不同含油量油梨果肉的转录组、生理生化和细胞学水平差异分析

Differencial analysis of transcriptome,physio-biochemical andcytological levels of avocado mesocarp with different oil contents

【目的】分析不同含油量油梨果肉的转录组、生理生化和细胞学水平差异,为挖掘油梨果肉脂肪酸合成过程的相关基因及油梨新品种选育提供参考依据。【方法】以3个油梨品种Hass、RL-1和RL-2成熟果肉为试验材料,采用IlluminaHiSeq2500测序平台完成测序,以|log2FoldChange|>1和P<0.05为标准,通过DEGseq(v2)获得差异表达Unigenes,通过韦恩图和火山图分析3个比较组(RL-1vsHass、RL-2vsHass和RL-2vsRL-1)差异表达Unigenes,并进行GO功能注释、KEGG信号通路富集和聚类分析,采用实时荧光定量PCR验证转录组测序结果,测定3个油梨品种的含油量,采用激光共聚焦技术在40倍物镜下观察油梨成熟果肉中的油体。【结果】3个油梨品种成熟果肉的转录本中,在至少2个样品间有显著差异的Unigenes共19007条(P<0.05,下同);在3个比较组间相对表达量有明显差异的Unigenes共3000个。在RL-2 vs Hass比较组中有9个功能条目显著富集,RL-1 vs Hass比较组中只有细胞组分类别下的3个功能条目显著富集;仅2个KEGG信号通路在RL-2 vs Hass比较组中显著富集。筛选到32条参与油梨果肉脂肪酸合成的差异表达Unigenes;聚类分析结果显示,差异表达Unigenes表达模式分为2组。实时荧光定量PCR验证结果表明转录组测序数据可靠。3个油梨品种成熟果肉含油量排序为Hass>RL-2>RL-1,油梨果肉油体差异观察结果显示,Hass成熟果肉中油体较大,最大的直径达15~20μm。【结论】筛选到与油梨果肉脂肪酸成分合成相关的32条差异表达Unigenes,其中5条Unigenes[PaPDH(E1β)、PaACP4-1、PaACP4-2、PaSAD(FAB2)和PaFaTA]在3个油梨品种成熟果肉中的相对表达量较高。

【Objective】The aim was to analyze the differences in transcriptome,physio-biochemical and cytological levels of avocado mesocarp with different oil contents,so as to provide reference basis for mining related genes in fatty acid synthesis process of avocado mesocarp and breeding new varieties of avocado.【Method】The mature mesocarp of three avocado varieties Hass,RL-1 and RL-2 were used as experimental materials,and the Illumina HiSeq 2500 sequencing platform was used to complete the sequencing.With|log2 Fold Change|>1 and P<0.05 as the standard,the differentially expressed unigenes were obtained by DEGseq(v2).The differentially expressed unigenes in the three comparison groups(RL-1 vs Hass,RL-2 vs Hass and RL-2 vs RL-1)were analyzed by Venn diagram and volcano diagram.Through GO functional annotation,KEGG signaling pathway enrichment and cluster analysis,real-time fluorescence quantitative PCR was used to verify the transcriptome sequencing results,and the oil contents of three avocado varieties were determined.The oil body in the mature mesocarp of avocado was observed under a 40-time objective lens by laser confocal technique.【Result】Among transcripts of three varieties of mature avocado mesocarps,a total of 19007 unigenes were found to be significantly different between at least 2 transcripts of samples(P<0.05,the same below).There were 3000 unigenes with obvious differences in relative expression among three comparison groups.In the RL-2 vs Hass comparison group,there were 9 functional items and they were significantly enriched.In the RL-1 vs Hass comparison group,only 3 functional items under the cell component category were significantly enriched.Only two KEGG signaling pathways were significantly enriched in the RL-2 vs Hass comparison group.A total of 32 differentially expressed unigenes involved in fatty acid synthesis in avocado mesocarp were screened.The results of cluster analysis showed that the expression patterns of differentially expressed unigenes were divided into two groups.The results of real-time fluorescence quantitative PCR showed that the transcriptome sequencing data were reliable.The oil contents of the mature mesocarp of the three avocados varieties were ranked as Hass>RL-2>RL-1.The observation results of the oil body difference of the avocado mesocarp showed that the mature mesocarp of Hass had larger oil bodies,and the maximum diameter was 15-20μm.【Conclusion】A total of 32 differentially expressed unigenes related to the synthesis of fatty acid components in avocado mesocarp are screened,among which five unigenes[PaPDH(E1β),PaACP4-1,PaACP4-2,PaSAD(FAB2)and PaFaTA]had higher relative expression levels in mature mesocarp of three avocado varieties.