【摘要】：NAC轉(zhuǎn)錄因子是一個(gè)植物特有、功能多樣性的基因家族,它們?cè)谥参锷L(zhǎng)發(fā)育、衰老、激素響應(yīng)以及生物應(yīng)答過(guò)程中發(fā)揮重要的作用。本研究基于對(duì)青桿基因PwNAC1的研究,找到擬南芥同源基因ANAC018,并對(duì)ANAC018的轉(zhuǎn)錄活性抑制域研究及ANAC018的功能研究。我們從擬南芥中分離鑒定出ANAC018基因,穩(wěn)定遺傳轉(zhuǎn)化至擬南芥中驗(yàn)證其在植物中的功能。結(jié)果顯示,在擬南芥中過(guò)表達(dá)ANAC018可以在紅光下改變花期,改變莢果衰老時(shí)間,增強(qiáng)擬南芥對(duì)干旱和鹽脅迫的耐受性,具體結(jié)果如下：1、將青桿PwNAC1及擬南芥ANAC018在擬南芥中穩(wěn)定表達(dá),觀察PwNAC1及ANAC018的亞細(xì)胞定位情況。結(jié)果表明,PwNAC1及ANAC018主要分布于根尖細(xì)胞的細(xì)胞核中,這與其作為轉(zhuǎn)錄因子在細(xì)胞核發(fā)揮功能一致,而只轉(zhuǎn)化GFP的對(duì)照則在根尖細(xì)胞的細(xì)胞質(zhì)和細(xì)胞核中均有分布。2、轉(zhuǎn)錄激活實(shí)驗(yàn)證實(shí)PwNAC1及ANAC018在酵母系統(tǒng)中沒(méi)有轉(zhuǎn)錄激活活性,預(yù)示PwNAC1及ANAC018可能以轉(zhuǎn)錄抑制形式發(fā)揮作用：酵母單雜交試驗(yàn)驗(yàn)證了PwNAC1及ANAC018及其同源蛋白的C端具有轉(zhuǎn)錄激活活性,而全長(zhǎng)不具有轉(zhuǎn)錄激活活性。分別將PwNAC1及ANAC018分為9段,通過(guò)酵母單雜實(shí)驗(yàn),最終確定轉(zhuǎn)錄抑制結(jié)構(gòu)分別存在于PwNAC1的126/136氨基酸及ANAC018的128/138氨基酸,抑制了C端轉(zhuǎn)錄激活能力。3、通過(guò)系統(tǒng)進(jìn)化樹(shù)找到了青桿基因PwNAC1擬南芥同源基因ANAC018、 ANAC025、ANAC029和ANAC047,酵母雙雜交結(jié)果表明ANAC018自身不能體外互作,推測(cè)其不能形成同源二聚體；但能與ANAC029體外互作,猜測(cè)ANAC018可能與ANAC029形成異源二聚體,參與轉(zhuǎn)錄激活或行使其他功能。4、擬南芥組織特異性表達(dá)試驗(yàn)中,ANAC018的表達(dá)量隨著擬南芥的成熟衰老而升高,在多種激素和非生物脅迫處理后發(fā)現(xiàn),ANAC018的表達(dá)量可被Eth誘導(dǎo)升高。5、將ANAC018在擬南芥中過(guò)表達(dá),得到的轉(zhuǎn)基因植株與野生型及突變植株進(jìn)行逆境實(shí)驗(yàn),觀察植株對(duì)鹽及干旱脅迫的響應(yīng),并統(tǒng)計(jì)數(shù)據(jù)。結(jié)果表明,幼苗階段,過(guò)表達(dá)ANAC018通過(guò)升高POD及SOD酶活性提高幼苗抗逆性；成苗階段,過(guò)表達(dá)ANAC018的抗旱能力較強(qiáng),而在突變體對(duì)非生物脅迫的敏感性較高；.在高鹽處理下,過(guò)表達(dá)植株成活率明顯高于突變體。6、擬南芥中過(guò)表達(dá)ANAC018可以使植株在衰老過(guò)程中相關(guān)基因表達(dá)量降低,特別是SAGs基因下降特別明顯,推測(cè)過(guò)表達(dá)ANAC018通過(guò)調(diào)控?cái)M南芥衰老相關(guān)基因SAG12、SAG18、SAG20、SEN4表達(dá)量下降來(lái)延緩莢果的衰老；進(jìn)一步實(shí)驗(yàn),將不同植株的離體葉片在3μM ABA溶液中培養(yǎng)3天的環(huán)境后,過(guò)表達(dá)植株葉片衰老程度低于突變體。推測(cè),過(guò)表達(dá)ANAC018調(diào)控衰老屬于ABA依賴型。7、紅光照射實(shí)驗(yàn)結(jié)果表明,過(guò)表達(dá)擬南芥開(kāi)花時(shí)間較野生型及突變體晚。過(guò)表達(dá)ANAC018可以升高植株抑制開(kāi)花基因FLC的表達(dá),降低促進(jìn)開(kāi)花基因SOC1、FT表達(dá)。推測(cè)ANAC018參與了植物光形態(tài)建成,從而影響植物的開(kāi)花時(shí)間。
[Abstract]:NAC transcription factor is a plant-specific and functional family of genes, which play an important role in plant growth and development, senescence, hormone response and biological response. Based on the study of green rod gene PwNAC1, the homologous gene ANAC018, of Arabidopsis thaliana was found and the transcriptional activity inhibitory domain of ANAC018 and the function of ANAC018 were studied in this study. ANAC018 gene was isolated and identified from Arabidopsis thaliana and transformed into Arabidopsis thaliana to verify its function in plants. The results showed that overexpression of ANAC018 in Arabidopsis thaliana could change flowering stage, change pod senescence time and enhance tolerance of Arabidopsis thaliana to drought and salt stress under red light. The results were as follows: 1. The subcellular localization of PwNAC1 and ANAC018 was observed by stable expression of PwNAC1 and Arabidopsis ANAC018 in Arabidopsis thaliana. The results showed that PwNAC1 and ANAC018 were mainly distributed in the nucleus of root tip cells, which was consistent with their function as transcription factors in the nucleus, while the control only transformed with GFP was distributed in the cytoplasm and nucleus of root tip cells. 2. Transcriptional activation assay confirmed that PwNAC1 and ANAC018 had no transcriptional activation activity in yeast system. It is suggested that PwNAC1 and ANAC018 may play a role in the form of transcriptional inhibition: yeast one-hybrid test confirmed that the C-terminal of PwNAC1 and ANAC018 and their homologous proteins had transcriptional activation activity, but the full-length protein had no transcriptional activation activity. PwNAC1 and ANAC018 were divided into 9 segments, and the transcriptional inhibitory structure was confirmed to exist in 126 鈮，

本文編號(hào)：2497430