杂交小麦抗坏血酸过氧化物酶基因克隆及其在种子老化中的潜在功能分析

以BS型二系杂交小麦BS1453/11GF5135及其BS1453(母本)、11GF5135(父本)的种子为材料,在BS1453/11GF5135中通过同源克隆获得抗坏血酸过氧化物酶基因TaAPX。该基因包含一个832 bp的ORF,共编码277个氨基酸。通过进一步生信分析预测miRNA与TaAPX基因的互作关系,发现TaAPX基因可能受miR396等抗逆及种子活力相关miRNAs的调控。另外通过蛋白互作预测分析,发现APX蛋白主要与氧化还原相关的酶互作反应。通过对不同老化时间的父母本及杂交种的胚进行qPCR及酶活性分析,发现TaAPX基因在杂交种及亲本中的表达趋势是先被诱导上调表达,后下调表达,但杂交种子内TaAPX基因的表达量在第7天才开始下降,而亲本种子内TaAPX基因的表达量在第5天开始下降,且miR396与TaAPX互为拮抗作用。随着老化时间的延长,亲本种子内APX酶活性表现为下降趋势,杂交种子内APX酶活性在第3天呈短暂下降趋势,随后上升,第9天开始下降,表明在老化条件下杂交种子内APX酶清除体内过氧化物的能力高于亲本,即杂交种抗老化能力高于其父母本,且TaAPX基因对种子活力的调控是一个复杂的多因素参与的调控过程。本研究为进一步研究BS型杂交小麦种子活力的分子调控机理奠定了一定的基础。

Cloning and potential function analysis of ascorbic peroxidase gene of hybrid wheat in seed aging

In this study,the seeds of two-line hybrid wheat of BS-type BS1453/11GF5135 and its parents BS1453(female parent)and 11GF5135(male parent)were used as materials,and the ascorbate peroxidase gene(TaAPX)was obtained by homologous cloning in BS1453/11GF5135.The gene contains an open reading frame(ORF)of 832 bp,which encoded a total of 277 amino acids.The interaction between miRNAs and TaAPX was predicted by bioinformatics analysis,and the results showed that TaAPX may be regulated by miR396 and other miRNAs that related to stress resistance and seed viability.In addition,it was found that APX protein mainly interacted with redox-related enzymes via protein interaction prediction analysis.qPCR and enzyme activity analysis were conducted on embryos of parents and hybrid at different aging time,it was found that the expression trend of TaAPX gene in hybrid and parents were first up-regulated and then down-regulated.However,the expression of TaAPX gene in hybrid seeds peaked on the seventh day,and it began to decline in parents seeds on the fifth day.There was an antagonistic effect between miR396 and TaAPX.With the increase of aging time,the activity of APX enzyme in parents seeds showed a downtrends,while it showed a temporary downtrends on the third day in hybrid seeds,then it increased and began to decline on the ninth day.It was suggested that the ability of APX enzyme to remove peroxides in the hybrid seeds was higher than that in the parents seeds under the aging condition,that is,the anti-aging ability of hybrid was higher than that of the parents.Moreover,the regulation of seed activity by TaAPX gene was a complex multi-factor regulation process.This study laid a foundation for the further research on the molecular regulation mechanism of the seed vigor of BS type hybrid wheat.