普通小麦胁迫相关蛋白基因TaSAP17的克隆与功能分析

发布时间：2018-03-05 21:09

本文选题：小麦(Tricticum　切入点：aestivum　出处：《西北农林科技大学》2017年硕士论文　论文类型：学位论文

【摘要】：小麦(Tricticum aestivum L.)是主要粮食作物,在粮食安全中占有举足轻重的地位。然而,随着全球气候变化以及不合理的灌溉导致土壤盐渍化的愈发严重,与其他作物一样,小麦的生长发育与生产都受到了一定的影响。利用生物技术等手段发掘优异抗逆基因资源,培育抗逆高产的小麦品种,是打破盐渍化制约的重要途径之一。胁迫相关蛋白(stress associated protein,SAP)基因是一类广泛存在于植物中的多基因家族,可以响应多种生物与非生物胁迫,在植物抗逆过程中发挥着重要的作用。然而,对于SAPs多基因家族在小麦中的相关信息还知之甚少。鉴于该家族在植物中的重要作用,论文利用隐马尔可夫模型检索到小麦中的SAP基因,以小麦品种旱选10号为材料扩增目标基因,通过实时定量PCR分析在胁迫处理下SAP基因的表达模式,筛选出显著响应逆境胁迫的基因TaSAP17,分析其功能并进行转基因功能验证,以期为小麦分子育种提供理论依据和基因资源。论文取得的主要研究结果如下:1.克隆了小麦胁迫相关蛋白基因TaSAP17。该基因包含两个外显子和一个内含子,具有SAP基因家族独特的结构域,两个保守的AN1和C2H2结构域,属于AN1/AN1型SAP基因。小麦TaSAP17基因高度保守,A、B、D三个基因组中的等位基因在cDNA水平上的相似度达98.35%,在氨基酸水平上的相似度达99.03%。2.TaSAP17-D在小麦生长的各个时期和组织中均有表达。TaSAP17-D在苗期叶片中的表达量最高,在茎秆节间中的表达量最低;TaSAP17-D响应高盐、外源ABA、低温和PEG胁迫处理,其中对高盐胁迫的响应最强,而对外源ABA、低温和PEG处理的响应较为微弱;在耐盐品种“泰山23”中TaSAP17-D的表达量整体上高于不耐盐品种“农大20074”,进一步说明TaSAP17-D响应高盐胁迫。亚细胞定位表明,TaSAP17-D在细胞核、细胞质和细胞膜中均有表达。3.TaSAP17-D增强转基因拟南芥的耐盐性。在高盐胁迫下,转基因拟南芥的存活率、细胞膜稳定性、游离脯氨酸含量、叶绿素含量均高于野生型(WT),渗透势、离体叶片失水速率低于WT。4.转录激活试验和酵母文库筛选。TaSAP17-D没有转录激活活性;通过筛选小麦全长cDNA酵母文库,得到七个可能与TaSAP17-D互作的候选蛋白。综合以上研究结果,TaSAP17-D能显著增强转基因拟南芥的耐盐性是改良农作物耐盐与抗旱性的重要候选基因。
[Abstract]:Wheat Tricticum aestivum L.is the main food crop and plays an important role in food security. However, as global climate change and irrational irrigation lead to soil salinization, like other crops, The growth, development and production of wheat have been affected to some extent. Stress associated protein (SAP) gene is one of the most important ways to break down the restriction of salinization. Stress associated protein (SAP) gene is a kind of polygene family that exists widely in plants and can respond to multiple biological and abiotic stresses. Play an important role in plant stress resistance. However, little is known about the SAPs polygene family in wheat. In this paper, the SAP gene in wheat was retrieved by hidden Markov model, and the target gene was amplified from wheat cultivar Hanxuan 10. The expression pattern of SAP gene was analyzed by real-time quantitative PCR analysis under stress. TaSAP17, a gene that was significantly responsive to stress, was screened, its function was analyzed and the transgenic function was verified. In order to provide theoretical basis and genetic resources for wheat molecular breeding, the main results obtained in this paper are as follows: 1. The wheat stress-related protein gene TaSAP17 was cloned. The gene contains two exons and one intron. There are two conserved domains of AN1 and C2H2, which are unique domain of SAP gene family. The alleles of the three genomes of wheat TaSAP17 gene highly conserved are 98.35 at cDNA level and 99.03 at amino acid level. 2. TaSAP17-D has a table in all stages and tissues of wheat growth. The expression of TaSAP17-D in seedling leaves was the highest. The lowest expression level of TaSAP17-D in stem Internode responded to high salt stress, exogenous ABA, low temperature and PEG stress, among which the response to high salt stress was the strongest, while to exogenous ABA, low temperature and PEG treatment was weak. The expression of TaSAP17-D in salt tolerant variety "Taishan 23" was higher than that in intolerant variety "Nongda 20074", which further indicated that TaSAP17-D was responsive to high salt stress. Subcellular localization showed that TaSAP17-D was in the nucleus. The expression of TaSAP17-D in cytoplasm and cell membrane enhanced the salt tolerance of transgenic Arabidopsis thaliana. Under high salt stress, the survival rate, cell membrane stability, free proline content and chlorophyll content of transgenic Arabidopsis thaliana were higher than that of wild type Arabidopsis thaliana, and the osmotic potential was higher than that of wild type Arabidopsis thaliana. The rate of water loss in leaves in vitro was lower than that in WT.4.Transcriptional activation test and yeast library screening. TaSAP17-D had no transcriptional activation activity. Seven candidate proteins which may interact with TaSAP17-D were obtained. TaSAP17-D can significantly enhance salt tolerance of transgenic Arabidopsis thaliana and is an important candidate gene for improving crop salt tolerance and drought resistance.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S512.1

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