress.(6) Changes in physiological parameters of tobacco under high temperature stressTo investigate the effect of ZmHSP16.9over-expression in tobacco on the physiologicalresponses to stress, a panel of physiological parameters, including the accumulation of H2O2,O2, antioxidant enzyme activities, antioxidants content, Malondialdehyde (MDA) contentsand relative electrolytic leakage (REL) were examined. All these physiological parametersreflect normally plant responses or tolerance to stress environment, thus serving as the plantphysiological index under stress. As shown in the result, heat stress induced the accumulationof H2O2, O2in the WT and transgenic tobacco, but this accumulation was more in WT.Through maintaining relatively higher antioxidant enzyme activities and antioxidants content,ZmHSP16.9decreased the accumulation of ROS to reduce the damage to cells. After hightemperature treatment, compared with WT plants, the MDA content was significantly lowerin ZmHSP16.9over-expressing plants. Transgenic plants had a lower electrolyte leakage thanWT under high temperature. These results indicate over-expressing ZmHSP16.9tobaccocould enhance the tolerance to the high temperature compared with WT plants.(7) ZmHSP16.9over-expressing tobacco improved tolerance to oxidative stressUnder oxidative stress, ZmHSP16.9over-expressing tobacco showed a significantlyhigher germination rate and the seedling growth was better than WT plants. H2O2in differentconcentration was used to estimate the effects of the oxidative stress on the growth of the WTand transgenic tobacco plants. ZmHSP16.9over-expressing plants showed higher chlorophyllcontent than WT plants under both high and low H2O2concentrations. These data suggest theadvantages of anti-oxidative characterization in the ZmHSP16.9over-expressing tobaccolines.更多还原