花生铁螯合酶1基因的克隆与及其在盐胁迫下的功能分析

发布时间：2018-08-01 08:19

【摘要】：花生(Arachis hypogaea L)是我国乃至世界上重要的油料作物之一,因含有丰富的油脂和蛋白质而具有极高的营养价值以及经济价值。工业污染加剧以及农业灌溉措施和化肥使用量的不当等诸多原因,造成次生盐碱地面积日益扩大,已经并将继续威胁旱作花生的种植区,严重影响了花生的产量和品质。因此,如何保证花生的稳产、高产和优质成为育种的重大目标和技术难题。为了实现这一宏达的目标,加速花生品种的改良进程,研究者逐渐将研究方向转移至耐盐分子机理的研究,以期通过基因工程技术手段对现有品种进行遗传改良,选育出耐盐的花生新品种,为提高花生产量和品质提供实际应用的参考价值。铁螯合酶催化亚铁血红素的合成,为呼吸链中的复合物以及过氧化物酶提供辅基。研究表明,亚铁血红素很可能作为一种沟通质体和细胞核的信号分子,参与干旱等逆境胁迫的响应。本课题意在克隆花生铁螯合酶1基因并研究其在盐胁迫下的功能。我们以拟南芥铁螫合酶1基因(AtFC1, GI:145358447)为探针,比对搜索NCBI中花生EST数据库获得两条同源的EST序列并予以拼接,根据拼接后的序列设计引物,以盐胁迫处理的花生叶片为试材,利用RT-PCR和RACE技术克隆获得花生铁螯合酶1基因,对其进行了生物信息学分析和功能预测,通过在拟南芥原生质体中瞬时表达其与绿色荧光蛋白(GFP)的融合基因确定其在细胞中的位置,并通过在烟草中过表达该基因研究其在盐胁迫响应中的功能。研究结果如下：(1)从盐胁迫处理的花生叶片中克隆获得完整的花生铁螯合酶1基因的cDNA序列,命名为AhFC1,已在Genbank注册为KU560625。该基因cDNA序列全长为1965 bp,5’端非翻译区为157 bp,开放阅读框长1449 bp,3’端非翻译区为359 bp,编码的蛋白质含482个氨基酸。(2)采用多种方法对其编码蛋白进行结构域预测,发现AhFC1蛋白具有高度保守的铁螫合酶N端和C端结构域,在这两个结构域上分布有多个酶活性位点,在其C端还有一个跨膜区,属于不依赖于ATP的螯合酶II超家族成员,位于叶绿体区室中。在进化上,AhFC1蛋白和黄瓜的铁螯合酶1亲缘关系最近。(3)表达模式分析表明,干旱胁迫处理能够迅速诱导AhFCl的表达量上升,6h后即可达到最大值,随后开始缓慢降低。相对于干旱胁迫处理,盐胁迫诱导的AhFCl呈缓慢上升趋势,达到峰值所需的时间也相对延后,至12h才达到表达峰值,然后开始下降。(4)盐胁迫下,转AhFC1基因烟草的萌发率显著高于野生型烟草。虽然转基因烟草和野生型烟草叶片中的亚铁血红素均有所下降,但转基因烟草的亚铁血红素含量显著高于野生型。转基因烟草叶片的ROS含量、MDA、电导率明显低于野生型植株,而以亚铁血红素为辅基的CAT、APX等过氧化物酶活性较野生型烟草显著升高。尽管转基因烟草与野生型烟草叶片中的SOD活性均受到盐胁迫诱导升高,但是二者并无明显差异。以上结果表明,过量表达AhFCl基因提高了转基因烟草的亚铁血红素含量和ROS清除能力,缓解了由盐分引起的氧化胁迫,从而增强了转基因烟草的耐盐性。
[Abstract]:Arachis hypogaea L is one of the most important oil crops in our country and in the world. Because of its rich oil and protein, it has high nutritional value and economic value. The aggravation of industrial pollution and the improper use of agricultural irrigation measures and chemical fertilizer have caused the increase of the area of secondary saline and alkali land. The crop yield and quality of peanuts are seriously affected by the continued threat of peanut planting areas. Therefore, how to ensure stable yield, high yield and high quality of peanuts has become a major goal and technical problem for breeding. In order to achieve this goal and accelerate the improvement process of peanut varieties, researchers have gradually shifted the research direction to the molecular mechanism of salt tolerance. The study is aimed at genetic improvement of existing varieties by genetic engineering techniques and selecting new peanut varieties with salt tolerance to provide practical application for improving the yield and quality of peanut. Iron chelating enzyme catalyzes the synthesis of heme, which provides an auxiliary basis for the complex in the respiratory chain and the peroxidase. Hemoglobin is likely to be a signaling molecule to communicate plastids and nuclei and participate in drought stress response. This lesson is intended to clone peanut iron chelase 1 gene and study its function under salt stress. We use the Arabidopsis iron chelase 1 gene (AtFC1, GI: 145358447) as a probe to compare the peanut EST database in the search NCBI Two homologous EST sequences were obtained and the primers were designed according to the sequence after the splicing. The peanut leaves treated with salt stress were used as the test materials. The peanut iron chelating enzyme 1 gene was cloned by RT-PCR and RACE technology, and the bioinformatics analysis and function prediction were carried out. The gene was transiently expressed in the Arabidopsis protoplast. The fusion gene of fluorescent protein (GFP) determines its location in the cell and studies its function in salt stress response by overexpressing the gene in tobacco. The results are as follows: (1) a complete cDNA sequence of peanut iron chelating enzyme was cloned from the peanut leaves treated with salt stress, named AhFC1, which has been registered in Genbank KU560625. the full length of cDNA sequence is 1965 BP, the 5 'end non translation region is 157 BP, the open reading frame length is 1449 BP, the 3' end non translation region is 359 BP, the encoded protein contains 482 amino acids. (2) a variety of methods are used to predict the encoding protein of the protein. It is found that AhFC1 egg white has a highly conserved iron chelase N end and C terminal domain. There are several enzyme active sites on the two domains, and there is a transmembrane region at its C end, belonging to the II superfamily of the chelating enzyme, which is not dependent on ATP, and in the chloroplast compartment. In evolution, the relationship between the AhFC1 protein and the iron chelating enzyme of cucumber is closest. (3) the expression model analysis shows that the drought stress treatment can induce AhFC quickly. The expression of L increased, and then reached the maximum after 6h, and then began to decrease slowly. Compared with drought stress treatment, the AhFCl induced by salt stress was slowly rising, and the time needed to reach the peak was also relatively delayed, and the peak value reached to 12h, and then began to decline. (4) the germination rate of transgenic tobacco was significantly higher than that in the wild under salt stress. The heme of transgenic tobacco and wild type tobacco were decreased, but the content of heme of transgenic tobacco was significantly higher than that of wild type. The ROS content of transgenic tobacco leaves, MDA, electrical conductivity were significantly lower than that of wild type plants, while the activity of CAT, APX and other peroxidase activities in the heme supplemented group Although the SOD activity in transgenic tobacco and wild type tobacco leaves increased by salt stress, there was no significant difference between the two. The above results showed that overexpression of AhFCl gene increased the heme content and ROS scavenging ability of transgenic tobacco and alleviated the oxidative stress caused by salt. It is forced to enhance the salt tolerance of transgenic tobacco.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.2;Q943.2

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