Transcriptome of Passiflora edulis Leaves under Cadmium Stress
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摘要:
目的 百香果对镉（Cd）具较强的吸收富集能力，严重影响果实的品质与商品价值，本研究通过模拟Cd胁迫的方法探讨百香果叶片响应Cd胁迫的分子机理，为百香果的安全生产和遗传育种改良提供依据。 方法 通过水培方式进行Cd中度胁迫（50 μmol·L−1）处理，采集不同处理时间的百香果叶片进行转录组测序，并采用qRT-PCR对测序的结果进行验证。 结果 与对照相比， Cd胁迫处理24、48、72 h后差异表达基因分别有 3465 、1262 和2039 个，获得63个特异性调控百香果响应镉胁迫的相关转录因子及蛋白。qRT-PCR 分析表明转录组测序结果有较高的可靠性。转录组GO功能富集和KEGG代谢通路分析结果表明，差异基因主要富集在细胞结构、催化活性及转录调节等功能组以及光合作用和碳水化合物代谢通路。结论 Cd胁迫诱导的百香果叶片差异表达基因主要富集在代谢和生物合成相关途径，研究结果为百香果叶片响应Cd胁迫的分子网络研究提供依据。 Abstract:Objective Molecular mechanism of Passiflora edulis leaves in respond to cadmium (Cd) stress was analyzed for genetic improvement on resisting the heavy metal absorption of the plant. Method Transcriptome of the leaves of passion fruit plants under a moderate Cd stress of 50 μmol·L−1 for varied durations in a hydroponic experiment was sequenced. qRT-PCR was applied to verify and analyze the results. Results There were 3,465, 1,262, and 2,039 expressed genes in the samples under the Cd stress for 24, 48, and 72h, respectively. Sixty-three specific transcription factors and proteins that responded to the stress were obtained. The transcriptome sequencing was confirmed highly reliable by a qRT-PCR analysis. The GO functional enrichment and KEGG metabolic pathways analyses on the transcriptions showed the differential expression genes (DEGs) to be mainly associated with cell structure, catalytic activity, and transcriptional regulations as well as the photosynthesis and carbohydrate metabolism pathways. Conclusion The DEGs in passion fruit leaves responding to Cd stress were mainly enriched in the metabolic and biosynthesis-related pathways. The information would pave the way to studies on curtailing Cd contamination that seriously affects the quality and commercial value of the fruit. -
图 3 差异表达基因的 GO 功能注释
A：T24 vs T0差异表达基因的GO功能注释散点图；B：T48 vs T0差异表达基因的GO功能注释散点图；C：T72 vs T0差异表达基因的GO功能注释散点图。富集因子代表富集到 GO功能注释 DEG 数量与 DEGs 总数的比值。
Figure 3. Go functional annotation of DEGs
A: GO function annotation scatter plot of T24 vs. T0 DEGs enrichment; B: GO function annotation scatter plot of T48 vs. T0 DEGs enrichment；C: GO function annotation scatter plot of T72 vs. T0 DEGs enrichment. Rich factor indicated ratio of number of enriched DEGs in GO function annotations to total number of DEGs.
图 4 KEGG 富集散点图
A：T24 vs T0差异表达基因的KEGG富集散点图；B：T48 vs T0差异表达基因的KEGG富集散点图； C：T72 vs T0差异表达基因的KEGG富集散点图。富集因子表示富集到 KEGG 通路的 DEG 数量与 DEGs 总数的之比。
Figure 4. KEGG enrichment scatter plot
A: KEGG pathway scatter plot of T24 vs. T0 DEGs enrichment; B: KEGG pathway scatter plot of T48 vs. T0 DEGs enrichment；C: KEGG pathway scatter plot of T72 vs. T0 DEGs enrichment. Rich factor indicated ratio of number of enriched DEGs in KEGG pathways to total number of DEGs.
图 5 不同时间点 Cd 处理下的连续性差异表达基因及部分转录因子的差异表达基因
A：不同时间点Cd处理下的差异表达基因韦恩图；B和C ：部分差异表达基因的表达热图。
Figure 5. Continuous and partial DEGs under Cd stress at different times
A: Venn diagram of DEGs treated with Cd at different time point; B, C: heat map of partial DEGs.
图 6 部分差异表达基因的 qRT-PCR 验证
*：P ＜ 0.05 水平差异显著； **： P ＜ 0.01 水平差异极显著。
Figure 6. Verification of DEGs by qRT-PCR
*: significant difference at P＜0.05; **: extremely significant difference at P＜0.01.
表 1 实时荧光定量PCR的引物序列
Table 1. Sequences of qRT-PCR primers applied
基因代码
Gene ID正向序列
Forward sequences (5′–3′)反向序列
Reverse sequences (5′–3′)60S AGGTGGGTAACAGGATTATC TGGCTGTCTTTTGGTGCTG WRKY31 CAGGTTCAACGTTGTGCAGA AGCAAGGAAGGATGGCTCTT NAC100 AAAGCCTCTGATCAACCCCA TGCAGCTTCTCCATGACAGA MYB306 TGCAGGCTTAGATGGACCAA GTCTTGTGTCAGAGGGTCCA bHLH87 GCCGAACGTTCATCCAAAGA TTCTGGGTCAGCTGGTTCTT HMA3 AGTGGTAGGAACAATCGCCA CCGGTTTCTGCTATGACTGC 
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