【摘要】：NAC轉錄因子是一個植物特有、功能多樣性的基因家族,它們在植物生長發(fā)育、衰老、激素響應以及生物應答過程中發(fā)揮重要的作用。本研究基于對青桿基因PwNAC1的研究,找到擬南芥同源基因ANAC018,并對ANAC018的轉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)定遺傳轉化至擬南芥中驗證其在植物中的功能。結果顯示,在擬南芥中過表達ANAC018可以在紅光下改變花期,改變莢果衰老時間,增強擬南芥對干旱和鹽脅迫的耐受性,具體結果如下：1、將青桿PwNAC1及擬南芥ANAC018在擬南芥中穩(wěn)定表達,觀察PwNAC1及ANAC018的亞細胞定位情況。結果表明,PwNAC1及ANAC018主要分布于根尖細胞的細胞核中,這與其作為轉錄因子在細胞核發(fā)揮功能一致,而只轉化GFP的對照則在根尖細胞的細胞質和細胞核中均有分布。2、轉錄激活實驗證實PwNAC1及ANAC018在酵母系統(tǒng)中沒有轉錄激活活性,預示PwNAC1及ANAC018可能以轉錄抑制形式發(fā)揮作用：酵母單雜交試驗驗證了PwNAC1及ANAC018及其同源蛋白的C端具有轉錄激活活性,而全長不具有轉錄激活活性。分別將PwNAC1及ANAC018分為9段,通過酵母單雜實驗,最終確定轉錄抑制結構分別存在于PwNAC1的126/136氨基酸及ANAC018的128/138氨基酸,抑制了C端轉錄激活能力。3、通過系統(tǒng)進化樹找到了青桿基因PwNAC1擬南芥同源基因ANAC018、 ANAC025、ANAC029和ANAC047,酵母雙雜交結果表明ANAC018自身不能體外互作,推測其不能形成同源二聚體；但能與ANAC029體外互作,猜測ANAC018可能與ANAC029形成異源二聚體,參與轉錄激活或行使其他功能。4、擬南芥組織特異性表達試驗中,ANAC018的表達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,ANAC018的表達量可被Eth誘導升高。5、將ANAC018在擬南芥中過表達,得到的轉基因植株與野生型及突變植株進行逆境實驗,觀察植株對鹽及干旱脅迫的響應,并統(tǒng)計數據。結果表明,幼苗階段,過表達ANAC018通過升高POD及SOD酶活性提高幼苗抗逆性；成苗階段,過表達ANAC018的抗旱能力較強,而在突變體對非生物脅迫的敏感性較高；.在高鹽處理下,過表達植株成活率明顯高于突變體。6、擬南芥中過表達ANAC018可以使植株在衰老過程中相關基因表達量降低,特別是SAGs基因下降特別明顯,推測過表達ANAC018通過調控擬南芥衰老相關基因SAG12、SAG18、SAG20、SEN4表達量下降來延緩莢果的衰老；進一步實驗,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,過表達植株葉片衰老程度低于突變體。推測,過表達ANAC018調控衰老屬于ABA依賴型。7、紅光照射實驗結果表明,過表達擬南芥開花時間較野生型及突變體晚。過表達ANAC018可以升高植株抑制開花基因FLC的表達,降低促進開花基因SOC1、FT表達。推測ANAC018參與了植物光形態(tài)建成,從而影響植物的開花時間。
[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：2497430