论文部分内容阅读
【目的】谷子(Setaria italica L.)具有显著耐旱性。研究旨在通过反向遗传学方法分析并鉴定在干旱条件下影响植物萌发过程的重要调控因子,为研究作物干旱条件下种子萌发的调控机制创造条件。【方法】使用Clustal X 2.0和MEGA 5.05软件对谷子SiNAC18蛋白序列及其同源序列进行多序列比对,并构建系统进化树;利用real-time PCR方法检测SiNAC18在不同胁迫条件下的表达模式;通过瞬时转化的方法分析SiNAC18蛋白亚细胞定位;在拟南芥中过表达SiNAC18,分析SiNAC18的生物学功能;分析SiNAC18在转基因拟南芥中可能控制的下游基因。【结果】SiNAC18全长1 074 bp,编码由357个氨基酸组成的亲水性蛋白,分子量约为38.8 k D;系统进化树分析表明SiNAC18属于NAC转录因子家族第Ⅰ组的NAP亚组,与拟南芥基因At NAC29同源性最高;氨基酸序列比对结果显示,SiNAC18与其他物种包括水稻、拟南芥、大豆和玉米中同源性最高的NAC类转录因子蛋白的N端都具有A、B、C、D和E这5个保守结构域,蛋白C端具有高度多态性,证明SiNAC18的N端序列与其结合下游基因启动子元件相关;real-time PCR结果显示,SiNAC18在干旱(PEG)、ABA、高盐(Na Cl)及过氧化氢(H2O2)处理条件下的表达量明显上升;亚细胞定位结果表明SiNAC18蛋白定位于细胞核中;基因功能分析结果显示,在ABA和PEG胁迫处理下,SiNAC18转基因拟南芥与野生型种子的萌发率存在明显差异:在正常生长条件下,野生型拟南芥WT和SiNAC18转基因拟南芥的萌发率基本一致,在PEG浓度为10%和15%的MS培养基上,SiNAC18转基因拟南芥的萌发率显著高于WT。在2和5μmol·L-1 ABA处理条件下,转基因拟南芥的萌发率显著低于WT;下游基因表达分析结果显示,ABA信号途径相关基因At RD29A,脯氨酸合成相关基因At P5CR和At PRODH以及过氧化物酶基因At PRX34在SiNAC18转基因株系中的表达量高于WT中的表达量,表明SiNAC18通过调控这些下游基因影响转基因植物在干旱条件下的萌发率。【结论】谷子NAC类转录因子基因SiNAC18可能通过ABA信号途径、氧化胁迫调控等途径正向调控植物在干旱条件下的萌发过程。
【Objective】 The yield of millet (Setaria italica L.) is significantly drought-tolerant. The aim of this study is to analyze and identify the important regulatory factors that affect plant germination under drought conditions through reverse genetics and to create conditions for studying the regulatory mechanism of seed germination under drought conditions in crops. 【Method】 Multiple sequence alignments of SiNAC18 protein sequence and its homologous sequences in millet were carried out using Clustal X 2.0 and MEGA 5.05 software, and the phylogenetic tree was constructed. The expression pattern of SiNAC18 under different stress conditions was detected by real-time PCR. The subcellular localization of SiNAC18 protein was analyzed by transient transformation. The expression of SiNAC18 was overexpressed in Arabidopsis. The biological functions of SiNAC18 were analyzed. The possible downstream genes of SiNAC18 in transgenic Arabidopsis were analyzed. 【Results】 The full length of SiNAC18 was 1 074 bp, encoding a hydrophilic protein consisting of 357 amino acids with a molecular weight of 38.8 kD. Phylogenetic tree analysis indicated that SiNAC18 belonged to the NAP subgroup of NAC transcription factor family Ⅰ, The amino acid sequence alignment of AtNAC29 in Arabidopsis thaliana showed the highest homology. The results of amino acid sequence alignment showed that the N-terminal NACs of SiNAC18 and other species including rice, Arabidopsis, soybean and maize with the highest homology have A and B C, D and E. The results showed that the N-terminal sequence of SiNAC18 was associated with its downstream promoter element. Real-time PCR results showed that SiNAC18 was highly expressed in PEG , ABA, NaCl and hydrogen peroxide (H2O2). The results of subcellular localization indicated that SiNAC18 protein localized in the nucleus. The results of gene function analysis indicated that under the stress of ABA and PEG treatment , The germination rate of SiNAC18 transgenic Arabidopsis thaliana and wild-type seeds was significantly different: Under the normal growth conditions, the germination rate of wild-type Arabidopsis thaliana WT and SiNAC18 transgenic Arabidopsis thaliana was basically the same, at the PEG concentration of 10% and 15% MS medium, The germination rate of SiNAC18 transgenic Arabidopsis was significantly higher than that of WT. Under 2 and 5μmol·L-1 ABA treatment, the germination rate of transgenic Arabidopsis thaliana was significantly lower than that of WT. The downstream gene expression analysis showed that ABA signaling pathway related genes At RD29A, proline synthesis related genes At P5CR and At PRODH and the peroxidase gene At PRX34 in SiNAC18 transgenic lines were higher than those in WT, indicating that SiNAC18 affects the germination rate of transgenic plants under drought conditions by regulating these downstream genes. 【Conclusion】 SiNAC18, a gene of cereal NAC transcription factor, may positively regulate plant germination under drought conditions through ABA signaling pathway and oxidative stress regulation.