玉米花药不同发育时期的转录组分析及其差异表达基因的鉴定

Transcriptome analysis and identification of differentially expressed genes in maize at different anther development stages

【目的】鉴定玉米花药角质层和花粉壁发育的关键基因与代谢途径,为玉米雄性育性分子机制解析和雄性不育种质资源创制奠定基础。【方法】以玉米自交系B73小孢子母细胞形成期(S6)、四分体时期(S8b)、成熟花粉粒形成期(S13)花药和苗期叶片为材料,利用转录组测序和生物信息学分析方法鉴定差异表达基因,通过GO和KEGG富集分析挖掘差异表达基因参与的生物学过程和生化代谢途径。采用已知基因的实时荧光定量PCR(qRT-PCR)分析验证转录组数据。【结果】不同发育时期花药差异表达基因KEGG分析显示,S8b时期是玉米花药角质层发育的关键时期,16个可能与角质和蜡质合成代谢途径相关的基因差异表达,编码产物涉及脂肪酰还原酶、乙酰转移酶、过加氧酶、脂肪酸羟化酶、醛脱羧酶和羟基脂肪酸脱氢酶。S8b时期花药与其他3组样品之间存在3555个共同的差异表达基因,其中1348个基因在S8b时期花药中均上调表达。GO分析表明,共同上调表达差异基因在花粉壁发育相关生物学过程显著富集,包含脂肪酰还原酶基因、脂肪酸羟化酶基因、α-吡喃酮还原酶基因、多聚半乳糖醛酸酶基因、异胡豆苷合酶基因、查尔酮合成酶基因和4-香豆酰辅酶A连接酶基因等11个基因。191个共同上调表达基因被注释为转录因子和转录调节因子。【结论】鉴定到16个基因(Zm00001d049950,Zm00001d048337,Zm00001d012274,Zm00001d042723,Zm00001d025833,Zm00001d017953,Zm00001d042814,Zm00001d012326,Zm00001d014055,Zm00001d051932,Zm00001d017251,Zm00001d032284,Zm00001d020238,Zm00001d051800,Zm00001d017528,Zm00001d002687)和11个基因(Zm00001d048337,Zm00001d046537,Zm00001d027837,Zm00001d024712,Zm00001d020680,Zm00001d031488,Zm00001d020970,Zm00001d002342,Zm00001d047858,Zm00001d019478,Zm00001d003702)分别参与玉米花药角质层和花粉壁发育过程,发现了一些新的代谢途径影响玉米花药角质层和花粉壁发育。

【Objective】The key genes and metabolic pathways for the development of the anther cuticle and pollen wall in maize were identified to lay the foundation for analysis of the molecular mechanism of male fertility and generation of male sterile germplasm resources.【Method】Inbred line B73 anthers at the microspore mother cell formation stage(S6),tetrad stage(S8b)and mature pollen grain formation stage(S13)as well as leaves at the seedling stage were selected as the experimental materials.Transcriptome sequencing and bioinformatics analyses were performed to identify differentially expressed genes(DEGs).The biological processes and biochemical metabolic pathways associated with DEGs were explored via GO and KEGG enrichment analyses.The transcriptome data were verified on the basis of a qRT-PCR assay.【Result】The KEGG analysis of DEGs at different developmental stages revealed that S8b was the key stage for anther cuticle development in maize,with 16 DEGs possibly related to cutin and wax biosynthesis.The proteins encoded by these DEGs included fatty acyl reductase,acetyltransferase,peroxygenase,fatty acid hydroxylase,aldehyde decarboxylase and hydroxy fatty acid dehydrogenase.A total of 3555 common DEGs were detected between in the anthers at S8b and in the other three groups,of which 1348 genes were up-regulated in the anthers at S8b.The GO analysis indicated these 1348 genes,which were significantly associated with pollen wall development,included 11 genes like fatty acyl reductase,fatty acid hydroxylase,α-pyrone reductase,polygalacturonase,strictosidine synthase,chalcone synthase and 4-coumarate-CoA ligase.Additionally,191 of these genes were annotated as transcription factors or transcription regulatory factors.【Conclusion】The findings of this study suggest 16 genes(Zm00001d049950,Zm00001d048337,Zm00001d012274,Zm00001d042723,Zm00001d025833,Zm00001d017953,Zm00001d042814,Zm00001d012326,Zm00001d014055,Zm00001d051932,Zm00001d017251,Zm00001d032284,Zm00001d020238,Zm00001d051800,Zm00001d017528,Zm00001d002687)and 11(Zm00001d048337,Zm00001d046537,Zm00001d027837,Zm00001d024712,Zm00001d020680,Zm00001d031488,Zm00001d020970,Zm00001d002342,Zm00001d047858,Zm00001d019478,Zm00001d003702)may be involved in the development of the maize anther cuticle and pollen wall,respectively.Furthermore,this study clarified the contributions of some new metabolic pathways to the development of the anther cuticle and pollen wall in maize.