PEG和NaCl胁迫下毛竹萌发种子的MicroRNAs表达谱分析

王晓静; 李潞滨; 杨凯; 王涛

doi:10.12403/j.1001-1498.20220211

PEG和NaCl胁迫下毛竹萌发种子的MicroRNAs表达谱分析

Identification of MicroRNAs during Seed Germination and Its Response to PEG and NaCl Stresses

摘要

摘要:
目的鉴定和分析不同干旱和盐胁迫下毛竹种子在露白时期的microRNAs（miRNAs）及其表达模式。
方法分别使用聚乙二醇（PEG6000）和氯化钠（NaCl）模拟干旱和盐胁迫，构建H₂O、10% PEG、15% PEG、50 mmol·L⁻¹ NaCl和100 mmol·L⁻¹ NaCl处理下毛竹露白时期种子的small RNA文库，通过高通量测序和生物信息学分析揭示样本中的miRNA及其表达谱。
结果通过small RNA测序共鉴定了246条已知miRNAs的成熟体序列，并预测得到262条novel miRNAs的成熟体序列；在毛竹种皮破裂阶段的种子中，丰度最高的已知miRNA为miR166，其次是miR159、miR6478、miR319等；根据miRNA靶基因预测结果，靶基因数量最多的已知miRNA属于MIR396家族，PH02Gene13935（GAMYB）被预测受到MIR159、MIR319、MIR396家族共28个miRNAs的调控；在6个比较组中共鉴定了181个差异表达miRNA，与对照组相比，10% PEG、15% PEG、50 mmol·L⁻¹ NaCl和100 mmol·L⁻¹ NaCl胁迫下表达水平最高且显著差异表达的已知miRNA分别为phe-miR171e-5p、phe-miR3630-3p、phe-miR171e-5p和phe-miR159a.1；差异表达miRNA靶基因能够显著富集在不同的GO和KEGG途径中；对10个差异表达miRNAs进行qRT-PCR验证，荧光定量结果的整体趋势和测序数据一致。
结论毛竹种皮破裂阶段种子中积累了大量的miR159、miR6478、miR319等已知miRNA，且在对照和胁迫处理组中均具有高表达水平，可能在毛竹种子萌发中具有保守调控作用；与对照组相比，phe-miR171e-5p、phe-miR3630-3p、phe-miR171e-5p和phe-miR159a.1在 10% PEG、15% PEG、50 mmol·L⁻¹ NaCl、100 mmol·L⁻¹ NaCl胁迫下分别显著差异表达，能够在毛竹种子露白阶段响应PEG或NaCl胁迫。

Abstract:
Objective To identify microRNAs (miRNAs) and reveal its expressional pattern in seed coat rupture stage of Moso bamboo seeds (Phyllostachys edulis) under different drought and salt stresses.
MethodsPolyethylene glycol (PEG6000) and NaCl were used to simulate drought and salinity stress, respectively. Small RNA libraries were separately built for Moso bamboo seeds germinated under H₂O, 10% PEG, 15% PEG, 50 mmol·L⁻¹ NaCl and 100 mmol·L⁻¹ NaCl, and the seeds were all sampled at seed coat rupture stage. High throughput sequencing and bioinformatics analysis were used to explore the expressional pattern of miRNA.
ResultsA total of 246 known miRNAs and 262 novel mature miRNAs were identified in this study. The most abundant miRNAs in seed coat rupture stage of Moso bamboo was miR166, followed by miR159, miR6478, miR319, etc. According to miRNA target prediction, MIR396 family owned the largest number of target genes, and ph02gene13935 (GAMYB) could to be regulated by 28 miRNAs of MIR159, MIR319 or MIR396; A total of 181 differentially expressed miRNAs (DEmiRNA) were identified in six comparison groups; Compared with control group, in 10% PEG, 15% PEG, 50 mmol·L⁻¹ NaCl and 100 mmol·L⁻¹ NaCl treatments, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a were differentially expressed respectively with highest expressional level in known miRNAs; The target genes of DEmiRNA were significantly enriched in different GO and KEGG pathways; Ten DEmiRNAs were verified by qPCR, and the overall trend of qPCR results was consistent with the sequencing data.
Conclusion In seed coat rupture stage of moso bamboo, there exhibit high accumulation of known miRNAs such as miR159, miR6478, miR319 in all control and four treatment groups, which may play a conservative regulatory role in Moso bamboo seed germination. Compared with the control group, phe-miR171e-5p, phe-miR3630-3p, phe-miR171e-5p and phe-miR159a 1 are differentially expressed in 10% PEG, 15% PEG, 50 mmol·L⁻¹ NaCl and 100 mmol·L⁻¹ NaCl, respectively, which can respond to PEG or NaCl stress during seed coat rupture stage of Moso bamboo.

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