【作者】 苏丹；

【导师】 陈年来； 高天鹏；

【作者基本信息】 甘肃农业大学 ， 生态学， 2012， 博士

【摘要】 盐碱地是一种广泛分布且影响植物正常生长发育的土壤。随着全球环境的不断恶化，土壤盐碱化已严重威胁着人类赖以生存的有限的土壤资源。土壤盐分是由多种离子组成的混合物，不同盐离子对植物种子萌发及生长发育的影响很大。目前关于植物耐盐性的研究，多数研究人员以单一的NaCl代替土壤中的盐分进行胁迫处理，以说明土壤盐分对植物的影响，这可能会限制研究结论的准确性。因此，本实验以典型耐盐性植物盐节木（Halocnermumstrobilaceum(Pall.)Bieb.)为材料，比较NaCl和土壤盐分胁迫时，盐节木种子萌发、幼苗生长及其生理生化特性反应的差异性，同时对盐节木耐盐的分子机制进行探讨。首先，采用双因素交叉设计，在三个昼-夜温度（5~15℃、15~25℃、25~35℃）和十个盐溶液浓度水平下（0%、0.3%、0.6%、0.9%、1.2%、1.5%、2.0%、2.5%、3.0%、3.5%）研究了复合盐和NaCl对其种子萌发及幼苗生长发育影响的差异性。其次，在0%、0.9%、2.7%和5.4%四种浓度下，研究了生长20d，60d时复合盐和NaCl对其整株幼苗生物量、Na~+、K~+离子含量、丙二醛（Malonaldehyde,MDA）、脯氨酸（Prolin,Pro）、甜菜碱（GlycineBetaine,GB）含量以及超氧化物歧化酶（SuperoxideDismutase,SOD）、过氧化物酶（Peroxidase,POD）、过氧化氢酶（Catalase,CAT）活性的影响。最后，根据辽宁碱蓬（Suaedaliaotungensis,AF359282）、菠菜（Spinaciaoleracea,AY156694）、中亚滨藜(Atriplexcentralasiatica,AY093682）、甜菜（Betavulgaris,X58463）的BADH基因完整读码框设计引物，以cDNA为模板，扩增盐节木BADH基因开放阅读框(openreadingframe,ORF)，将目的DNA片段克隆到PMD182T测序载体上测序，得到盐节木BADH全序列，然后利用网上数据库(http://www.ncbi.Nlm.nih.gov/)进行BLAST分析，用在线工具SOPMA进行蛋白等电点及分子量相关分析，以DNAMAN软件对HsBADH基因进行蛋白序列聚类分析。获得主要研究结果如下：1.温度及盐浓度对盐节木种子萌发和幼苗生长的影响在15~25℃，盐节木种子萌发率、幼苗胚根长度及鲜重显著高于5~15℃和25~35℃两个处理（P<0.05）。15~25℃为种子萌发及幼苗生长的最适温度范围。在5~15℃范围内，当NaCl和复盐浓度分别低于0.6%和1.2%时盐节木种子萌发率、幼苗胚根和鲜重显著高于对照（P<0.05）；在15~25℃范围内，NaCl与复盐浓度分别低于0.6%和1.5%时，种子萌发率、幼苗胚根长度和鲜重显著高于对照（P<0.05）。在25~35℃范围内，当NaCl和复盐分别低于0.6%和0.9%时盐节木种子萌发率、幼苗胚根长度和鲜重显著高于对照（P<0.05）。在试验的三个温度下，随着NaCl和复盐浓度的升高，盐节木种子萌发率、幼苗胚根长度和鲜重均呈先升后降的趋势，当NaCl和复盐浓度小于0.6%时，盐溶液对种子萌发和幼苗生长具有显著促进作用（P<0.05）。在15~25℃，复盐胁迫后的种子萌发、幼苗胚根长度和鲜重分别是NaCl胁迫的1.23、1.27和1.28倍。同一温度相同浓度下，NaCl胁迫的盐节木种子萌发率、幼苗胚根长度和鲜重均显著小于复盐胁迫，NaCl对种子萌发及幼苗生长的抑制作用显著高于复盐（P<0.05）。在试验的三个温度下，当NaCl和复盐浓度分别大于0.9%、1.2%时种子萌发均有延迟现象。随着盐浓度的增加种子萌发逐渐延迟，盐浓度是影响种子萌发的关键因素之一。以上结果表明，应用NaCl进行单盐胁迫不能完全反映自然状态下植物对土壤盐分的适应规律，复合盐含有的钾、钙、硅等复合离子可能有助于提高种子的萌发及幼苗生长能力。2.盐胁迫对盐节木生理生化特性的影响盐节木被处理20d和60d后随着NaCl和复盐浓度增加，其整株幼苗的K~+/Na~+、K~+含量、MDA和PRO含量以及SOD、POD、CAT活性均呈现先升后降的变化趋势，且显著高于对照，当NaCl和复盐浓度为2.7%，其K~+、渗透调节物质含量和酶活性达到最大值。当NaCl和复盐为同一浓度时，随处理时间的延长，Na~+、K~+、Pro、GB含量逐渐升高，但MDA含量、抗氧化酶活性显著降低（P<0.05）；在同一盐浓度下，NaCl处理的幼苗K~+、PRO、GB含量及SOD、POD、CAT活性显著低于复盐处理，但MDA含量显著高于复盐处理（P<0.05）。上述结果表明，在盐节木处理20d时，膜质过氧化反应强烈，氧化伤害严重，抗氧化酶活性增强。随着盐胁迫时间的延长，植物表现出一定的适应性；在复盐不同浓度处理下，复盐所含的钾、硅、钙等复合离子，促进了盐节木对K~+的吸收速率，提高K~+/Na~+、PRO和GB含量，同时也增强了抗氧化酶活性，降低细胞膜过氧化程度，提高盐节木的耐盐能力。3.HsBADH基因的克隆、序列分析及同源性比较采用race技术克隆了盐节木的甜菜碱醛脱氢酶基因，命名为HsBADH。该基因读码框序列全长为1848bp，编码559个氨基酸，预测HsBADH蛋白分子量为6.46KD，等电点为9.26。氨基酸分析表明，该蛋白富含丙氨酸（Alanine，3.75%）、谷氨酸（Glutamic，3.03%）、甘氨酸（Glycine，5.89%）、亮氨酸（Leucine，7.14%）和缬氨酸（Valine，4.82%）；对HsBADH蛋白二级结构的分析结果表明，推测的HsBADH蛋白多肽含有26.12%的α-螺旋结构、27.01%的延伸链、36.14%的无规则卷曲。同源性分析表明，HsBADH与藜科植物BADH同源性高达90%以上，与水稻BADH的同源性达到53%。本研究对盐节木在复盐条件下的耐盐机理进行了初步的探讨，为能够克服自然条件下的盐害，寻找提高植物耐盐性的途径和方法提供理论依据。同时丰富了植物的耐盐基因库，为进一步利用盐节木BADH基因资源开展作物和林木的遗传改良，提高非耐盐生物的耐盐性奠定了基础。更多还原

【Abstract】 Soil salinity is the one of the main limitation factors affecting plants growth in the word, and it becomes more and more serious as pollution mounts up, which effect the growth and development of plants. Study on mechanism of salinity tolerance in plants is very important. There are different types of ions in soil. It is different for the effects of ions in soil on seed germination and seedling growth of plants. Most studies concerning effects of salts on seed germination and seedling growth were carried out by using NaCl, which may limit the accuracy of the conclusions, especially under natural conditions. In this work, we have studied the effect of NaCl or mixed salt from the soil on Halocnermum strobilaceum. Firstly, we analyzed the seed germination and early growth of H. strobilaceum, and measured seed accumulated germination rate, seedling length and seedling fresh weight under different temperature and concentrations of salts. Three night-day temperature regimes5～15℃,15～25℃25～35℃, and NaCl and mixed salt solutions at nine concentrations were applied and distilled water was used as the controled solution. Secondly, the effect of salt stress on physiological characteristics of H. strobilaceum were studied using four different salt concentrations,0%,0.9%,2.7%,5.4%of NaCl and mixed salt. The contents of Na+, K+, Glycine Betaine (GB), Proline (Pro), Malonaldehyde (MDA) and activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) were measured. Thirdly, we designed primers according to BADH completed reading frame of Suaeda liaotungensis (AF359282), Spinacia oleracea (AY156694), Atriplex centralasiatic (AY093682), Beta vulgaris(X58463). In order to clone HsBADH, we make objective cDNA to pMD18-T and got the BADH sequence.We blast analyzed HsBADH of cDNA using online database (http://www.ncbi.Nlm.Nih.Gov/) and analyzed the protein isoelectric point and the molecular weight with online tools SOPMA. HsBADH gene sequence of protein cluster was analyzed using DNAMAN.The results showed that the night-day temperature range of15～25℃was optimum for seed germination, seedling length and seedling fresh weight. In the three night-day temperatures conditions, seed germination, seedling length and seedling fresh weight were increased firstly and then decreased as the concentration of NaCl and mixed salt increased. Low-concentration of NaCl or mixed salt could promote germination of seeds and growth of seedings. In5～15℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration NaCl or mixed salt was below0.9%or1.5%(P<0.05). Under15～25℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration of NaCl or mixed salt was below0.6%or1.2%(P<0.05). At25～35℃, seed germination, seedling length and seedling fresh weight were much higher than those of control when concentration NaCl or mixed salt was below0.6%or0.9%(P<0.05). Seed germination rate, seedling length and seedling fresh weight were higher in mixed salt than in NaCl (P<0.05). At15～25℃, seed germination, seedling length and seedling fresh weight in mixed salt were23%,27%and28%higher than those in NaCl. This experiment showed that NaCl was more harmful than mixed salt solution to accumulated germination, seedling length and seedling fresh weight of H.Strobilaceum. The accumulated seed germination rates, height and fresh weight of seedlings under NaCl treatment were higher than under mixed salt treatment. As salt concentration increased, seed germination was delayed under5～15℃,15～25℃,25～35℃conditions. So concentration of NaCl and mixed salt was one of key factors to seed germination. All that may lead to different results of salt-tolerant behaviors of plants between natural environments and laboratories. Potassium ion, calcium ion and silicon ion in mixed salt solutions may improve seed germination and promoted early growth of H. strobilaceum seedlings.Under NaCl and mixed salt stress, after treatment of20d and60d, K+/Na+and the content of K+, MDA, Pro and antioxidant enzyme activities (SOD, POD, CAT) of H. Strobilaceum first rose and then declined with increased concentration, which were highest when salt concentration was2.7%and then decreased. The content of Na+, K+MDA, Pro all gradually increased, but contents of MDA and antioxidant enzyme activities gradually decreased as prolonged growth time. At the concentration of NaCl and mixed salt, the content of K+, Pro and antioxidant enzyme activities were higher in mixed salt than in NaCl, however, content of MDA was lower in mixed salt than in NaCl. Potassium ion, calcium ion and silicon ion (mixed-irons) from mixed salt may decrease the content of MDA, and enhanced K+, K+/Na+, Pro, GB and antioxidant enzyme activities. The effect potassium ion, calcium ion and silicon ionalleviates salt deficit of H. strobilaceum by preventing the oxidative membrance damge and may be associated with plant osmotic adjustment.In this work we isolated cDNAs of betaine aldehyde dehydrogenase from H. Strobilaceum. We named the betaine aldehyde dehydrogenase of H. strobilaceum was HsBADH. The HsBADH DNA contained a completed open reading frame of1848bp, coding for a polypepitde of549amino acids. GenBank landing number is EF4382. we would forecast that HsBADH molecular weight of protein was6.46KD, isoelectric point of9.26. It included Alanine3.75%, Glutamic3.03%, Glycine5.89%, Leucine7.14%and Valine4.82%. HsBADH protein peptide contains26.12%of the alpha helix structure,27.01%of the extension,36.14%of no rules of chain curly. The homology of H. Strobilaceum are90%of Halostachys Caspica, and53%of Rice Latin. So the equivalent of plant evolution degree is similar in the plants evolving process.The study could provide primary reference for mixed salt tolerance of H. strobilaceum. We are looking forword to getting new way and method to salt resistance of plant. The HsBADH gene would enrich the resources of salt resistance of plant. It is very important to improve salt tolerance in plants by making good use of gene resoureces of the species.更多还原