【作者】 张路；

【导师】 张启翔；

【作者基本信息】 北京林业大学 ， 园林植物与观赏园艺， 2014， 博士

【摘要】 报春花属植物种类繁多,花色丰富而艳丽,具有很高的观赏价值,被誉为“世界三大高山花卉”之一,可用于花坛、花镜,高山景观、岩石园景观、野趣园以及沼泽园的构建,亦可作为盆栽观赏。但由于其为典型的暖温带植物,生长在低纬度高海拔区域,温度是影响其广泛应用的主要限制因素之一,提高其耐热性是引种及育种成功的关键。因此,报春耐热性的研究对扩大其生存适应范围,使报春花属植物在园林绿化中广泛的应用具有重要意义。在已有灰岩皱叶报春热激差减文库研究的基础上,克隆得到3个编码热激蛋白的基因PfHSP17.1,PfHSP17.2,及PfHSP21.4,为进一步研究验证3个基因的功能及其热激应答机制分别对3个基因进行功能研究,主要结论如下：1、为研究PfHSP17.1、PfHSP17.2、及PfHSP21.4,基因在活体中的作用,构建pET43.1a-PfHSP17.1、pEF43.1a-PfHSP17.2、pEF43.1a-PfHSP21.4,重组质粒,转入大肠杆菌BL21,以转pET43.1a空载的大肠杆菌为对照。通过SDS-PAGE和Western blot确定融合蛋白在大肠杆菌中的正确表达。对转目的基因大肠杆菌和pET43.1a空载的大肠杆菌进行胁迫实验,结果表明小分子热激蛋白的转入明显提高了大肠杆菌对高温、低温、盐和干旱胁迫的耐受能力。2、从灰岩皱叶报春中克隆得到PfHSp17.1,通过同源比对和系统发育分析,以及洋葱表皮细胞定位,将PfHSP17.1归类于细胞质Class Ⅰ小分子热激蛋白。热激条件下,PfHSP17.1的表达具有组织特异性,其在叶片中的表达量明显高于在根、茎、花等器官中的表达量。该小分子热激蛋白基因也受干旱、盐和氧化胁迫等的诱导。在拟南芥中过量表达PfHSP17.1提高了转基因植株对热、盐和干旱胁迫耐受能力。3、从灰岩皱叶报春中克隆得到pfhSp17.2,通过同源比对和系统发育分析,以及洋葱表皮细胞定位,将PfHSP17.2归类于细胞质Class Ⅱ小分子热激蛋白。热激条件下,PfHSP17.2在幼叶中的表达量略高于老叶,且明显高于其在根、茎、花等器官中的表达量,在种子萌发和发育中也检测到该基因的表达。此外,该小分子热激蛋白基因在低温、盐和氧化胁迫下也大量表达,而在干旱和ABA处理下表达较为微弱。PfHSP17.2基因在拟南芥中过量表达提高了转基因植株对热、盐和低温胁迫的耐受能力。4、从灰岩皱叶报春中克隆得到叶绿体小分子热激蛋白基因,PfHSP21.4。该基因能迅速被热激诱导,也参与了种子萌发的过程。热激条件下,PfHSP21.4在拟南芥中过量表达有效的维持了较高的叶绿素含量和光合系统Ⅱ的效率,细胞膜受到的损伤小于同等胁迫下的野生型株系,同时,pfHSP21.4的表达也提高了过氧化物酶和超氧化物歧化酶的活性,从而减少了活性氧对细胞的损害,热激后游离脯氨酸在转基因株系中的积累也更丰富。此外,pfHSP21.4可能参与激活HSP101,HSP70，P5CS和APX的表达,从而提高转基因植株的耐热性。本研究对小分子激蛋白基因在灰岩皱叶报春中的表达模式进行了分析,并将PfHSP17.1、PfHSP17.2、PfHSP21.4,三个不同亚家族的小分子热激基因转入拟南芥和大肠杆菌中进行较为全面的功能分析,为今后的报春耐热性研究奠定了基础。更多还原

【Abstract】 Because of the number of varieties grown and its hold over the hearts and minds of the public, Primula is praised as one of the three great garden genera, only Rhododendron and Rosa can compare with it. With great wealth of species and high ornamental value, it is wildly sold as potted flowers; however, it could be also used in many other kinds of landscape decoration, such as parterre, rock garden, and bog and marsh garden. Approximately all species in this genus are distributed throughout the moister and cooler regions of the northern hemisphere where they are covered by snow during much of their resting period. Thus, high temperature becomes the primary barrier to cultivating them in warm areas. Therefore, it is interesting to research how to enhance the tolerance of plants to heat and other stresses. In our previous study, we found that Primula forrestii displayed the highest heat-resistance under heat stress compared with P. malacoides, P. obconica, P. veris, P. saxatilis and P. denticulatete. Through the suppression subtractive hybridization (SSH) method, three sHSP genes, PfHSP17.1, PfHSP17.2and PfHSP21.4, were highly expressed in the leaves of P. forrestii under high temperature treatment. In this study, we analyzed their expression patterns in P. forrestii under various abiotic stress treatments using RT-PCR. Then, the three genes were overexpressed in Arabidopsis thaliana and Escherichia coli to analyse their function.To elucidate the function of the three sHSP genes, recombinant plasmids expressing full-length PfHSP17.1, PJHSP17.2and PfHSP21.4were constructed. SDS-PAGE and Western blot analyses were used to confirm the expression of fusion proteins. The recombinant E. coli displayed enhanced viability under different stresses, such as high and low temperature, high salt and drought.PfHSP17.1was isolated from heat treated P. forrestii. Sequence alignments and phylogenetic analysis showed that PfHSP17.1belonged to sHSP cytosolic class I. Subcellular localization confirmed that PfHSP17.1localized in cytosol. The expression of PfHSP17.1was also triggered remarkably by salt, drought and oxidative stress conditions but was only slightly induced by abscisic acid (ABA). It was highly expressed in young leaves, old leaves, roots and stems, and a lesser extent in flowers under heat stress. Transgenic A. thaliana constitutively expressing PfHSP17.1displayed increased thermotolerance and higher resistance to salt and drought compared with wild-type plants.A novel small heat shock protein gene, PfHSP17.2, coding a protein of152amino acids was isolated from heat treated P. forrestii. Sequence alignments and phylogenetic analysis indicate that PfHSP17.2is a cytosolic class Ⅱ sHSP, which was further supported by PfHSP17.2-GFP fusion protein. PfHSP17.2was detected in developing and germinating seeds under normal conditions, and was highly expressed in leaves, roots, stems and flowers under heat stress. This gene was also strongly induced by cold, salt and oxidative treatments and to a lesser extent by drought and ABA stresses. Overexpression of PfHSP17.2in Arabidopsis enhanced tolerance to heat, cold and salt stresses.Here, expression analysis showed that the Primula Cp-sHSP gene, PfHSP21.4, was highly induced by heat stress in all vegetative and generative tissues in addition to constitutive expression in certain development stages. PfHSP21.4was introduced into Arabidopsis, and its function was analysed in transgenic plants. Under heat stress, the PfHSP21.4transgenic plants showed increased heat tolerance as shown by preservation of hypocotyl elongation, membrane integrity, chlorophyll content and photosystem II activity (Fv/Fm), increased seedling survival and increase in proline content. Alleviation of oxidative damage was associated with increased activity of superoxide dismutase and HSP101, HSP70, APX and P5CS under heat stress was more pronounced in transgenic plants than in wild-type plants.These results highlight the important roles that PfHSP17.1, PfHSP17.2and PfHSP21.4play in diverse physiological and biochemical processes related to adverse conditions. This study paves the way for Primula sHSP genes future utilization in plant resistance breeding and facilitates the HSP research expanded to ornamental plants.更多还原