小ERF转录因子参与调控拟南芥对ABA和盐胁迫的响应

发布时间：2018-07-26 08:23

【摘要】：乙烯(Ethylene)参与调控植物生长发育的多个方面,包括种子休眠的打破、幼苗生长调控、叶片脱落、花的开放及衰老和脱落、果实成熟、根瘤形成等;此外,乙烯也参与调控植物对病原菌侵染等生物胁迫,以及干旱、盐碱等非生物胁迫的响应。ERFs(Ethylene Response Factors)是植物中特有的转录因子,属于AP2(APETALA2)/ERF超家族。ERF家族中有4个小ERF蛋白,分别是ERF95、ERF96、ERF97和ERF98,这些蛋白序列高度相似,具有与AtERF1等其它ERFs的羧基端序列高度相似的DNA结合域,还都具有一个功能未知的结构域CMIX-1,但它们均不具备AtERF1等其它ERFs所具有的N末端转录因子活性区域。已有研究报道ERF95和ERF98参与了植物对非生物胁迫响应的调控,而ERF96和ERF97参与了植物对生物胁迫响应的调控。本文中则系统地研究了小ERFs在调控拟南芥对ABA响应中的作用。利用蛋白的氨基酸全序列的系统发育树分析结果显示ERF95、ERF96和ERF97聚为一簇,而ERF98与它们三个的关系比较远。qRT-PCR结果显示这些小ERFs的表达模式各不相同:ERF95、ERF97和ERF98在拟南芥植株的根、茎、叶、花以及成熟的种子中均有不同程度地表达,但ERF95在成熟的种子中表达量最高,ERF98在茎中表达量最高,而ERF97在花和种子中表达量都相对较高。ERF96也是在花和种子中表达量比较高,但在根中几乎检测不到ERF96的表达。蛋白亚细胞定位分析表明ERF95、ERF96、ERF97和ERF98均定位于细胞核中。叶肉细胞原生质体瞬时转染实验表明所有ERFs都具有转录激活活性,且EDLL基序是其转录激活活性所必需的。尽管三突变体erf95 erf96 erf97和erf96 erf97 erf98,及四突变体erf95 erf96erf97 erf98都与野生型的表型近似,但小ERFs的过表达拟南芥植株呈现出明显不同于野生型的表型。尤其是ERF95、ERF96和ERF98过表达拟南芥植株的莲座叶大小与野生型存在极显著的差异(p0.01),开花时间也明显延迟(p0.01)。ABA敏感性试验结果显示,拟南芥小ERFs过表达转基因植株对ABA敏感性增强,特别是ERF95、ERF96和ERF98过表达转基因植株,在ABA处理后,无论发芽率、绿苗率还是根的长度均远低于野生型。qRT-PCR结果表明,过表达ERFs转基因植株中ABA响应基因ABI5、ABF3、ABF4、RD29A、P5CS和COR15A的表达水平在ABA处理时显著高于Col野生型。生理实验结果表明,小ERFs过表达转基因植株失水速率显著低于Col野生型,气孔孔径小于Col野生型,而植株的瞬时叶片水分利用效率则要高于Col野生型植株。这些结果说明小ERFs均参与调控了拟南芥对ABA的响应。此外,我们还发现ERF96、ERF97也参与拟南芥对NaCl胁迫的响应。在NaCl处理条件下,ERF96和ERF97过表达转基因植株的幼苗生长状态均好于野生型,其地上部分鲜重显著高于野生型,侧根数目也显著多于野生型。NaCl处理可诱导Col野生型植株中RD29A、P5CS、COR15A、KIN1和RAB18等盐胁迫响应基因的表达,而过表达ERF96和ERF97转基因植株中这几个基因的表达水平均显著高于Col野生型。离子含量检测结果表明,小ERF96和ERF97过表达转基因植株中Na+含量相对较低,而K+含量较高,说明小ERFs可能通过影响Na+、K+代谢进而调控植物对盐胁迫的反应。
[Abstract]:Ethylene (Ethylene) participates in many aspects of plant growth and development, including seed dormancy break, seedling growth regulation, leaf abscission, flower opening and senescence and abscission, fruit ripening, nodule formation, etc. in addition, ethylene also participates in regulating plant biological stress such as pathogen infection, as well as response to abiotic stress such as drought, salt and other abiotic stresses,.ER Fs (Ethylene Response Factors) is a unique transcription factor in plants. It belongs to the AP2 (APETALA2) /ERF superfamily.ERF family with 4 small ERF proteins, which are ERF95, ERF96, ERF97, and respectively. These proteins are highly similar, and are highly similar to those of other carboxy terminal sequences. The domain CMIX-1, but they do not possess the N terminal transcriptional factor active regions of other ERFs, such as AtERF1 and other ERFs, has been reported that ERF95 and ERF98 participated in the regulation of plant response to abiotic stress, while ERF96 and ERF97 participated in the regulation of plant responses to biological stress. The effect of mustard on ABA response. The phylogenetic tree analysis of the whole sequence of amino acids showed that ERF95, ERF96 and ERF97 were clustered into one cluster, and the relationship between ERF98 and their three was far from.QRT-PCR results showed that the expression patterns of these small ERFs were different: ERF95, ERF97 and ERF98 in the roots, stems, leaves, flowers and maturity of Arabidopsis plants. The expression of ERF95 is the highest in the mature seeds, but the highest expression in the mature seeds, the highest expression of ERF98 in the stem, while the expression of ERF97 in the flowers and seeds is relatively high.ERF96 is also high in the flowers and seeds, but in the root, almost no expression of ERF96 is detected. Protein subcellular localization analysis indicates ERF95, E RF96, ERF97 and ERF98 were both located in the nucleus. The transient transfection experiment of mesophyll protoplasts showed that all ERFs had transcriptional activation activity, and the EDLL motif was necessary for its transcriptional activation activity. Although the three mutant erf95 erf96 erf97 and erf96 erf97 erf98, and the four mutant erf95, are all closely related to the wild type phenotype. However, the overexpression of Arabidopsis plants was obviously different from the wild type, especially in ERF95. Especially, there was a significant difference between the size of the lotus leaf and the wild type of ERF95, ERF96 and ERF98 (P0.01), and the flowering time was obviously delayed (P0.01).ABA sensitivity test results showed that the transgene of Arabidopsis small ERFs was overexpressed. The sensitivity of the plant to ABA was enhanced, especially ERF95, ERF96 and ERF98 overexpressed transgenic plants. After ABA treatment, the germination rate, the green seedling rate and the root length were far lower than those of the wild type.QRT-PCR. The expression level of ABA responsive genes in the over expressed ERFs transgenic plants was significantly higher than that of the ABA response genes. The results of physiological experiments showed that the water loss rate of the transgenic plants with small ERFs overexpression was significantly lower than that of the Col wild type, and the pore size of the plant was less than that of the wild type of Col, and the instantaneous leaf water use efficiency of the plant was higher than that of the Col wild type. These results indicated that the small ERFs was all involved in the response to ABA in Arabidopsis thaliana. In addition, we were able to regulate the response of the transgenic plants to the wild type of the Col. It was also found that ERF96 and ERF97 were also involved in the response of Arabidopsis to NaCl stress. Under the condition of NaCl treatment, the seedling growth of ERF96 and ERF97 over expressed transgenic plants was better than the wild type, and the fresh weight on the ground part was significantly higher than that of the wild type, and the number of the lateral roots was significantly more than the wild type.NaCl treatment could induce RD29A, P5CS, COR15A in the wild type Col. The expression of salt stress response genes, such as KIN1 and RAB18, and the expression level of these genes in ERF96 and ERF97 transgenic plants were significantly higher than those of Col wild type. The results of ion content detection showed that the content of Na+ in the small ERF96 and ERF97 overexpressed transgenic plants was relatively low, and the K+ content was higher, indicating that small ERFs may be influenced by Na+, K+. Metabolism then regulates plant responses to salt stress.
【学位授予单位】：东北师范大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q943.2

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