拟南芥锌指转录因子PLATZ5在盐胁迫响应中的功能研究

发布时间：2018-05-19 12:17

本文选题：PLATZ5 + SOS3　；参考：《山东农业大学》2017年硕士论文

【摘要】：土壤盐渍化是影响当今农作物产量和质量的一个重要因素。土壤盐分浓度过高会导致植物代谢紊乱,影响植物对必需元素的吸收,造成渗透胁迫及氧化胁迫等次生胁迫,进而影响植物正常的生长发育。如何提高植物的耐盐性已成为农业发展急需解决的问题。PLATZ(Plant A/T rich sequence-and zinc-binding protein)是一类植物特有的转录因子,2002年豌豆PLATZ1首次被报道能非特异地结合富含A/T碱基的序列并发挥转录抑制的作用。拟南芥中共有12个PLATZ成员,本研究对拟南芥AtPLATZ5进行了表达分析和功能鉴定,结果如下:(1)利用半定量RT-PCR和GUS染色的方法检测了AtPLATZ5的组织表达模式,结果表明AtPLATZ5在各个组织中均有表达,且表达差异不大。RT-PCR和qRT-PCR的结果同时证明了AtPLATZ5的表达量受盐胁迫诱导。(2)利用瞬时转化烟草的方法对PLATZ5的亚细胞定位进行了分析,结果表明PLATZ5除在细胞核中表达外,在细胞质中也有表达,这说明PLATZ5可能发挥多种功能。(3)利用双荧光素酶报告系统对PLATZ5的转录活性进行了分析,结果表明PLATZ5具有转录抑制活性。(4)对AtPLATZ5超表达株系进行100mM和150mM NaCl处理,发现AtPLATZ5超表达株系地上部鲜重比对照分别降低了30%与50%,而根部的鲜重比对照分别降低了20%与18%,这表明盐胁迫对AtPLATZ5超表达株系地上部抑制更明显。提高NaCl浓度到200mM时,超表达株系叶片白化,存活率显著低于野生型。这说明AtPLATZ5负调控幼苗对盐胁迫的抗性。AtPLATZ5超表达株系地上部也会对100mM NaNO3、100mM KCl和100mM KNO3胁迫处理表现出微弱敏感的表型,同时超表达株系对10mM LiCl超敏感而对甘露醇不敏感。以上结果表明AtPLATZ5主要响应离子胁迫。(5)插入到外显子区的platz5突变体对盐胁迫处理没有表型,表明PLATZ家族成员可能存在着功能冗余。(6)钠离子探针染色结果显示超表达株系在受到盐胁迫时根与叶片部积累的钠离子增多,表明超表达株系体内钠离子的吸收转运受到了影响。(7)qRT-PCR分析盐胁迫后超表达株系体内信号通路marker基因的变化,发现SOS途径的SOS3/CBL4及其同源基因CBL10表达量下调,而染色质免疫共沉淀实验证明PLATZ5能靶向SOS3启动子,这表明PLATZ5通过抑制SOS3的表达来响应盐胁迫。
[Abstract]:Soil salinization is an important factor affecting crop yield and quality. The high concentration of soil salt will lead to the disorder of plant metabolism, which will affect the absorption of essential elements, osmotic stress, oxidative stress and other secondary stresses, and then affect the normal growth and development of plants. How to improve plant salt tolerance has become an urgent problem in agricultural development. The plant A / T rich sequence-and zinc-binding protein is a kind of plant-specific transcription factor. In 2002, pea PLATZ1 was first reported to be able to combine the sequence with rich in A / T base and play an important role. Transcriptional inhibition. There are 12 PLATZ members in Arabidopsis thaliana. In this study, the expression and function of Arabidopsis AtPLATZ5 were analyzed. The results are as follows: 1) the expression pattern of AtPLATZ5 was detected by semi-quantitative RT-PCR and GUS staining. The results showed that AtPLATZ5 was expressed in all tissues, and the difference was not significant. The results of RT-PCR and qRT-PCR also proved that the expression of AtPLATZ5 was induced by salt stress. (2) the subcellular localization of PLATZ5 was analyzed by transient transformation of tobacco. The results showed that PLATZ5 was expressed not only in the nucleus, but also in the cytoplasm, which suggested that PLATZ5 might play a variety of functions.) the transcriptional activity of PLATZ5 was analyzed by using double luciferase report system. The results showed that PLATZ5 had transcriptional inhibitory activity. 4) 100mM and 150mM NaCl were used to treat AtPLATZ5 overexpression lines. The results showed that the fresh weight of AtPLATZ5 superexpression lines was 30% and 50% lower than that of the control, while the fresh weight of roots was 20% and 18% lower than that of the control, respectively, which indicated that salt stress had more obvious inhibition on the shoot of AtPLATZ5 overexpressed lines. When the concentration of NaCl was increased to 200mM, the overexpressed lines were albino, and the survival rate was significantly lower than that of wild type. This indicated that the shoots of the seedlings with negative AtPLATZ5 regulation showed weak phenotypic sensitivity to 100mM NaNO3100mM KCl and 100mM KNO3 stress, but the overexpressed lines were not sensitive to mannitol but hypersensitive to 10mM LiCl. The results indicated that AtPLATZ5 was mainly responsive to ion stress. The platz5 mutant inserted into the exon region had no phenotype under salt stress. The results indicated that the PLATZ family members might have functional redundancy. 6) the results of sodium probe staining showed that the accumulation of sodium ions in roots and leaves of the overexpression lines increased under salt stress. The results showed that the absorption and transport of sodium ions in the overexpression lines were affected by QRT-PCR. The changes of marker gene in the signaling pathway of the overexpression lines after salt stress were analyzed. It was found that the expression of SOS3/CBL4 and its homologous gene CBL10 in the SOS pathway was down-regulated. The chromatin immunoprecipitation assay showed that PLATZ5 could target the SOS3 promoter, which suggested that PLATZ5 responded to salt stress by inhibiting the expression of SOS3.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q943.2

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