青杆PwNAC1及其拟南芥同源基因ANAC018转录活性及功能分析

发布时间：2019-06-11 20:28

【摘要】：NAC转录因子是一个植物特有、功能多样性的基因家族,它们在植物生长发育、衰老、激素响应以及生物应答过程中发挥重要的作用。本研究基于对青杆基因PwNAC1的研究,找到拟南芥同源基因ANAC018,并对ANAC018的转录活性抑制域研究及ANAC018的功能研究。我们从拟南芥中分离鉴定出ANAC018基因,稳定遗传转化至拟南芥中验证其在植物中的功能。结果显示,在拟南芥中过表达ANAC018可以在红光下改变花期,改变荚果衰老时间,增强拟南芥对干旱和盐胁迫的耐受性,具体结果如下：1、将青杆PwNAC1及拟南芥ANAC018在拟南芥中稳定表达,观察PwNAC1及ANAC018的亚细胞定位情况。结果表明,PwNAC1及ANAC018主要分布于根尖细胞的细胞核中,这与其作为转录因子在细胞核发挥功能一致,而只转化GFP的对照则在根尖细胞的细胞质和细胞核中均有分布。2、转录激活实验证实PwNAC1及ANAC018在酵母系统中没有转录激活活性,预示PwNAC1及ANAC018可能以转录抑制形式发挥作用：酵母单杂交试验验证了PwNAC1及ANAC018及其同源蛋白的C端具有转录激活活性,而全长不具有转录激活活性。分别将PwNAC1及ANAC018分为9段,通过酵母单杂实验,最终确定转录抑制结构分别存在于PwNAC1的126/136氨基酸及ANAC018的128/138氨基酸,抑制了C端转录激活能力。3、通过系统进化树找到了青杆基因PwNAC1拟南芥同源基因ANAC018、 ANAC025、ANAC029和ANAC047,酵母双杂交结果表明ANAC018自身不能体外互作,推测其不能形成同源二聚体；但能与ANAC029体外互作,猜测ANAC018可能与ANAC029形成异源二聚体,参与转录激活或行使其他功能。4、拟南芥组织特异性表达试验中,ANAC018的表达量随着拟南芥的成熟衰老而升高,在多种激素和非生物胁迫处理后发现,ANAC018的表达量可被Eth诱导升高。5、将ANAC018在拟南芥中过表达,得到的转基因植株与野生型及突变植株进行逆境实验,观察植株对盐及干旱胁迫的响应,并统计数据。结果表明,幼苗阶段,过表达ANAC018通过升高POD及SOD酶活性提高幼苗抗逆性；成苗阶段,过表达ANAC018的抗旱能力较强,而在突变体对非生物胁迫的敏感性较高；.在高盐处理下,过表达植株成活率明显高于突变体。6、拟南芥中过表达ANAC018可以使植株在衰老过程中相关基因表达量降低,特别是SAGs基因下降特别明显,推测过表达ANAC018通过调控拟南芥衰老相关基因SAG12、SAG18、SAG20、SEN4表达量下降来延缓荚果的衰老；进一步实验,将不同植株的离体叶片在3μM ABA溶液中培养3天的环境后,过表达植株叶片衰老程度低于突变体。推测,过表达ANAC018调控衰老属于ABA依赖型。7、红光照射实验结果表明,过表达拟南芥开花时间较野生型及突变体晚。过表达ANAC018可以升高植株抑制开花基因FLC的表达,降低促进开花基因SOC1、FT表达。推测ANAC018参与了植物光形态建成,从而影响植物的开花时间。
[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，

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