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胡杨在盐碱胁迫下的生理响应及其钾离子转运基因HAK克隆和功能分析
张选
学位类型硕士
导师姚银安
2015
学位授予单位中国科学院大学
学位授予地点北京
学位专业植物学
关键词胡杨 盐碱胁迫 生理响应 钾离子转运基因pehak 基因克隆 酵母功能验证
摘要西北荒漠地区土壤盐碱含量较高,而土壤盐渍化及钾营养匮乏已成为影响植物正常生长发育的重要因素。盐渍化对植物的伤害主要是离子毒害,植物体内离子平衡调节成为植物抗盐的重要策略。因此,与离子调节相关的转运蛋白正在被广泛关注,尤其是HAK(high-affinity potassium transporter) 类蛋白正受到研究者们越来越多的重视,其功能也逐渐被揭示。研究表明HAK 类蛋白在调节植物细胞内稳定的K+/Na+ 比,K+ 、Na+ 吸收及K+ 转运等起着重要作用。 本研究以典型的盐生植物胡杨为实验材料,通过对新疆杨(Populus bolleana)和胡杨(Populus euphratica)进行盐碱处理(土壤 pH 8.5-9.0, 100 mM Na+),以新疆杨为对照,研究胡杨不同成熟度叶片的离子分配、生理响应以及钠钾转运相关基因的表达分析。同时对11个HAK基因盐胁迫下在三种杨树根中的表达分析,以新疆杨和俄罗斯杨作为对照,筛选出胡杨中差异表达钾离子转运基因PeHAK3与PeHAK8进行克隆,并构建酵母转化重组质粒,对其转化酵母进行功能验证。以期为揭示胡杨的耐盐碱机制提供理论依据,以及培育在低钾、盐渍化土壤上生长的作物新品种提供优良的功能基因资源。 获得以下研究结果: (1) 胡杨在盐碱胁迫下的生理响应 通过对胡杨(P. euphratica)在盐碱胁迫下的生理响应,以新疆杨(P. bolleana)为对照,研究发现,在碱胁迫下两种杨树均将过多Na+分配到成熟老叶中,而且胡杨幼叶和老叶中的Na+ 含量均高于新疆杨的幼叶和老叶,而胡杨老叶中的K+ 含量也大幅高于新疆杨老叶中的K+ 含量。且胡杨幼叶和老叶中的Na+/K+ 比均高于新疆杨幼叶和老叶中的Na+/K+ 比。而盐碱胁迫下,二价离子(Ca2+、Mg2+)含量在新疆杨的幼叶和老叶中均降低,但在胡杨的幼叶和老叶中均升高。两种杨树这些指标的差异表明:胡杨较新疆杨更耐盐碱胁迫。研究表明这种有利于幼叶的离子分配策略是两种杨树抵抗盐碱胁迫的重要生存策略,对于其在荒漠地区的生存具有重要意义。 (2) 200 mM NaCl盐胁迫下11个HAK基因在胡杨根部的表达模式分析 通过实时荧光定量qRT-PCR对盐胁迫下11个HAK基因在三种杨树根部的表达模式分析发现,11个基因均对盐胁迫有表达量上的响应,且在三种杨树中的表达响应不相同。以新疆杨和俄罗斯杨为对照,筛选出胡杨中的差异表达基因PeHAK3和PeHAK8进行进一步的深入研究。 (3) 胡杨PeHAK3、PeHAK8的克隆及其序列的生物信息学分析 利用电子克隆和RT-PCR技术克隆得到胡杨PeHAK3、PeHAK8包含完整ORF的cDNA,分别得到全长为2343 bp,编码780个氨基酸;2379 bp,编码792个氨基酸。生物信息学及分子系统进化树分析表明,可能在胡杨的耐盐机制中发挥着独特的功能。 (4) 转化酵母进行功能验证 重组质粒PeHAK3-pYES2.0、PeHAK8-pYES2.0转化酵母突变菌株CY162和G19,以期对PeHAK3、PeHAK8转运蛋白进行功能验证。结果表明,PeHAK8介导低亲和K+ 吸收,同时PeHAK8也介导Na+ 的吸收,此结果进一步证实了PeHAK8转运蛋白调节K+/Na+平衡。
其他摘要The content of salt and alkali in soil in northwest desert region of China was high. Salinity and potassium shortage affected plant growth and development, and in which ion toxicity of salinity is the main harmful factor to plant. So it is essential to regulate the ion homeostasis for enhancing the salt tolerance of plant. Therefore, ion transporter proteins are widely concerned, especially high affinity K+ transport proteins (HAK), and its function was also gradually found. Findings showed that HAK played an impotrant role in regulating osmotic pressure, K+/Na+ ratio, and transferring potassium. The experiment took halophyte P. euphratica as test organism. To investigate the salt-alkali tolerance mechanism of P. euphratica, taken Populus bolleana as control, we analyzed the ion distribution, physiological responses and the expression of sodium-potassium transport genes in different maturity leaves of P. euphratica, which grown with soil pH 8.5-9.0 subjected to 100 mM Na+. And Real-time RT-PCR analyses the expression mode of eleven HAKs under 200 mM salt stress in roots of P. euphratica, P. bolleana and P. russkii. Taken P. bolleana and P. russkii as control, screening the differential expression gene PeHAK3 and PeHAK8 in P. euphratica, cloned them and made recombiant plasmid for yeast transformation and function analysis. The study aimed to shed light upon understanding the resisiting salt-alkali mechanism and provide good gene resources for new crop varieties. The findings as above: (1) Physiological response of P. euphratica to salt-alkali stress Taken P. bolleana as control, we analyses the physiological response of P. euphratica under salt-alkali stress. Findings showed that Na+ was mainly allocated in old leaves in two populus species, and Na+ contents in young and old leaves in P. euphratica were higher than those in P. bolleana. Moreover, the content of K+ in old leaves in P. euphratica was higher than that in P. bolleana. The Na+/K+ ratio in young or old leaves in P. euphratica was higher than that in P. bolleana. Under alkali stress, the contents of Ca2+ or Mg2+ in young and old leaves in P. bolleana decreased, but it increased in P. euphratica. Above all, P. euphratica was more resistant to salt-alkali stress than P. bolleana. The study indicated that the ion allocation of P. bolleana being in favor of young leaves was an important survival strategy to resist salt-alkali stress and it was of great importance for its survival in desert region. (2) The eleven HAK expression mode analysis in root of P. euphratica under 200 mM NaCl stress Real-time RT-PCR showed that eleven of HAKs made reponse to salt stress in roots in P. euphratica, P. bolleana and. This illustrated that the eleven HAKs showed different expression responses in three populus species. Taken P. bolleana and P. russkii as control, screening the differential expression gene PeHAK3 and PeHAK8 in P. euphratica and made further study for them. (3) The cloning and bioinformatics analysis of PeHAK3 and PeHAK8 We obtain PeHAK3 and PeHAK8 genes by electronic cloning and RT-PCR from P. euphratica. The sequences analysis of PeHAK3 and PeHAK8 indicated that the cDNA of PeHAK3 contained 2343 bp ORF, which encodes 780 amino acids; and the cDNA of PeHAK8 contained 2379 bp ORF, encoding 792 amino acids. The analysis of PeHAK3 and PeHAK8 bioinformatics and molecular phylogenetic tree showed that PeHAK3 and PeHAK8 might play important roles in salt tolerance mechanism of P. euphratica. (4) The transformation and function analysis into yeast mutant CY162 and G19 The ORF of PeHAK3 and PeHAK8 were constructed into pYES2.0. The combiant of PeHAK3-pYES2.0 and PeHAK8-pYES2.0 were transferred into the mutant yeast CY162 and G19 in order to analyse the function of PeHAK3 and PeHAK8. The results showed that PeHAK8 transporter mediated the absorption of K+ and Na+.
学科领域植物学
语种中文
文献类型学位论文
条目标识符http://ir.xjlas.org/handle/365004/14681
专题研究系统_荒漠环境研究室
作者单位中科院新疆生态与地理研究所
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张选. 胡杨在盐碱胁迫下的生理响应及其钾离子转运基因HAK克隆和功能分析[D]. 北京. 中国科学院大学,2015.
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