【摘要】：乙烯(Ethylene)參與調(diào)控植物生長發(fā)育的多個方面,包括種子休眠的打破、幼苗生長調(diào)控、葉片脫落、花的開放及衰老和脫落、果實成熟、根瘤形成等;此外,乙烯也參與調(diào)控植物對病原菌侵染等生物脅迫,以及干旱、鹽堿等非生物脅迫的響應(yīng)。ERFs(Ethylene Response Factors)是植物中特有的轉(zhuǎn)錄因子,屬于AP2(APETALA2)/ERF超家族。ERF家族中有4個小ERF蛋白,分別是ERF95、ERF96、ERF97和ERF98,這些蛋白序列高度相似,具有與AtERF1等其它ERFs的羧基端序列高度相似的DNA結(jié)合域,還都具有一個功能未知的結(jié)構(gòu)域CMIX-1,但它們均不具備AtERF1等其它ERFs所具有的N末端轉(zhuǎn)錄因子活性區(qū)域。已有研究報道ERF95和ERF98參與了植物對非生物脅迫響應(yīng)的調(diào)控,而ERF96和ERF97參與了植物對生物脅迫響應(yīng)的調(diào)控。本文中則系統(tǒng)地研究了小ERFs在調(diào)控擬南芥對ABA響應(yīng)中的作用。利用蛋白的氨基酸全序列的系統(tǒng)發(fā)育樹分析結(jié)果顯示ERF95、ERF96和ERF97聚為一簇,而ERF98與它們?nèi)齻�的關(guān)系比較遠(yuǎn)。qRT-PCR結(jié)果顯示這些小ERFs的表達模式各不相同:ERF95、ERF97和ERF98在擬南芥植株的根、莖、葉、花以及成熟的種子中均有不同程度地表達,但ERF95在成熟的種子中表達量最高,ERF98在莖中表達量最高,而ERF97在花和種子中表達量都相對較高。ERF96也是在花和種子中表達量比較高,但在根中幾乎檢測不到ERF96的表達。蛋白亞細(xì)胞定位分析表明ERF95、ERF96、ERF97和ERF98均定位于細(xì)胞核中。葉肉細(xì)胞原生質(zhì)體瞬時轉(zhuǎn)染實驗表明所有ERFs都具有轉(zhuǎn)錄激活活性,且EDLL基序是其轉(zhuǎn)錄激活活性所必需的。盡管三突變體erf95 erf96 erf97和erf96 erf97 erf98,及四突變體erf95 erf96erf97 erf98都與野生型的表型近似,但小ERFs的過表達擬南芥植株呈現(xiàn)出明顯不同于野生型的表型。尤其是ERF95、ERF96和ERF98過表達擬南芥植株的蓮座葉大小與野生型存在極顯著的差異(p0.01),開花時間也明顯延遲(p0.01)。ABA敏感性試驗結(jié)果顯示,擬南芥小ERFs過表達轉(zhuǎn)基因植株對ABA敏感性增強,特別是ERF95、ERF96和ERF98過表達轉(zhuǎn)基因植株,在ABA處理后,無論發(fā)芽率、綠苗率還是根的長度均遠(yuǎn)低于野生型。qRT-PCR結(jié)果表明,過表達ERFs轉(zhuǎn)基因植株中ABA響應(yīng)基因ABI5、ABF3、ABF4、RD29A、P5CS和COR15A的表達水平在ABA處理時顯著高于Col野生型。生理實驗結(jié)果表明,小ERFs過表達轉(zhuǎn)基因植株失水速率顯著低于Col野生型,氣孔孔徑小于Col野生型,而植株的瞬時葉片水分利用效率則要高于Col野生型植株。這些結(jié)果說明小ERFs均參與調(diào)控了擬南芥對ABA的響應(yīng)。此外,我們還發(fā)現(xiàn)ERF96、ERF97也參與擬南芥對NaCl脅迫的響應(yīng)。在NaCl處理條件下,ERF96和ERF97過表達轉(zhuǎn)基因植株的幼苗生長狀態(tài)均好于野生型,其地上部分鮮重顯著高于野生型,側(cè)根數(shù)目也顯著多于野生型。NaCl處理可誘導(dǎo)Col野生型植株中RD29A、P5CS、COR15A、KIN1和RAB18等鹽脅迫響應(yīng)基因的表達,而過表達ERF96和ERF97轉(zhuǎn)基因植株中這幾個基因的表達水平均顯著高于Col野生型。離子含量檢測結(jié)果表明,小ERF96和ERF97過表達轉(zhuǎn)基因植株中Na+含量相對較低,而K+含量較高,說明小ERFs可能通過影響Na+、K+代謝進而調(diào)控植物對鹽脅迫的反應(yīng)。
[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.
【學(xué)位授予單位】：東北師范大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：Q943.2

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