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【目的】核转录因子Y(nuclear transcription factor Y,NF-Y)属于一类广泛存在于真核生物中的转录因子基因家族,参与植物生长发育、胁迫响应等过程,但其参与低氮胁迫响应的研究尚未报道。从抗旱及耐瘠薄的谷子中分离出一个新的NF-Y类转录因子基因Si NF-YA6,对其表达模式和生物学功能进行分析。【方法】通过对谷子低氮处理前后的转录组差异表达谱分析,鉴定出一个受低氮诱导表达明显上调的的NF-Y类转录因子A亚基基因Si NF-YA6。利用DNAMAN和MEGA6.0软件对Si NF-YA6进行系统进化树分析;通过Real-time PCR方法分析Si NF-YA6在不同处理条件下的表达模式;并将Si NF-YA6蛋白进行亚细胞定位;通过Si NF-YA6转化拟南芥,对Si NF-YA6的功能进行鉴定。【结果】Si NF-YA6全长714 bp,编码237个氨基酸,包含一个由62个氨基酸组成的CBF保守域。系统进化树分析结果表明,NF-YA类蛋白家族一共分为6个组,Si NF-YA6属于第5组。Si NF-YA6与一个水稻NF-YA类蛋白(Os03g07880)同源性最高(77.4%)。Si NF-YA6表达模式分析结果显示,Si NF-YA6在受到低氮胁迫时,其表达量显著上调,在处理5 d时,其表达量达到最高,较处理前表达量上调约5倍。同时Si NF-YA6在高盐、干旱、ABA处理条件下的表达量也呈现上升趋势。Si NF-YA6对于SA、GA处理无明显响应。组织特异性表达分析显示,Si NF-YA6主要在谷子的根部表达。蛋白亚细胞定位结果显示,Si NF-YA6主要定位于植物的细胞核及细胞膜上。低氮胁迫抗性试验结果显示,在未处理条件下,Si NF-YA6转基因拟南芥与野生型拟南芥长势无明显差异,但在低氮(总氮含量为1 mmol·L-1)处理条件下,Si NF-YA6转基因拟南芥的总根长及主根长均明显高于野生型拟南芥,鲜重也明显大于野生型植株,并达到显著性差异(P<0.01),表明过表达Si NF-YA6显著提高转基因植物耐低氮胁迫的能力。下游基因表达分析结果显示在Si NF-YA6转基因拟南芥中氮转运相关基因(NRT2.1和NRT2.4)的表达量显著提高。同时,Si NF-YA6转基因拟南芥对于高盐与干旱胁迫的抗性与野生型拟南芥无明显差异。【结论】过表达谷子NF-Y类转录因子基因Si NF-YA6通过提高氮转运基因的表达显著提高转基因植株在低氮条件下的抗性。
【Objective】 Nuclear transcription factor Y (NF-Y) belongs to a family of transcription factors widely found in eukaryotes and participates in the process of plant growth and development, stress response, but it is involved in the response to low nitrogen stress The study has not been reported. A novel NF-Y transcription factor gene, Si NF-YA6, was isolated from drought-resistant and infertile millet and its expression patterns and biological functions were analyzed. 【Method】 A NF-Y transcription factor A subunit gene Si NF-YA6, which was significantly up-regulated by low nitrogen induction, was identified by differential expression profiling of transcriptome of low-nitrogen treatments. The phylogenetic tree analysis of Si NF-YA6 was carried out by using DNAMAN and MEGA 6.0 software. The expression pattern of Si NF-YA6 under different processing conditions was analyzed by Real-time PCR. The subcellular localization of Si NF-YA6 protein was also performed. Arabidopsis thaliana was transformed with Si NF-YA6 to verify the function of Si NF-YA6. 【Result】 Si NF-YA6 was 714 bp in length and encoded 237 amino acids. It contained a 62 amino acid conserved CBF domain. Phylogenetic tree analysis showed that the NF-YA protein family was divided into six groups and Si NF-YA6 belonged to the fifth group. Si NF-YA6 has the highest homology (77.4%) with a rice NF-YA protein (Os03g07880). Si NF-YA6 expression pattern analysis showed that Si NF-YA6 significantly up-regulated the expression of Si NF-YA6 when exposed to low nitrogen stress. The expression level of Si NF-YA6 was up to 5-fold up-regulated after 5 days of treatment. Meanwhile, the expression level of Si NF-YA6 in the condition of high salt, drought and ABA also showed an upward trend. Si NF-YA6 had no obvious response to SA and GA treatment. Tissue-specific expression analysis showed that Si NF-YA6 is mainly expressed in the roots of millet. Protein subcellular localization results show that Si NF-YA6 is mainly located in plant nucleus and cell membrane. The results of low nitrogen stress resistance test showed that there was no significant difference between Si NF-YA6 transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana under untreated conditions, but under low nitrogen (total nitrogen content of 1 mmol·L-1) The total root length and root length of Si NF-YA6 transgenic Arabidopsis thaliana were significantly higher than that of wild type Arabidopsis thaliana under fresh conditions, and the fresh weight of Si NF-YA6 transgenic Arabidopsis thaliana was also significantly higher than that of wild type (P <0.01) Overexpression of Si NF-YA6 significantly increased the ability of transgenic plants to tolerate low nitrogen stress. Downstream gene expression analysis results showed that the expression of nitrogen transport related genes (NRT2.1 and NRT2.4) in Si NF-YA6 transgenic Arabidopsis significantly increased. Meanwhile, the resistance of Si NF-YA6 transgenic Arabidopsis to high salt and drought stress was not significantly different from that of wild type Arabidopsis thaliana. 【Conclusion】 Si NF-YA6, an overexpressed cereal NF-Y transcription factor, significantly increased the resistance of transgenic plants under low nitrogen conditions by increasing the expression of nitrogen transporter genes.