中国农业科学 ›› 2014, Vol. 47 ›› Issue (10): 1878-1893.doi: 10.3864/j.issn.0578-1752.2014.10.002

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

人工劣变处理对玉米种胚差异基因表达的影响

 杨伟飞, 张景龙, 吕伟增, 曹广灿, 陈军营   

  1. 河南农业大学农学院,郑州450002
  • 收稿日期:2013-11-22 出版日期:2014-05-20 发布日期:2014-01-15
  • 通讯作者: 陈军营,E-mail:chenjunying3978@126.com
  • 作者简介:杨伟飞,E-mail:yangweifei4115@163.com
  • 基金资助:

    国家自然科学基金项目(31371709)

Study on the Differential Genes Expression in Maize Embryo Treated by a Controlled Deterioration Treatment

 YANG  Wei-Fei, ZHANG  Jing-Long, 吕Wei-Zeng , CAO  Guang-Can, CHEN  Jun-Ying   

  1. College of Agronomy, Henan Agricultural University, Zhengzhou 450002
  • Received:2013-11-22 Online:2014-05-20 Published:2014-01-15

摘要: 【目的】利用数字基因表达谱技术(digital gene expression tag profiling,DGE)探索人工控制劣变(controlled deterioration treatment,CDT)条件下玉米种胚差异基因的表达变化,为揭示种子劣变的分子机理提供依据。【方法】以玉米杂交种郑单958种子为材料,采用高温(45℃)高湿(相对湿度100%)方法处理72 h,以未处理材料为对照,利用DGE分别进行高通量测序,测序结果与参考基因组和参考基因数据库比对获得表达基因,利用RPKM(Reads Per Kb per Million reads)方法计算基因的表达量,根据FDR(false discovery rate)<0.001和|log2 ratio(T/CK)|≥1的标准筛选差异表达的基因,对获得的差异表达基因(digital expression genes,DEGs)进行GO(gene ontology)和KEGG(kyoto encyclopedia of genes and genomes)数据库功能注释,并对注释结果进行富集分析处理。【结果】对照玉米种胚中检测到32 000多种mRNAs。CDT处理后差异表达基因有4 713个。其中上调表达2 874个,下调表达1 839个。GO富集分析表明,这些基因涉及细胞组分、分子功能和生物学过程方面,其编码的蛋白主要分布在细胞器和细胞膜上,参与能量代谢、信号转导、刺激响应和衰老死亡等过程,具有结合、催化和抗氧化等功能。这些基因中,能被KEGG数据库注释的有2 470个,参与了288条代谢通路,其中有16条通路存在着显著性富集。这些通路中,参与能量代谢的基因共有113个,分别参与糖酵解/糖异生过程(59个)、磷酸戊糖途径(31个)和丙酮酸代谢过程(50个);其中,调节糖酵解/糖异生过程的烯醇化酶基因、3-磷酸甘油醛脱氢酶基因等、丙酮酸代谢中的丙酮酸激酶基因等和磷酸戊糖途径中的α-L-岩藻糖苷酶基因等上调幅度最大。还发现,调节NADH代谢相关的基因有25个(9个上调,16个下调);NADPH代谢的有10个(4个上调,6个下调)。这些基因的表达变化能够调节活性氧的产生和积累。【结论】DGE可以作为研究种子劣变和活力丧失的有效手段。CDT能够影响玉米种胚差异基因的表达,进而影响细胞能量代谢相关过程,主要表现在糖酵解途径受到抑制,导致ROS产生和积累,从而加速玉米种胚细胞的衰老及死亡,最终造成种子的劣变和活力丧失。在这个过程中,差异表达基因可能扮演重要角色。

关键词: 玉米 , 干种胚 , 人工控制劣变 , 差异表达基因

Abstract: 【Objective】 In this work, digital gene expression tag profiling (DGE) was employed to investigate the differentially expressed genes (DEGs) in the embryo of maize seeds treated by a controlled deterioration treatment (CDT) to provide a basis for a better understanding of essential molecular mechanism underlying seed deterioration. 【Method】 In this study, maize (Zea mays L.) cultivar (zhengdan 958) seeds were used as a model and treated by CDT (45℃, 100% relative humidity) for 72 h (T), untreated seeds were used as control (CK). DGE was performed and the high-quality sequences were mapped to the reference genome and maize genes database to obtain the expression genes. The expression level of each gene was calculated by RPKM method. A combination of FDR<0.001 and the absolute value of |log2 ratio (T/CK)|≥1 was used as the threshold to determine the significance of gene expression difference. Finally, GO and pathway enrichment analysis were used to identify the significantly enriched function classification and metabolic pathways in DEGs.【Result】About 3 2000 mRNAs were detected in dry maize embryos (CK). A total of 4 713 DEGs, including 2 874 up-regulated and 1 839 down-regulated, were identified under CDT for 72 h. GO enrichment analysis revealed that the DEGs involved in three GO categories, i.e., cellular component, molecular function and biological process. The proteins coded by these genes were distributed on organelle/membrane in cells and participated in some metabolic processes, signaling transduction, response to stimulus and death process, etc. They would have binding, catalytic activity, and antioxidant activity, etc. There were 2 470 annotated DEGs that participated in 288 KEGG pathways in which 16 pathways were significantly enriched. Among these pathways, there were 113 genes involved in energy metabolism, i.e, 59 genes in glycolysis /gluconeogenesis, 50 genes in pyruvate metabolism, and 31 genes in pentose phosphate metabolism, respectively. The genes encoding enolase and glyceraldehyde 3-phosphate dehydrogenase in glycolysis /gluconeogenesis,pyruvate kinase in pyruvate metabolism,and alpha-L-fucosidase in pentose phosphate metabolism were up-regulated at highest levels. There were 25 genes that regulate the metabolism of NADH (9 up-regulated and 16 down-regulated genes) and 10 genes regulate the metabolism of NADPH (4 up-regulated and 6 down-regulated genes) were detected. They may regulate reactive oxygen species (ROS) production and accumulation. 【Conclusion】DGE provided an innovative and powerful tool for investigating the molecular mechanism of seed deterioration or vigor loss during aging. CDT could affect DEGs expression in dry maize embryos and then energy metabolism in cells. They would inhibit glycolytic pathway and promote ROS production and accumulation, then, accelerate cells aging or death in seed embryos, and ultimately lead to seed deterioration and vigor loss. DEGs might play a critical role in the process.

Key words: maize , the dry maize embryo , CDT , DEGs