‘鸭梨’及其自交亲和性芽变‘金坠梨’花粉转录组测序比较分析

Sequencing analysis of pollen transcriptome of 'Yali' and its spontaneous self-compatible mutant 'Jinzhui'

【目的】系统研究‘鸭梨’及其花粉部分自交亲和性突变体‘金坠梨’花粉基因表达情况,筛选二者之间的差异表达基因,为进一步揭示‘金坠梨’自交亲和性突变的分子机制提供数据支持。【方法】以‘鸭梨’和‘金坠梨’花粉为材料,采用Illumina高通量测序技术进行转录组测序分析,利用生物信息学方法分析二者的差异表达基因,并利用q RTPCR验证转录组测序结果。【结果】‘鸭梨’和‘金坠梨’花粉转录组测序的干净读序(clean reads)分别为44 778 208条和44 995 100条。2个样品Reads与参考基因组的比对效率分别为66.35%和66.08%,并且唯一比对位置的数量都超过55.02%。数据分析显示,‘鸭梨’花粉样品与‘金坠梨’花粉样品显著差异表达基因的数目是136个,其中上调表达数量是76个,下调表达基因数量是60个,涉及一些自交不亲和、泛素化、抗逆境胁迫、RNA降解及转录等相关基因。q RTPCR结果表明,转录组测序数据可信度很高。挖掘转录组和q RT-PCR数据,发现了一个差异表达在40倍以上的泛素结合酶E2同源基因(基因号:103966960)。【结论】明确了‘鸭梨’和‘金坠梨’花粉部分自交亲和性突变中差异表达的基因,为进一步大量挖掘在梨属果树自交不亲和机制中发挥重要作用的基因奠定基础。

【Objective】‘Jinzhui＇（JZ） is a spontaneous self-compatible mutant of‘Yali＇（Pyrus bretschneideri Rehd., YL） with a typical S-RNase-based gametophytic self-incompatibility（GSI）. The phenotypic changes of the pollen-part mutation（PPM）‘Jinzhui＇might be due to a natural mutation in the pollen-S gene. However, the molecular mechanisms behind these phenotypic changes remain unclear. To understand the possible mechanisms in response to SI, a comparative transcriptomic analysis with pollenes of YL and JZ was performed in order to provide valuable information for analyzing the candidate self-incompatibility associated genes of P. bretschneideri Rehd.【Methods】Pollen samples of YL and JZ were collected as experimental materials and high-throughput next generation sequencing technology RNA-seq was used to conduct sequencing. Sequence comparison with designated reference genome was performed to obtain mapped data. From the comparison of transcriptomic data of YL pollenes and JZ pollenes, differentially expressed genes（DEGs） were identified and the regulated models were analyzed. To better understand the distribution of gene functions at the macro level, the GO function classi-fication of the DEGs were analyzed using the WEGO online tool. To further investigate the influence of the DEGs on pathways, statistical pathway enrichment analysis of DEGs was performed based on KEGG database. To identify differentially expressed genes associated with self-incompatibility（SI） in P. bretschneideri Rehd., the expression levels of SI related DEGs were measured based on the fragments per kb per million of the mapped reads（FPKM） value.【Results】Through transcriptome sequencing data analysis, totally 44 778 208 and 44 995 100 clean reads were generated in the YL and JZ libraries after removing adaptor, ambiguous and low-quality reads, and the GC contents were 47.5% and47.7% respectively. The Q30 contents of two samples were both over 95%, indicating the high quality of transcriptome sequencing and the high accuracy of the data. Of these high-quality reads, 66.35% and66.08% were aligned to reference genome or gene sequences, respectively, and for unique alignment position the sequence alignment efficiency between reads of both samples and reference genome and genes was over 55.02%. For these aligned genes in YL and JZ, FPKM method was used to get the standard measure. The significance of gene expression differences of YL and JZ was determined using the threshold of FDR≤ 0.001 and ｜log2 Ratio｜≥ 1. Totals of 136 differentially expressed genes（DEGs） were obtained between samples YL and JZ. Specifically, the expression levels of 76 genes were up-regulated and 60 genes were down-regulated in sample YL compared with sample JZ. For these up-and downregulated genes, GO and KEGG analysis were performed. When the DEGs matched to the GO terms, a total of 68 of these DEGs were associated with 33 subcategories belonging to 3 categories, biological process, cellular component and molecular function. Among the biological process category,＂metabolic process＂and＂cellular process＂were the main functional groups, which were followed by＂single organism process＂and＂response to stimulus＂. In terms of cellular component,＂cell part＂and＂cell＂were the most highly represented subcategories. For the molecular function category,＂catalytic activity＂and＂binding＂were the two main groups. To further investigate biological behavior, the DEGs were assigned to the biochemical pathways described in the KEGG database. A total of 86 DEGs were assigned to the 49 KEGG pathways, including metabolic pathways, biosynthesis of secondary metabolites, plant-pathogen interaction, signal transduction, ubiquitin-mediated proteolysis, and RNA degradation. To identify differentially expressed genes associated with SI in YL and JZ, the expression levels of 41 DEGs were measured based on the fragments per kb per million of the mapped reads（FPKM）value. Several notable genes were potentially involved in SI responses, such as those involved in pollen tube growth, RNA degradation and stress resistance, polyubiquitin, ubiquitin conjugating enzyme complex member, vesicle-mediated transporter were identified. Some defense-related genes were upregulated in JZ pollen sample, which might function not only in defense but also in response to recognition process. Notably, a gene ubiquitin-conjugating enzyme E2 variant 1 D-like（103966960） was found for over 40 times difference of the gene expression. Further study is necessary on those genes that might be associated with SI.【Conclusion】The results of transcriptome analysis suggested that multiple genes might be associated with SI in P. bretschneideri Rehd. We hypothesized that the gene, ubiquitin-conjugating enzyme E2 variant 1 D-like, might be crucial for self-recognition. However, further studies are required to fully understand the role of the candidate gene in SI. Our study provides a pool of SI-related genes in P. bretschneideri Rehd. and offers a valuable resource for elucidating the mechanisms of SI in Pyrus.