作　　者： (窦祎凝）; (秦玉海）; (闵东红）; (张小红）; (王二辉）; (刁现民）; (贾冠清）; (徐兆师）; (李连城）; (马有志）; (陈明）;

机构地区： 中国农业科学院作物科学研究所、农作物基因资源与基因改良国家重大科学工程、农业部麦类生物学与作物遗传育种重点实验室,北京100081

出　　处： 《中国农业科学》 2017年第16期3071-3081,共11页

摘　　要： 【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� [Objective] Foxtail millet （Setaria italica L.） has strong tolerance to drought stress. The objective of this research is to screen key regulatory factors affecting the germination process under drought conditions in plants through reverse genetics method, which will contribute for further research of regulation mechanism of seed germination under drought condition. [Method] Multiple sequence alignment of SiNAC18 protein sequences and millet homologous sequences were made by using ClustalX 2 and MEGA 5.05 softwares, and the phylogenetic tree was constructed. The expression patterns of SiNAC18 under the stress condition were analyzed using the real-time PCR method. SiNAC18 protein subcellular localization was analyzed by transient transfection method. Biological function of SiNAC18 was analyzed by using overexpression of SiNAC18 in Arabidopsis thaliana. The expression of downstream genes of SiNAC18 was analyzed by real-time PCR. [Result] The length of SiNAC18 is 1 074 bp encoding a hydrophilic protein with polypeptide of 357 amino acids, and its molecular weight is about 38.8 kD. Phylogenetic tree analysis indicated that SiNAC18 belongs to NAP subgroup of group I in the NAC transcription factors family and has the highest homology with Arabidopsis gene AtNAC29. The amino acid sequence alignment results show that the N-terminal of SiNAC18 and the highest homology transcription factors of SiNAC18 in other species, including rice, Arabidopsis thaliana, soybean and maize, has A, B, C, D and E five conserved domains. The C-terminal of the protein has a high degree of polymorphism, demonstrating that the N-terminal sequence of SiNAC18 is associated with its downstream promoter. Real-time PCR results showed that SiNAC18 were induced by drought （PEG）, high salt （NaCl） and hydrogen peroxide （H2O2） treatment. Subcellular localization results showed that SiNAC18 protein is localized in the nucleus. Gene function analysis showed that in the ABA and PEG stress treatments, the germination rate of SiNAC18 transge

关 键 词： 谷子 类转录因子 种子萌发 干旱胁迫 信号途径