海滨锦葵盐胁迫转录组分析和抗盐基因研究

发布时间：2017-12-31 02:19

本文关键词：海滨锦葵盐胁迫转录组分析和抗盐基因研究　出处：《中国科学院烟台海岸带研究所》2016年博士论文　论文类型：学位论文

【摘要】：随着全球土地盐渍化程度的不断加深和盐渍化土地面积的不断扩张,土壤盐胁迫发展成为制约粮食产量和质量的主要限制因素。盐胁迫能够抑制植物的光合作用,造成植物体生长发育的迟缓和停滞。同时还能对植物体造成渗透胁迫和离子毒害,导致植物体水分平衡的失调和各种代谢活动的紊乱。面对盐胁迫对植物体多方面的影响,植物特别是耐盐植物进化出了多种不同层次的响应机制和策略。揭示这些机制对于人类充分利用植物资源、保护与生态恢复人类空间环境、增加与改善食品营养以及充分利用盐渍土资源实现现代农业可持续发展都具有重大理论与实践意义。本论文以耐盐植物海滨锦葵为研究材料,围绕海滨锦葵在盐胁迫下的应答响应分子机理展开研究工作。针对海滨锦葵超强的耐盐特性,研究海滨锦葵的抗盐机制和关键的抗盐基因,并对抗盐基因的功能进行了深入研究。(1)对海滨锦葵幼苗进行盐胁迫处理、取材、提取RNA、高通量转录组测序。通过对3个样品文库的测序获得了大量海滨锦葵的转录本信息,每个样品文库均有多达6千万个转录本被测序,总测序长度达170多亿个碱基。通过不同文库之间的比较分析发现,盐胁迫诱导了海滨锦葵数千甚至上万个转录本的改变。借助已有物种数据库的数据信息,对海滨锦葵转录组数据分析发现,盐胁迫下海滨锦葵生长发育和光合途径相关基因被明显抑制,而编码离子通道、载体蛋白的基因发生明显上调。通过KEGG数据库比对分析发现,淀粉、蔗糖分解代谢,氨基酸、脂肪代谢以及激素代谢和信号传导相关基因发生明显上调。最后qPCR技术对转录组结果的验证,证明结果是准确可靠的。(2)通过不同测序文库之间的比较分析,筛选出海滨锦葵对盐胁迫响应明显的基因。进一步利用分子生物学技术克隆获得了两个盐胁迫响应基因的全长,并分别命名为KvLEA和KvTIP3。对KvLEA基因编码蛋白的分析发现,该蛋白具有许多已报道LEA蛋白的特性,与拟南芥中的两个LEA蛋白基因At2g36640和At3g53040具有很高的同源性。组织表达模式显示,该基因在海滨锦葵根中具有较高的表达水平。另外KvLEA基因的表达能够被盐胁迫明显诱导,随着盐离子浓度的升高表达水平逐渐递增。同时该基因还受ABA、高温和干旱的诱导表达。因此,该基因与海滨锦葵的抗逆性密切相关。对KvTIP3基因的分析表明,其编码了一个水通道蛋白,分子量的大小,NPA基序以及保守的跨膜结构域均与典型水通道蛋白的特征相一致。组织表达模式显示,其编码了海滨锦葵的一个根部特异的水通道蛋白。同时非生物胁迫下的表达模式显示,其不仅参与了海滨锦葵对盐胁迫的响应,同时低温和ABA处理也均能诱导其表达,是海滨锦葵一个重要的抗逆基因。(3)KvHSP70是从海滨锦葵中分离获得的另一个盐胁迫响应基因,生物信息学分析表明该基因编码了一个胞质定位的热激蛋白,在氨基酸序列上与其它物种中的HSP70蛋白具有高达96%的相似性。洋葱表皮细胞瞬时转化实验证明,该蛋白定位于细胞的胞质中。表达分析结果表明,它不仅对盐胁迫有响应,对高温和ABA也都具有响应。利用基因工程技术对该基因的生物学功能研究发现,其在烟草中的表达增强了转基因烟草盐胁迫下各项生理生化指标。证明该基因具有提高植物抵抗高盐等非生物胁迫的生物学功能。
[Abstract]:With the continuous expansion of the global land salinization degree deepening and salinization land area, soil salt stress development has become the main restrict factors of grain yield and quality. Salt stress inhibited the photosynthesis of plants, resulting in plant growth retardation and stagnation. But also the plant caused by osmotic stress and ion toxicity, resulting in water the plant body balance disorders and metabolic activity disorder. In the face of the effect of salt stress on plants in many aspects, especially in plant salt tolerant plants have evolved a variety of response mechanisms and Strategies of different levels. These reveal the mechanism for the human to make full use of plant resources, protection and restoration of human environment, increase and improve food nutrition and make full use of the resources of saline soil is of great theoretical and practical significance to realize the sustainable development of modern agriculture. In this paper, salt tolerance Plant kostelezkyavirginica as research material, on the response of Kosteletzkya virginica under salt stress response mechanism to start the research. Strong salt tolerance for virginica, salt resistance mechanism and the key of kosteletzkyavirginica salt resistant gene, and the salt resistance gene function were studied. (1) of Kosteletzkya virginica seedlings for treatment, salt stress were extracted from RNA, high-throughput transcriptome sequencing. The sequencing of 3 samples obtained a library of transcription of kosteletzkyavirginica this information, each sample library has as many as 60 million transcripts were sequenced, the total sequencing length of about 17000000000 nucleotides. Through the comparison between different library analysis discovery, salt stress induced kosteletzkyavirginica thousands or even tens of thousands of transcripts. The change of the existing species database data of Kosteletzkya virginica transcriptome data analysis found that the salt sea Jin Bin Sunflower Growth and significant inhibition of photosynthetic pathway related genes, and encoding ion channels, carrier protein gene was up-regulated by KEGG. Database analysis found that starch, sucrose metabolism, amino acid, fat metabolism and hormone metabolism and signal transduction related genes were up-regulated. To verify the final qPCR on the transcriptome results the results are accurate and reliable. (2) through the analysis and comparison between different sequencing library and screened response were gene of Kosteletzkya virginica under salt stress. Further cloning obtained two salt stress response gene sequences by molecular biology technology, and were named to analysis of the KvLEA gene encoding protein KvLEA and KvTIP3., the protein LEA protein has been reported to have many characteristics, has high homology with the Arabidopsis LEA protein two gene At2g36640 and At3g53040 group. Show the fabric expression pattern, the gene has a higher expression level in Kosteletzkya virginica root. KvLEA gene expression also can be significantly induced by salt stress, salt ion concentration increases with the expression level increased gradually. At the same time the gene is also affected by ABA expression induced by high temperature and drought. Therefore, the gene and the resistance of kosteletzkyavirginica closely related. Analysis of KvTIP3 gene showed that the encoding of a water channel protein, molecular weight, consistent with characteristics of NPA motifs and conserved transmembrane domains with typical aquaporins. Tissue expression pattern showed that the water channel protein encoding a specific root of kosteletzkyavirginica at the same time. Non display expression patterns of biological stress, which not only in kosteletzkyavirginica in response to salt stress, while low temperature and ABA treatment can induce the expression of kosteletzkyavirginica, is an important base of resistance . (3) KvHSP70 was isolated and another salt obtained from kosteletzkyavirginica in stress response genes, bioinformatics analysis showed that the gene encoding heat shock protein a cytoplasmic localization, and other species in the amino acid sequence of HSP70 protein with similarity as high as 96%. The instantaneous onion epidermal cells the transformation experiment proved that the protein located in the cytoplasm. The expression analysis results show that it is not only the salt stress response, both in response to high temperature and ABA. The study of biological function by gene engineering technology to the discovery of the gene, its expression in tobacco enhances the physiological and biochemical indexes of the transgenic tobacco salt stress. The gene can improve the biological function of high salt and other abiotic stress resistance in plants.

【学位授予单位】：中国科学院烟台海岸带研究所
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q945.78

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