海滨锦葵盐胁迫转录组分析和抗盐基因研究
汤晓丽
学位类型博士
导师邵宏波
2016-05-16
学位授予单位中国科学院大学
学位授予地点北京
关键词海滨锦葵 盐胁迫 转录组 Kvlea Kvtip3 Kvhsp70
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KvLEAKvTIP3
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。
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。
其他摘要
With the continuous expansion of saline soil and severity of soil salinization, salinity is becoming the most important limiting factor for food quantity and quality all over the world. Salt stress can restrain photosynthesis and lead to growth retardation and stagnation to plant. In addition, the excess ions in plant can bring about osmotic stress and ion toxicity, resulting in water imbalance and metabolic disorders. In face of the adverse effects, plant especially the salt-tolertant plant has developed various efficient regulation mechanisms and strategies. To reveal these mechanisms and strategies has great significance in theory and practice, which could help to take full advantage of plant resources, protect and restore ecology environment, increase and improve food nutrition as well as promote the sustainable development of modern agriculture.
In this paper we selected Kosteletzkya virginica as study material to carry out our salt stress research. Our work focused on the molecular mechanisms of K. virginica under salt stress. On account of its resistance to high salinity, we studied the salt resistance mechanism and salt-risisting gene in K. virginica. We also studied the gene function futher at the same time.
(1) High-throughput transcriptome sequencing was performed after the seedlings were stressed, sampled and extracted RNA. Large amount of data was abtained and there were as many as more than sixty million transcripts in every single library. In addition, the total length of the sequencing reached up to seventeen billion. The comparative analysis among different libraries revealed that the expressions of thousands of genes were altered in K. virginica after salt stress.
Basing on the existing data from other species, the genes with changes in expression were given classifications, predictions of protein function and analysis of participated metabolic pathways. The results suggested that the genes involved in growth,development and photosynthesis were inhibited significantly, while the genes encoding ion channels and transporters were up-regulated. Expression analysis of the genes with KEGG database indicated that most of the genes involved in starch and sucrose decomposition reactions, amino acid and fatty acid metabolism as well as phytohormone metabolism and signal transduction were increased. Finally, qPCR was performed to verify the results of RNA-Seq and the result showed the outcome of RNA-Seq was credible.
(2) The genes responding to salt stress were screened out by differential analysis among libraries. The full sequences of two genes were acquired by molecular cloning and they were named KvLEA and KvTIP3, respectively.
The analysis on protein encoded by KvLEA discovered that this protein possessed many common properties belonging to LEA proteins. It showed high similarity to two LEA genes At2g36640 and At3g53040 in Arabidopsis. The tissue expression pattern displayed that this gene had a higher expression in root. This gene was induced by salt stress and in addition, with the increase of NaCl concentration, the expression of the gene increased synchronously. Meanwhile, research also showed that high temperature, chilling and ABA could induce the expression of this gene, too. Thus, this gene should have close relationship with abiotic stress resistance in K. virginica.  
The analysis of KvTIP3 revealed that it coded an aquaporin. The molecular weight, conserved functional motifs and transmembrane domains of this protein were the same with the typical features of aquaporins. The tissue expression profiles indicated that this protein should be a root-specific aquaporin. Moreover, this gene was not noly induced by salt stress, but it also responsed to chilling and ABA treatment. Thus, it was an important stress resistance gene of K. virginica.
(3) KvHSP70 gene is another gene isolated from K. virginica. Bioinformatic analysis displayed that it encoded a HSP70 located in cytoplasm. The predicted protein of this gene had up to 96% similarity with other known HSP70s. The transient transformation in onion epidermal cells proved that this protein situated in cytoplasm. The expression analysis discovered that it responsed to salt, heat as well as ABA. Gene engineering technology was applied to study its biological function. The transgenic tobacco expressing this gene was endowed with enhanced salt stress resistance. A number of physiological and biochemical parameters performed better than control under salt stress. Therefore, KvHSP70 was capable of advancing stress resistance of plant.   
文献类型学位论文
条目标识符http://ir.yic.ac.cn/handle/133337/13838
专题中国科学院烟台海岸带研究所知识产出_学位论文
作者单位中国科学院烟台海岸带研究所
第一作者单位中国科学院烟台海岸带研究所
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汤晓丽. 海滨锦葵盐胁迫转录组分析和抗盐基因研究[D]. 北京. 中国科学院大学,2016.
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